CN105687205B - A kind of medical usage of triterpene compound - Google Patents

A kind of medical usage of triterpene compound Download PDF

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CN105687205B
CN105687205B CN201610131508.4A CN201610131508A CN105687205B CN 105687205 B CN105687205 B CN 105687205B CN 201610131508 A CN201610131508 A CN 201610131508A CN 105687205 B CN105687205 B CN 105687205B
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compound
influenza virus
mass spectrometric
triterpene compound
spectrometric data
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CN105687205A (en
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郭宝林
方伟
岑山
余利岩
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Institute of Medicinal Biotechnology of CAMS
Institute of Medicinal Plant Development of CAMS and PUMC
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Institute of Medicinal Plant Development of CAMS and PUMC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

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Abstract

The present invention provides triterpene compound, its stereoisomer, racemic modification, solvate, polymorphs body shown in a kind of structural formula I, or the pharmaceutically acceptable salt of triterpene compound shown in structural formula I, its stereoisomer, racemic modification, solvate, polymorphs body is preparing the purposes in anti-influenza virus medicament, wherein R1~R6It is defined in the description.

Description

A kind of medical usage of triterpene compound
Technical field
The invention belongs to medicine and pharmaceutical technology, and in particular to a kind of pentacyclic triterpenoid it is new pharmaceutical On the way.
Background technique
Influenza A virus is common influenza virus, to human pathogenic's height, not only causes seasonal influenza, also Zeng Duoci Cause it is worldwide be very popular, such as big influenza of Spain in 1918, the Asia influenza of nineteen fifty-seven, the Mao flu of nineteen sixty-eight, with And in April, 2009 is initially from Mexico and U.S.'s outburst and pandemic Influenza A H1N1 in the world.Due to first The highly pathogenic and high mortality of type influenza, either seasonal or worldwide prevalence, can all cause serious financial burden With society's fear.
Although vaccine is the most effective means for preventing viral infection, but since influenza A virus is easy variation and vaccine Production needs some cycles, causes the preventive and therapeutic effect of influenza vaccines not fully up to expectations.Drug therapy is still the master for the treatment of of influenza Want means, especially use anti-influenza virus medicament to treat as early as possible in initial infection, for patient especially old man, children, have The prognosis of the Susceptible population of the underlying diseases such as angiocarpy is significant.
Clinically have at present for the drug of resisiting influenza virus: (one) ion channel M2 retarding agent, such as amantadine (Amantadine), Rimantadine (Rimantadine) etc., such medicine clinical application time is more permanent, drug resistance strain drug resistance compared with It is more.(2) neuraminidase inhibitor, such as: Oseltamivir (Oseltamivir), zanamivir (Zanamivir), Peramivir (Peramivir) etc., it is the anti-influenza virus medicament of current mainstream.Although report is less, also there is influenza virus pair The clinical report of such Drug-resistant.In face of the variation and drug resistance of virus, current anti-influenza virus medicament meets far away clinic Needs.Therefore, the good anti-influenza virus medicament of new effective safety is found.
Saikosaponin a (Saikosaponin A, SSa), saikoside c (SSc) and saikoside d (SSd) etc. are from bavin The main active that the under ground portion of Hu platymiscium is isolated, they are all the pentacyclic triterpene chemical combination with epoxy ehter bond Object, wherein saikosaponin a and saikoside d isomers each other.Jianxin Chen etc. reports saikosaponin a in vitro and in vivo Has the function of anti-influenza A virus: where saikosaponin a inhibits H1N1PR8, H9N2 and H5N1 strains of influenza viruses in A549 The IC replicated in cell50Respectively 1.98,2.21 and 2.07 μM (Jianxin Chen, Mubing Duan, et al.Saikosaponin a inhibits influenza A virus replication and lung Immunopathology [J], Oncotarget, 2015,6 (40): 42541-42556).But it is also draped over one's shoulders simultaneously in this document Cytotoxicity is gone out to A549 cells show under dew external 48 hours 7.6 μM of concentration of saikosaponin a；And the isomery of saikosaponin a The cytotoxicity concentration of body saikoside d is lower.Therefore, the studies above show using saikosaponin a, d as representative it is this kind of at Divide safe dose window narrows, is not ideal new drug development candidate compound.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a kind of triterpene compound anti influenzas The new medical use of virus.Saikosaponin a is compared, such saponin compound cytotoxicity is small or even without cytotoxicity, peace Full window is big, is more suitable for the candidate compound of Tamiflu exploitation.
In order to achieve the above-mentioned object of the invention, present invention employs the following technical solutions:
Triterpene compound, its stereoisomer, racemic modification, solvate, polymorphic shown in a kind of structural formula I The pharmacy of triterpene compound shown in body or structural formula I, its stereoisomer, racemic modification, solvate, polymorphs body Upper acceptable salt is preparing the purposes in anti-influenza virus medicament,
Wherein, R1Selected from H, linear chain or branched chain C1-C6One of alkoxy, formoxyl and acetyl group, or it is selected from 0~2 A hydroxyl is by C1-C6One of rhamnose, glucose, fucose and the xylose that fatty acyl group replaces, or selected from by 0~2 Hydroxyl is by C1-C6Any disaccharides, trisaccharide or the tetrose that rhamnose, glucose, fucose, the xylose of fatty acyl group substitution connect into；
R2、R5And R6Selected from methyl or-CH2R7, wherein R7Selected from hydroxyl, substituted or unsubstituted linear chain or branched chain C1-C6Alkane Oxygroup, formyloxy or acetoxyl group；
R3And R4It is each independently selected from hydroxyl, substituted or unsubstituted linear chain or branched chain C1-C6Alkoxy, formyloxy Or acetoxyl group.
Preferably, R1Selected from H or 0~2 hydroxyl by C1-C4Rhamnose, the glucose, fucose of fatty acyl group substitution One of with xylose, or selected from by 0~2 hydroxyl by C1-C4Fatty acyl group replace rhamnose, glucose, fucose, Any disaccharides or trisaccharide that xylose connects into.
Preferably, R1Selected from H or 0~1 hydroxyl by C1-C4Rhamnose, the glucose, fucose of fatty acyl group substitution One of with xylose, or selected from by 0~1 hydroxyl by C1-C4Fatty acyl group replace rhamnose, glucose, fucose, Any disaccharides or trisaccharide that xylose connects into.
It is furthermore preferred that R1Selected from H or 0~1 hydroxyl by C1-C4Following monosaccharide, the disaccharides and three of fatty acyl group substitution One of pool is a variety of:
It is further preferred that R1Selected from H or 0~1 hydroxyl by C1-C4Following monosaccharide, the disaccharides of fatty acyl group substitution With one of three pools or a variety of:
Preferably, R7Selected from hydroxyl, linear chain or branched chain C1-C4Alkoxy, formyloxy or acetoxyl group.
It is furthermore preferred that R7Selected from hydroxyl, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, formyloxy or acetoxyl group.
Preferably, R3And R4It is each independently selected from hydroxyl, linear chain or branched chain C1-C4Alkoxy, formyloxy or acetyl oxygen Base.
It is furthermore preferred that R3And R4It is each independently selected from hydroxyl, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, formoxyl Or acetyl group.
As preferred embodiment, the triterpene compound of the structural formula I is selected from the compound having the following structure One of or it is a variety of:
As a kind of embodiment that can substitute of the present invention, the triterpene compound A ring and B ring of structural formula I can be with It is following structure:
Wherein, R3And R4As defined above, R8Selected from H, hydroxyl, linear chain or branched chain C1-C4Alkoxy.
Preferably, R8Selected from H, hydroxyl, methoxyl group, ethyoxyl, propoxyl group or isopropoxy；More it is selected as H, hydroxyl or methoxy Base.
It is above-mentioned can in alternate embodiments in, the triterpene compound that preferably has the following structure:
The triterpene compound of the structural formula I provided by the present invention is in the purposes for preparing anti-influenza virus medicament Refer to triterpene compound, its stereoisomer, racemic modification, solvate, polymorphs body or the structural formula I of the structural formula I Shown in triterpene compound, its stereoisomer, racemic modification, solvate, polymorphs body pharmaceutically acceptable Salt is as sole active agent, or with other substances with resisiting influenza virus effect collectively as active constituent, with pharmacy The anti-influenza virus medicament is prepared by conventional method in upper acceptable auxiliary material.
Other substances with resisiting influenza virus effect are selected from amantadine, Rimantadine, Oseltamivir, Zha Na meter One of Wei and Peramivir are a variety of.
The pharmaceutically acceptable auxiliary material is selected from solvent (such as water, ethyl alcohol), (such as starch, pregelatinated form sediment filler Powder, Icing Sugar, dextrin, lactose, microcrystalline cellulose, calcium sulfate etc.), adhesive (such as water, ethyl alcohol, starch slurry, dextrin, Icing Sugar, syrup, Rubber cement, polyethylene glycol, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose etc.), disintegrating agent (such as dried starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, cross-linked carboxymethyl cellulose Sodium, sodium bicarbonate, citric acid, magnesium stearate, talcum powder etc.) and one of coating material or a variety of.
The Tamiflu includes oral preparation and non-oral formulation.The oral preparation include but is not limited to tablet, Capsule, pill, suspension, syrup, oral solution and granule.The non-oral formulation include but is not limited to injection, Spray, nasal drop and suppository.
Triterpene compound of the present invention shows stronger anti-influenza virus activity in vitro, wherein representativenessization Close the EC for inhibiting influenza virus WSN33 outside object50Both less than Ribavirin, and the cytotoxicity of above compound is both less than radix bupleuri Saponin(e a.Therefore, the present invention is expected to provide new selection for clinical treatment influenza.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows each test-compound anti-influenza virus activity of embodiment 2, and ordinate is opposite Gluc activity, horizontal Coordinate is the number and title of test-compound.
Fig. 2 shows each test-compound cytotoxicity of embodiment 2, and ordinate is versus cell activity, and abscissa is The number and title of test-compound.
Fig. 3 shows anti-influenza virus activity and cytotoxicity under the test-compound various concentration of number 2 in embodiment 3 Measurement result.Wherein, influenza virus relative activity under various concentration is shown in 3A, and cell under various concentration is shown in 3B Relative activity.
Fig. 4 shows anti-influenza virus activity and cytotoxicity under the test-compound various concentration of number 3 in embodiment 3 Measurement result.Wherein, influenza virus relative activity under various concentration is shown in 4A, and cell under various concentration is shown in 4B Relative activity.
Fig. 5 shows anti-influenza virus activity and cytotoxicity under the test-compound various concentration of number 5 in embodiment 3 Measurement result.Wherein, influenza virus relative activity under various concentration is shown in 5A, and cell under various concentration is shown in 5B Relative activity.
Fig. 6 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 19 in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 6A, and cell under various concentration is shown in 6B Relative activity.
Fig. 7 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 11 in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 7A, and cell under various concentration is shown in 7B Relative activity.
Fig. 8 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 15a in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 8A, and cell under various concentration is shown in 8B Relative activity.
Fig. 9 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 15b in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 9A, and cell under various concentration is shown in 9B Relative activity.
Figure 10 shows anti-influenza virus activity and cell under the test-compound various concentration of number 22b in embodiment 3 Toxicity test result.Wherein, influenza virus relative activity under various concentration is shown in 10A, and 10B is shown under various concentration The relative activity of cell.
Figure 11 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 23 in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 11A, and 11B is shown under various concentration carefully The relative activity of born of the same parents.
Figure 12 shows anti-influenza virus activity and cell toxicant under the test-compound various concentration of number 27 in embodiment 3 Property measurement result.Wherein, influenza virus relative activity under various concentration is shown in 12A, and 12B is shown under various concentration carefully The relative activity of born of the same parents.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples Material raw material, reagent material etc. are commercially available products unless otherwise specified.Wherein, portion of reagent and Chinese medicine buy situation It is as follows:
Bupleurum marginatum var. Stenophyllum (Wolff) shan et Li (Bupleurum marginatum var.stenophyllum (Wolff) Shan et Li) produces From Gansu Weiyuan.
Dimethyl sulfoxide (DMSO) is purchased from Sigma company；DMEM, 0.25% pancreatin, FBS containing EDTA are purchased from Invitrogen company；Coelenterazine-h is purchased from Promega company；CCK-8 kit is purchased from the green skies biology in Shanghai Science and Technology Ltd..
293T-Gluc cell and strains of influenza viruses A/WSN/33 are provided by Chinese Academy of Medical Sciences's biotechnology research institute.
Embodiment 1The preparation of triterpene compound
8.2kg Bupleurum marginatum var. Stenophyllum (Wolff) shan et Li dry root is heated to 70% ethyl alcohol (containing 0.5% ammonium hydroxide) leaching of boiling with 10 times of amounts after crushing Bubble extracts 2 times, each 48h.Merge extracting solution twice, be concentrated under reduced pressure into no alcohol taste, adds upper macroporous resin column D101 after water dispersion, Respectively with 10% ethyl alcohol, 95% ethanol gradient elution, 10% alcohol elution is discarded, 95% alcohol elution is total saposins (400g)。
Take total saposins 300g to mix MCI column on sample (aperture resin), respectively with 20% methanol, 40% methanol, 60% methanol, 90% methanol, 100% methanol elution gradient.Silica gel chromatographic column on sample (200-300 mesh) is mixed in 90% methanol elution position (150g) Separation, with methylene chloride-methanol gradient elution (25: 1~0: 1), merge after TLC is detected same composition obtain 7 fractions (A, B, C, D, E, F, G).Pressure ODS post separation (50% methanol~100% methanol) obtains 4 fractions (D-1, D-2, D- in fraction D warp 3, D-4), wherein fraction D-2 through HPLC preparative separation (stationary phase: unitary C-18 (250mm × 10mm, 5 μm), mobile phase: 45% acetonitrile) obtain compound 2 and compound 3.Pressure ODS separation (50% methanol~90% methanol) obtains 5 streams in fraction E warp Part (E1, E2, E3, E4, E5), wherein fraction E3 through HPLC preparative separation (stationary phase: unitary C-18 (250mm × 10mm, 5 μm), mobile phase: 38% acetonitrile) obtain compound 5 and compound 11.Silica gel on sample is mixed in 60% methanol elution position (120g) again Chromatographic column (200-300 mesh) separation, with methylene chloride-methanol gradient elution (25: 1~0: 1), merges identical after TLC is detected Component obtains 3 flow points (I, II, III).It is detected through TLC and HPLC, merges fraction E and fraction II to obtain fraction H1 (51.6g), fraction G and fraction III merge to obtain H2 (30.0g).Pressure MDS separation (60%~90% methanol) obtains 5 in H1 warp A fraction (H1-O-1~H1-O-5).Wherein fraction H1-O-4 obtains chemical combination through HPLC preparative separation (mobile phase: 75% methanol) Object 19, fraction H1-O-4-1 and fraction H1-O-4-2, wherein fraction H1-O-4-2 is through HPLC preparative separation (mobile phase: 64% first Alcohol) obtain compound 15a and 15b.Fraction H1-O-2 obtains compound 21 with HPLC preparative separation (mobile phase: 76% methanol).
ODS (mobile phase: 40% methanol~70% methanol) isolated 6 fraction (H2-O-1~H2-O- are pressed in H2 warp 6).Fraction H2-O-5 obtains compound 27 with HPLC preparative separation (mobile phase: 33% acetonitrile).Fraction H2-O-4 is prepared through HPLC Separation (mobile phase: 31% acetonitrile) obtains compound 23,25 and fraction H2-O-4-1；Fraction H2-O-4-1 is prepared through HPLC again Separation (mobile phase: 70% methanol) obtains compound 22b.Fraction H2-O-2 warp in pressure ODS separation (mobile phase: 50% methanol~ 90% methanol) obtain 6 components (H2-O-2-1~H2-O-2-6).Wherein fraction H2-O-2-5 is through (the flowing of HPLC preparative separation Phase: 27% acetonitrile) obtain compound 29；Fraction H2-O-2-3 obtains chemical combination through HPLC preparative separation (mobile phase: 25% acetonitrile) Object 32；Fraction H2-O-2-2 obtains compound 35 through HPLC preparative separation (22% acetonitrile).
The above-mentioned number 11 being prepared, 15a, 15b, 21, the triterpene compounds of 22b, 23,25,27,29,32,35 warp Glucosides enzyme hydrolysis is crossed, removes monosaccharide one by one, obtains secondary glucoside and aglycon.
The above-mentioned triterpene compound being prepared, the compound of number 2,3,5 and 19 are the radix bupleuri with epoxy ehter bond Saponins compound.
The Structural Identification data for each compound that the present embodiment is prepared are as follows:
Compound 2 (6 "-crotonyl-saikosaponin a)
Chemical formula C46H72O14, mass spectrum m/z 871 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.98 (1H, m, H-3 " '), 6.02 (1H, br d, J=10.8Hz, H-12), 5.91 (1H, m, H-2 " '), 5.68 (1H, dd, J=10.2,3.0Hz, H-11), 5.29 (1H, d, J=7.8Hz, H-1 "), 5.03 (1H, d, J=7.8Hz, H-1 '), 1.61 (3H, dd, J=6.6,1.2Hz, H-4 " '), 1.46 (3H, d, J=6.0Hz, H-6 '), 1.42 (3H, s, H-26), 1.12 (3H, s, H-27), 1.01 (3H, s, H-25), 0.95 (3H, s, H-29), 0.94 (3H, s, H- 30), 0.92 (3H, s, H-24)；.
13C-NMR(C5D5N, 600MHz) δ: 166.8 (C-1 " '), 145.5 (C-3 " '), 132.6 (C-11), 131.6 (C- 12), 123.5 (C-3 " '), 107.1 (C-1 "), 106.5 (C-1 '), 85.9 (C-3 '), 84.5 (C-13), 82.1 (C-3), 78.7 (C-4 "), 76.0 (C-2 "), 75.9 (C-5 "), 73.5 (C-28), 72.6 (C-4 '), 72.1 (C-3 "), 72.0 (C-2 '), 71.5 (C-5 '), 65.0 (C-6 "), 64.5 (C-16, C-23), 53.6 (C-9), 52.6 (C-18), 47.8 (C-5), 47.5 (C-17), 46.1 (C-14), 44.2 (C-4), 42.7 (C-8), 39.1 (C-1), 38.2 (C-19), 36.7 (C-15), 36.6 (C-10), 35.2 (C-21), 34.1 (C-29), 32.1 (C-7, C-20), 26.6 (C-2), 26.2 (C-22), 24.3 (C-30), 21.3 (C- 27), 20.5 (C-26), 19.2 (C-25), 18.2 (C-6), 18.0 (C-4 " '), 17.8 (C-6 '), 13.5 (C-24).
Compound 3 (saikosaponin a),
Chemical formula C42H68O13, mass spectrometric data m/z 803 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.02 (1H, d, J=10.8Hz, H-12), 5.68 (1H, dd, J=10.8, 3.0Hz, H-11), 5.37 (1H, d, J=7.8Hz, H-1 "), 5.00 (1H, d, J=7.8Hz, H-1 '), 1.46 (3H, d, J= 6.0Hz, H-6 '), 1.42 (3H, s, H-26), 1.12 (3H, s, H-27), 1.01 (3H, s, H-25), 0.95 (3H, s, H-29), 0.94 (3H, s, H-30), 0.92 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 132.6 (C-11), 131.6 (C-12), 107.2 (C-1 "), 106.5 (C- 1 '), 85.7 (C-3 '), 84.5 (C-13), 82.0 (C-3), 79.3 (C-3 "), 78.9 (C-5 "), 76.3 (C-2 "), 73.5 (C- 28), 72.6 (C-4 "), 72.3 (C-4 '), 72.0 (C-2 '), 71.5 (C-5 '), 64.5 (C-23), 64.4 (C-16), 63.2 (C- 6 "), 53.6 (C-9), 52.6 (C-18), 47.8 (C-5), 47.5 (C-17), 46.1 (C-14), 44.2 (C-4), 42.7 (C-8), 39.1 (C-1), 38.2 (C-19), 36.7 (C-10), 36.6 (C-15), 35.2 (C-21), 34.1 (C-24), 32.0 (C-7, C- 20), 26.6 (C-2), 26.2 (C-22), 24.3 (C-30), 21.3 (C-27), 20.5 (C-26), 19.2 (C-25), 18.0 (C- 6), 17.7 (C-6 '), 13.5 (C-24).
Compound 5 (saikoside e),
Chemical formula C42H68O12, mass spectrometric data m/z 787 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.00 (1H, d, J=10.8,2.4Hz, H-12), 5.70 (1H, dd, J= 10.8,3.0Hz, H-11), 5.44 (1H, d, J=7.8Hz, H-1 "), 4.78 (1H, d, J=7.8Hz, H-1 '), 1.50 (3H, d, J=6.6Hz, H-6 '), 1.39 (3H, s, H-26), 1.34 (3H, s, H-23), 1.20 (3H, s, H-27), 0.99 (6H, s, H- 25, H-29), 0.93 (6H, s, H-24, H-30).
13C-NMR(C5D5N, 600MHz) δ: 132.6 (C-11), 131.7 (C-12), 107.3 (C-1 "), 107.2 (C- 1 '), 89.1 (C-3), 85.6 (C-3 '), 84.5 (C-13), 79.2 (C-3 "), 78.9 (C-5 "), 76.3 (C-2 "), 73.5 (C- 28), 72.7 (C-4 "), 72.2 (C-2 '), 72.0 (C-4 '), 71.5 (C-5 '), 64.5 (C-16), 63.1 (C-6 "), 55.8 (C- 5), 53.4 (C-9), 52.6 (C-18), 47.5 (C-17), 46.1 (C-14), 42.7 (C-8), 40.2 (C-4), 39.1 (C-1), 38.2 (C-19), 36.8 (C-10), 36.6 (C-15), 35.2 (C-21), 34.2 (C-29), 32.3 (C-7), 32.1 (C-20), 28.3 (C-23), 27.1 (C-2), 26.2 (C-22), 24.3 (C-30), 21.4 (C-27), 20.5 (C-26), 18.7 (C-6), 18.4 (C-25), 17.8 (C-6 '), 16.8 (C-24).
Compound 11 (preceding saikoside d),
Chemical formula C36H58O8, mass spectrometric data m/z 641 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.74 (1H, dd, J=10.2,3.2Hz, H-11), 5.76 (1H, d, J= 10.2Hz, H-12), 5.02 (1H, d, J=7.2Hz, H-1 '), 1.70 (3H, s, H-27), 1.58 (3H, d, J=6.6Hz, H- 6 '), 1.07 (3H, s, H-26), 1.05 (3H, s, H-25), 1.01 (3H, s, H-29), 0.95 (3H, s, H-30), 0.91 (3H, S, H-24).
13C-NMR(C5D5N, 600MHz) δ: 136.6 (C-13), 133.5 (C-18), 126.8 (c-12), 126.7 (C- 11), 106.8 (C-1 '), 82.2 (C-3), 76.0 (C-3 '), 73.5 (C-2 '), 73.3 (C-4 '), 71.8 (C-5 '), 68.1 (C- 16), 65.2 (C-23), 64.9 (C-28), 54.5 (C-9), 48.0 (C-5), 45.8 (C-17), 44.2 (C-4), 42.4 (C- 14), 41.6 (C-8), 39.5 (C-19), 38.9 (C-1), 37.0 (C-10), 36.0 (C-21), 33.1 (C-20), 33.0 (C- 15), 32.8 (C-7), 32.4 (C-30), 26.6 (C-2), 25.6 (C-29), 24.9 (C-22), 22.4 (C-27), 19.4 (C- 6), 18.8 (C-25), 18.0 (C-26), 17.8 (C-6 '), 13.6 (C-24)
Compound 11-1: obtained aglycon, mass spectrometric data m/z 495 [M+Na] are hydrolyzed by compound 11+.
Compound 15a (saikoside g),
Chemical formula C42H68O13Mass spectrometric data m/z 803 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 5.77 (1H, d, J=5.4Hz, H-11), 5.65 (1H, d, J=5.4Hz, H- 12), 5.36 (1H, d, J=7.8Hz, H-1 "), 4.98 (1H, d, J=7.8Hz, H-1 '), 1.45 (3H, d, J=6.0Hz, H- 6 '), 1.31 (3H, s, H-27), 1.30 (3H, s, H-25), 1.29 (3H, s, H-26), 1.00 (3H, s, H-30), 0.99 (3H, S, H-29), 0.89 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 155.5 (C-9), 145.8 (C-13), 121.6 (c-12), 116.5 (C-11), 107.2 (C-1 "), 106.5 (C-1 '), 85.7 (C-3 '), 81.9 (C-3), 79.3 (C-3 "), 78.9 (C-5 "), 76.3 (C- 2 "), 72.7 (C-4 "), 72.3 (C-4 '), 72.0 (C-2 '), 71.5 (C-5 '), 69.8 (C-28), 67.2 (C-16), 64.6 (C- 23), 63.1 (C-6 "), 47.4 (C-19), 44.2 (C-8), 44.0 (C-5), 43.8 (C-4), 43.6 (C-14), 43.1 (C- 18), 41.0 (C-17), 39.2 (C-10), 38.1 (C-1), 36.7 (C-15), 34.6 (C-21), 33.6 (C-29), 32.6 (C- 7), 31.5 (C-20), 27.4 (C-2), 26.6 (C-22), 26.5 (C-27), 24.5 (C-30), 21.7 (C-26), 21.5 (C- 25), 18.4 (C-6), 17.7 (C-6 '), 14.2 (C-24)
Compound 15a-1: the monoglycosides hydrolyzed by compound 15a, mass spectrometric data m/z 641 [M+Na]+.
Compound 15a-2: the aglycon hydrolyzed by compound 15a-1, mass spectrometric data m/z 495 [M+Na]+.
Compound 15b (3 β, 16 α, 23,28-tetradroxyolean-9 (11), 12-diene-3-O- β-D- Glucopyranosyl- (1 → 3)-β-D-fucopyranoside),
Chemical formula C42H68O13, mass spectrometric data m/z 803 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 5.84 (1H, d, J=5.4Hz, H-11), 5.78 (1H, d, J=5.4Hz, H- 12), 5.36 (1H, d, J=7.8Hz, H-1 "), 4.97 (1H, d, J=7.8Hz, H-1 '), 1.74 (3H, s, H-27), 1.45 (3H, d, J=6.0Hz, H-6 '), 1.32 (3H, s, H-25), 1.27 (3H, s, H-26), 1.13 (3H, s, H-30), 1.02 (3H, S, H-29), 0.99 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 155.5 (C-9), 147.5 (C-13), 121.5 (C-12), 116.6 (C-11), 107.3 (C-1 "), 106.5 (C-1 '), 85.8 (C-3 '), 82.0 (C-3), 79.3 (C-3 "), 78.9 (C-5 "), 76.3 (C- 2 "), 75.2 (C-16), 72.6 (C-4 '), 72.3 (C-4 "), 72.0 (C-2 '), 71.5 (C-5 '), 70.9 (C-28), 64.7 (C- 23), 63.2 (C-6 "), 49.0 (C-19), 44.2 (C-8), 44.1 (C-5), 41.3 (C-4), 41.2 (C-14), 41.1 (C- 18), 39.2 (C-10), 38.2 (C-1), 37.5 (C-15), 34.9 (C-21), 33.7 (C-29), 32.8 (C-7), 31.8 (C- 20), 31.3 (C-20), 27.5 (C-2), 26.7 (C-22), 25.3 (C-27), 21.8 (C-26), 21.6 (C-25), 18.5 (C- 6), 17.7 (C-6 '), 14.2 (C-24).
Compound 15b-1: the monoglycosides hydrolyzed by compound 15b, mass spectrometric data m/z 641 [M+Na]+.
Compound 15b-2: the aglycon hydrolyzed by compound 15b-1, mass spectrometric data m/z 495 [M+Na]+.
Compound 19 (saikoside d),
Chemical formula C42H68O13, mass spectrometric data m/z 803 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.06 (1H, d, J=10.2Hz, H-12), 5.72 (1H, dd, J=10.2, 3.0Hz, H-11), 5.36 (1H, d, J=7.8Hz, H-1 "), 5.00 (1H, d, J=7.8Hz, H-1 '), 1.65 (3H, s, H- 27), 1.46 (3H, d, J=6.0Hz, H-6 '), 1.38 (3H, s, H-26), 1.05 (6H, s, H-25, H-29), 0.98 (3H, s, H-30), 0.96 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 132.5 (C-11), 132.4 (C-12), 107.2 (C-1 "), 106.5 (C- 1 '), 85.7 (C-3 '), 85.4 (C-13), 82.2 (C-3), 79.3 (C-3 "), 78.9 (C-5 "), 78.3 (C-28), 77.6 (C- 16), 76.3 (C-2 "), 72.7 (C-4 '), 72.3 (C-2 '), 72.0 (C-4 "), 71.5 (C-5 '), 64.6 (C-23), 63.2 (C- 6 "), 53.5 (C-9), 51.9 (C-18), 47.9 (C-5), 45.8 (C-17), 44.2 (C-4), 44.1 (C-14), 42.4 (C-8), 39.2 (C-1), 38.9 (C-19), 37.3 (C-21), 36.8 (C-10), 36.0 (C-15), 34.3 (C-29), 32.4 (C-20), 32.0 (C-7), 31.8 (C-22), 26.6 (C-2), 24.9 (C-30), 20.1 (C-26), 19.3 (C-25), 18.6 (C-27), 18.1 (C-6), 17.8 (C-6 '), 13.6 (C-24).
(the saikoside b of compound 213),
Chemical formula C43H72O14, mass spectrometric data m/z 835 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 5.86 (1H, br s, H-11), 5.55 (1H, d, J=3.0Hz, H-12), 5.36 (1H, d, J=7.8Hz, H-1 "), 5.00 (1H, d, J=7.8Hz, H-1 '), 3.25 (3H, s, OCH3), 1.43 (3H, d, J= 6.6Hz, H-6 '), 1.42 (3H, s, H-26), 1.13 (6H, s, H-27), 1.09 (3H, s, H-25), 1.02 (3H, s, H-29), 0.98 (3H, s, H-30), 0.91 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 148.8 (C-13), 123.0 (C-12), 107.2 (C-1 "), 106.5 (C- 1 '), 85.8 (C-3 '), 82.2 (C-3), 79.3 (C-5 "), 78.9 (C-3 "), 76.4 (C-11), 76.3 (C-2 "), 72.7 (C- 4 "), 72.3 (C-4 '), 72.1 (C-2 '), 71.5 (C-5 '), 69.0 (C-28), 66.7 (C-16), 64.7 (C-23), 63.2 (C- 6 "), 54.5 (- OCH3), 52.6 (C-9), 48.1 (C-5), 47.5 (C-19), 44.5 (C-14), 44.4 (C-18), 44.3 (C- 17), 44.1 (C-4), 41.5 (C-8), 40.6 (C-1), 38.6 (C-10), 37.3 (C-15), 34.7 (C-21), 33.8 (C-7), 33.7 (C-29), 31.6 (C-20), 27.0 (C-27), 26.8 (C-2), 26.4 (C-22), 24.5 (C-30), 18.9 (C-6), 18.8 (C-26), 18.4 (C-25), 17.7 (C-6 '), 14.2 (C-24).
Compound 21-1: obtained monoglycosides, mass spectrometric data m/z 673 [M+Na] are hydrolyzed by compound 21+.
Compound 21-2: the aglycon hydrolyzed by compound 21-1, mass spectrometric data m/z 527 [M+Na]+.
Compound 22b (nepasaikosaponin k),
Chemical formula C48H80O18, mass spectrometric data m/z 967 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 5.51 (1H, d, J=3.0Hz, H-12), 5.26 (1H, brs, H-1 " '), 5.02 (1H, d, J=7.8Hz, H-1 "), 4.95 (1H, d, J=7.8Hz, H-1 '), 3.21 (3H, s ,-OCH3), 1.38 (3H, d, J= 7.2Hz, H-6 '), 1.37 (3H, s, H-26), 1.09 (6H, s, H-27), 1.05 (3H, s, H-25), 0.98 (3H, s, H-29), 0.94 (3H, s, H-30), 0.86 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 144.4 (C-13), 123.1 (C-12), 82.7 (C-3), 67.0 (C-16), 65.0 (C-28, C-23), 48.0 (C-19), 47.6 (C-9), 47.5 (C-5), 44.9 (C-18), 44.3 (C-14), 44.0 (C- 4), 41.5 (C-8), 40.6 (C-17), 39.2 (C-1), 37.3 (C-15), 37.2 (C-15), 34.7 (C-21), 33.8 (C- 29), 33.1 (C-7), 31.5 (C-20), 27.6 (C-27), 26.6 (C-22), 26.4 (C-2), 24.5 (C-30), 24.4 (C- 11), 18.6 (C-6), 17.4 (C-26), 16.8 (C-25), 14.1 (C-24)；3-glu, δ: 106.3 (C-1 '), 75.6 (C- 2 '), 77.3 (C-3 '), 80.2 (C-4 '), 76.0 (C-5 '), 69.3 (C-6 ')；4 '-rha, δ: 103.5 (C-1 "), 73.0 (C- 2 "), 73.2 (C-3 "), 74.3 (C-4 "), 71.0 (C-5 "), 19.0 (C-6 ")；6 '-glu, δ: 105.6 (C-1 " '), 75.3 (C- 2 " '), 78.9 (C-3 " '), 71.8 (C-4 " '), 79.0 (C-5 " '), 63.0 (C-6 " ').
Compound 22b-1: mass spectrometric data m/z 821 [M+Na]+.
Compound 22b-2: mass spectrometric data m/z 805 [M+Na]+.
Compound 22b-3: mass spectrometric data m/z 659 [M+Na]+.
Compound 22b-4: mass spectrometric data m/z 497 [M+Na]+.
Compound 23 (saikoside n),
Chemical formula C48H78O18, mass spectrometric data m/z 965 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.52 (1H, dd, J=10.2,2.4Hz, H-11), 5.66 (1H, d, J= 10.2Hz, H-12), 5.05 (1H, d, J=7.8Hz, H-1 '), 4.96 (1H, d, J=7.8Hz, H-1 "), 1.67 (3H, d, J= 6.6Hz, H-6 "), 1.06 (3H, s, H-26), 0.99 (3H, s, H-25), 0.96 (3H, s, H-29), 0.95 (3H, s, H-30), 0.89 (3H, s, H-24), 0.86 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 136.9 (C-13), 133.8 (C-18), 127.6 (C-11), 126.2 (C- 12), 82.5 (C-3), 77.1 (C-16), 64.7 (C-23), 55.0 (C-9), 47.8 (C-5), 44.9 (C-17), 44.8 (C- 14), 44.2 (C-4), 41.0 (C-8), 38.9 (C-1), 38.7 (C-19), 37.0 (C-10), 35.7 (C-15), 35.4 (C- 21), 33.2 (C-20), 32.9 (C-30), 32.8 (C-7), 30.5 (C-22), 26.5 (C-2), 25.3 (C-29), 22.5 (C- 27), 19.3 (C-6), 18.7 (C-25), 17.5 (C-26), 13.7 (C-24)；3-glu, δ: 106.3 (C-1 '), 75.7 (C- 2 '), 77.3 (C-3 '), 80.2 (C-4 '), 76.0 (C-5 '), 69.3 (C-6 ')；4 '-rha, δ: 103.5 (C-1 "), 73.0 (C- 2 "), 73.1 (C-3 "), 74.3 (C-4 "), 71.1 (C-5 "), 19.0 (C-6 ")；6 '-glu, δ: 105.6 (C-1 " '), 75.3 (C- 2 " '), 78.9 (C-3 " '), 72.0 (C-4 " '), 79.0 (C-5 " '), 63.1 (C-6 " ').
Compound 23-1: mass spectrometric data m/z 819 [M+Na]+.
Compound 23-2: mass spectrometric data m/z 803 [M+Na]+.
Compound 23-3: mass spectrometric data m/z 657 [M+Na]+.
Compound 25 (saikoside i),
Chemical formula C48H78O17, mass spectrometric data m/z 949 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 5.68 (1H, d, J=5.4Hz, H-11), 5.63 (1H, d, J=5.4Hz, H- 12), the 4.98 (- rha-H-1 of 1H, d, J=7.8Hz, 4 ' "), 4.81 (1H, d, J=7.8Hz, 3-glu-H-1 '), 4.77 (1H, br S, 6 '-glu-H-1 " '), the 1.68 (- rha-H-6 of 3H, d, J=6.0Hz, 4 ' "), 1.35,1.29,1.27,1.15,1.04, 1.03,0.94 (each 3H, s, 7 × CH3)。
13C-NMR(C5D5N, 600MHz) δ: 155.3 (C-9), 145.8 (C-13), 121.6 (C-12), 116.7 (C-11), 89.3 (C-3), 69.5 (C-28), 67.2 (C-16), 52.3 (C-5), 47.5 (C-19), 43.7 (C-8), 43.6 (C-14), 43.1 (C-18), 41.1 (C-17), 40.1 (C-4), 39.2 (C-10), 37.9 (C-1), 36.6 (C-15), 34.6 (C-21), 33.7 (C-29), 32.9 (C-7), 31.5 (C-20), 28.8 (C-23), 27.7 (C-2), 26.6 (C-22), 25.9 (C-27), 24.5 (C-30), 21.7 (C-26), 21.5 (C-25), 19.0 (C-6), 17.6 (C-24)；3-glu, δ: 107.1 (C-1 '), 75.5 (C-2 '), 77.3 (C-3 '), 80.2 (C-4 '), 76.0 (C-5 '), 69.7 (C-6 ')；4 '-rha, δ: 103.5 (C-1 "), 73.0 (C-2 "), 73.2 (C-3 "), 74.3 (C-4 "), 71.0 (C-5 "), 19.0 (C-6 ")；6 '-glu, δ: 105.7 (C-1 " '), 75.3 (C-2 " '), 78.9 (C-3 " '), 71.8 (C-4 " '), 79.0 (C-5 " '), 63.0 (C-6 " ').
Compound 25-1: mass spectrometric data m/z 803 [M+Na]+.
Compound 25-2: mass spectrometric data m/z 787 [M+Na]+.
Compound 25-3: mass spectrometric data m/z 641 [M+Na]+.
Compound 25-4: mass spectrometric data m/z 479 [M+Na]+.
Compound 27 (saikoside h),
Chemical formula C48H78O17, mass spectrometric data m/z 949 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.52 (1H, dd, J=10.2Hz, 2.4Hz, H-11), 5.66 (1H, d, J= 10.2Hz, H-12), 5.05 (1H, d, J=7.8Hz, H-1 '), 4.96 (1H, d, J=7.8Hz, H-1 "), 1.67 (3H, d, J= 6.6Hz, H-6 "), 1.06 (3H, s, H-26), 0.99 (3H, s, H-25), 0.96 (3H, s, H-29), 0.95 (3H, s, H-30), 0.89 (3H, s, H-24), 0.86 (3H, s, H-24).
13C-NMR(C5D5N, 600MHz) δ: 136.7 (C-13), 133.9 (C-18), 127.4 (C-12), 126.2 (C- 11), 89.5 (C-3), 64.4 (C-23), 55.7 (C-9), 54.7 (C-5), 44.9 (C-17), 44.7 (C-4), 40.9 (C-14), 40.1 (C-8), 38.9 (C-19), 38.6 (C-1), 36.9 (C-10), 35.7 (C-21), 35.3 (C-20), 33.1 (C-15), 33.0 (C-15), 32.7 (C-7), 30.4 (C-30), 28.4 (C-2), 27.0 (C-29), 25.3 (C-22), 22.5 (C-27), 19.0 (C-6), 18.9 (C-25), 17.4 (C-26), 16.9 (C-24)；3-glu, δ: 107.3 (C-1 '), 75.7 (C-2 '), 77.2 (C-3 '), 80.3 (C-4 '), 76.0 (C-5 '), 69.6 (C-6 ')；4 '-rha, δ: 103.5 (C-1 "), 73.0 (C-2 "), 73.2 (C-3 "), 74.3 (C-4 "), 71.1 (C-5 "), 18.7 (C-6 ")；6 '-glu, δ: 105.6 (C-1 " '), 75.3 (C- 2 " '), 78.9 (C-3 " '), 71.9 (C-4 " '), 79.0 (C-5 " '), 63.0 (C-6 " ').
Compound 27-1: mass spectrometric data m/z 803 [M+Na]+.
Compound 27-2: mass spectrometric data m/z787 [M+Na]+.
Compound 27-3: mass spectrometric data m/z 641 [M+Na]+.
Compound 27-4: mass spectrometric data m/z 479 [M+Na]+.
Compound 29 (saikoside s),
Chemical formula C48H78O18, mass spectrometric data m/z 965 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.69 (1H, br d, J=10.8Hz, H-11), 5.69 (1H, d, J= 10.8Hz, H-12), the 4.96 (- rha-H-1 of 1H, d, J=7.8Hz, 4 ' "), 4.84 (1H, br s, 3-glu-H-1 '), 4.74 (1H, br s, 6 '-glu-H-1 " '), the 1.67 (- rha-H-6 of 3H, d, J=6.6Hz, 4 ' "), 1.66 (3H, s, H-27), 1.07 (3H, s, H-26), 1.01 (3H, s, H-25), 0.99 (3H, s, H-29), 0.97 (3H, s, H-30), 0.89 (3H, s, H-24)；.
13C-NMR(C5D5N, 600MHz) δ: 136.5 (C-13), 133.5 (C-18), 126.7 (C-12), 126.6 (C- 11), 82.5 (C-3), 68.1 (C-16), 65.2 (C-28), 64.8 (C-23), 54.4 (C-9), 47.8 (C-5), 45.8 (C- 17), 44.1 (C-4), 42.4 (C-14), 41.6 (C-8), 39.5 (C-19), 38.8 (C-1), 37.0 (C-10), 35.9 (C- 21), 33.1 (C-20), 33.0 (C-15), 32.8 (C-7), 32.4 (C-30), 26.5 (C-2), 25.6 (C-29), 24.9 (C- 22), 22.4 (C-27), 19.4 (C-6), 19.0 (C-25), 17.7 (C-26), 13.7 (C-24)；3-glu, δ: 106.3 (C- 1 '), 75.6 (C-2 '), 77.3 (C-3 '), 80.2 (C-4 '), 76.0 (C-5 '), 69.3 (C-6 ')；4 '-rha, δ: 103.5 (C- 1 "), 73.0 (C-2 "), 73.1 (C-3 "), 74.3 (C-4 "), 71.1 (C-5 "), 18.7 (C-6 ")；6 '-glu, δ: 105.6 (C- 1 " '), 75.2 (C-2 " '), 78.9 (C-3 " '), 71.9 (C-4 " '), 79.0 (C-5 " '), 63.0 (C-6 " ').
Compound 29-1: mass spectrometric data m/z 819 [M+Na]+.
Compound 29-2: mass spectrometric data m/z 803 [M+Na]+.
Compound 29-3: mass spectrometric data m/z 657 [M+Na]+.
Compound 29-4: mass spectrometric data m/z 492 [M+Na]+.
Compound 32 (3 β, 16 β, 23,28,30-pentahydroxy-11,13 (18)-diene-3-O- β-D- Glucopyra-nosyl- (1 → 3)-β-D-fucopyranoside),
Chemical formula C42H68O14, mass spectrometric data m/z 819 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.62 (1H, d, J=10.2Hz, H-12), 5.70 (1H, br d, J= 10.2Hz, H-11), 5.37 (1H, d, J=7.8Hz, H-1 "), 5.03 (1H, d, J=7.8Hz, H-1 '), 1.48 (3H, d, J= 6.0Hz, H-6 '), 1.13 (3H, s, H-27), 1.10 (3H, s, H-29), 0.98 (3H, s, H-25), 0.93 (3H, s, H-24), 0.88 (3H, s, H-26).
13C-NMR(C5D5N, 600MHz) δ: 137.1 (C-13), 133.7 (C-18), 127.4 (C-12), 126.3 (C- 11), 107.3 (C-1 "), 106.6 (C-1 '), 85.7 (C-3 '), 82.0 (C-3), 79.3 (C-5 "), 78.9 (C-3 "), 76.8 (C-2 "), 76.3 (C-30), 73.6 (C-30), 72.6 (C-4 "), 72.3 (C-2 '), 72.0 (C-4 '), 71.5 (C-5 '), 64.7 (C-28), 64.4 (C-23), 63.2 (C-6 "), 55.0 (C-9), 47.7 (C-5), 45.4 (C-17), 44.8 (C-4), 44.2 (C- 14), 40.9 (C-8), 38.9 (C-1), 38.8 (C-20), 37.0 (C-10), 35.4 (C-19), 33.9 (C-15), 32.8 (C- 7), 30.4 (C-21), 29.9 (C-2), 26.6 (C-22), 22.4 (C-27), 21.3 (C-29), 19.2 (C-25), 18.6 (C- 6), 17.7 (C-26, C-6 '), 17.5 (C-26, C-6 '), 13.6 (C-24).
Compound 32-1: mass spectrometric data m/z 657 [M+Na]+.
Compound 32-2: mass spectrometric data m/z 511 [M+Na]+.
Compound 35 (saikoside 1),
Chemical formula C42H68O14, mass spectrometric data m/z 819 [M+Na]+, nuclear magnetic data:
1H-NMR(C5D5N, 600MHz) δ: 6.80 (1H, br d, J=10.8Hz, H-11), 5.72 (1H, d, J= 10.8Hz, H-12), 5.38 (1H, d, J=7.8Hz, H-1 "), 5.03 (1H, d, J=7.8Hz, H-1 '), 1.48 (3H, d, J= 6.6Hz, H-6 '), 1.74,1.27,1.02,0.94,0.92 (each 3H, s, 5 × CH3)。
13C-NMR(C5D5N, 600MHz) δ: 136.8 (C-13), 133.4 (C-18), 126.9 (C-12), 126.6 (C- 11), 107.3 (C-1 "), 106.6 (C-1 '), 85.8 (C-3 '), 82.1 (C-3), 79.4 (C-5 "), 78.9 (C-3 "), 76.3 (C-2 "), 73.9 (C-30), 72.7 (C-4 "), 72.3 (C-2 '), 72.0 (C-4 '), 71.5 (C-5 '), 68.3 (C-16), 65.5 (C-28), 64.5 (C-23), 63.2 (C-6 "), 54.5 (C-9), 47.8 (C-5), 46.3 (C-17), 44.2 (C-4), 42.4 (C- 14), 41.6 (C-8), 38.9 (C-1), 38.7 (C-20), 37.0 (C-10), 34.5 (C-19), 32.8 (C-15), 32.4 (C- 7), 30.7 (C-21), 26.7 (C-2), 24.6 (C-22), 22.4 (C-27), 21.6 (C-29), 19.4 (C-25), 18.8 (C- 6), 17.8 (C-26, C-6 '), 13.6 (C-24).
Compound 35-1: mass spectrometric data m/z 657 [M+Na]+.
Compound 35-2: mass spectrometric data m/z 511 [M+Na]+
Embodiment 2The anti-influenza virus activity Preliminary Determination of triterpene compound of the invention
1. test-compound
(1) triterpene compound that embodiment 1 is prepared
(2) positive control drug: Ribavirin (Ribavirin)
2. experimental method
(1) compound solution is prepared
Each test-compound (including positive control drug) is dissolved in DMSO respectively, is configured to the storage that concentration is 20mM Liquid is spare.Used time is diluted to suitable concentration.
(2) anti-influenza virus activity measures
By well-grown 293T-Gluc cell with 2.0 × 105/ mL concentration is inoculated in 96 orifice plates, every 100 μ L of hole.Training After supporting for 24 hours, 1 μ L test-compound solution is added in every hole, while it is respectively negative and positive control that DMSO and Ribavirin, which is arranged, Group.WSN33 infection cell (MOI=0.5) is utilized after 1h, after continuing culture for 24 hours, every hole takes 10 μ L supernatants, in multifunctional enzyme mark Instrument (BertholdCentro LB 960) measures Gluc activity.
(3) cytotoxicity experiment
By 293T-Gluc cell inoculation in 96 orifice plates, after culture for 24 hours, 1 μ L test-compound solution is added in every hole, together When setting DMSO and Ribavirin be respectively negative and positive controls.After for 24 hours, 10 μ L CCK-8 are added in every hole, and incubator is incubated for 450nm light absorption value is detected after 1h.
3. experimental result
The anti-influenza activity measurement result of 3.1 test-compounds
It is active by Gluc in measurement viral supernatants when each compound effects concentration that embodiment 1 is prepared is 20 μM, It is control with DMSO, the anti-influenza activity of each compound is calculated.Under normal circumstances, opposite Gluc value is lower, and compound is anti- Influenza Activity is stronger.Concrete outcome is shown in Fig. 1.
Fig. 1 is shown, under 20 μM of concentration, the inhibiting rate > 90% for the compounds on viral that number of the invention is 11, and table Good anti-influenza virus activity is showed；Present invention number 15a, 15b, 21, the compounds of 22b, 25,23,27,29,32 and 35 To the inhibiting rate > 50% of virus, certain antiviral activity is also shown.In addition, the triterpenes with epoxy diether linkage structure The compound infected by influenza of compound, such as number 2,3,5 and 19 also shows very strong inhibiting effect, right under 20 μM of concentration The inhibiting rate > 90% of virus.
3.2 test-compound toxicity test results
When each compound effects concentration that embodiment 1 is prepared is 20 μM, cell survival feelings are measured using CCK-8 method Condition is control with DMSO, and the relative activity that cell is descended in each test-compound effect for 24 hours is calculated.Concrete outcome is shown in Fig. 2.
Fig. 2 shows, present invention number 11,15b, 21, the compounds of 22b, 23,25,27,29,32 and 35 do not show substantially Cytotoxicity out, and the compound of number 15a then shows certain cytostatic effect.As a comparison, have The compound of the triterpene compound of epoxy diether linkage structure, such as number 2,3,5 and 19 all shows stronger cytotoxicity.
Therefore, with known triterpene compound (such as bavin with epoxy diether linkage structure with resisiting influenza virus effect Hu saponin(e a, d etc.) compare, the compound of the present invention has bigger safe dose window, more there is the value of new drug development.
Embodiment 3The activity of representative triterpene compound of the invention and the further research of cytotoxicity
1. test-compound 15a
(1) triterpene compound that embodiment 1 is prepared: 11,15a, 15b, 22b, 23,27 and 2,3,5 and 19.
(2) positive control drug: Ribavirin (Ribavirin)
2. experimental method
(1) compound solution is prepared
Each test-compound (including positive control drug) is dissolved in DMSO respectively, is configured to the storage that concentration is 20mM Liquid, be then diluted to respectively with DMSO 0.32 μM, 1.6 μM, 8 μM, 20 μM, 40 μM and 200 μM it is spare.
(2) anti-influenza virus activity measures
According to measuring under " anti-influenza virus activity measurement " item of embodiment 2, Gluc in vial supernatant is active.
(3) cytotoxicity experiment
According to measurement 450nm light absorption value under " cytotoxicity experiment " item of embodiment 2.
3. experimental result
(1) measurement result
The anti-influenza virus activity of each test-compound and cytotoxicity assay result are shown in Fig. 3-Figure 12 under various concentration.
(2)EC50、CC50And TI value calculates
In conjunction with said determination as a result, calculating each test-compound EC using GraphPad software50And CC50.Pass through CC50/ EC50Obtain TI value.Concrete outcome is shown in Table 1.
The EC of 1 test-compound of table50、CC50And TI
4. conclusion
(1) compound of number 2,3,5,19 all contains epoxy bond, and activity and cytotoxicity are strong, the small (TI of security window It is worth small), it is unsuitable for the preclinical exploitation of new drug.
(2) the compound EC of number 1150It is suitable with above compound, cash out very strong resisiting influenza virus conduct；And EC50And CC50Between window it is slightly larger (TI value be greater than the above-mentioned compound with epoxy ehter bond), and increased with concentration, activity mentions It rises quickly, in 20 μM of concentration, activity is extremely strong, is expected to carry out in-depth study as lead compound.
(3) compound of number of the invention 22b, 23,27, the EC of resisiting influenza virus50Less than positive control Ribavirin, more Interestingly do not find cytotoxic effect, thus also pole hold promise as resisiting influenza virus lead compound carry out after Continuous research and development, it is final to provide the new selection for the treatment of influenza for clinic.
In short, the present invention provides the new medical usages of the resisiting influenza virus of the triterpene compound with structural formula I. Compared with saikosaponin a and d, triterpene compound anti-influenza virus activity of the invention is suitable, but cytotoxicity is small, safety Property is more preferable, significant to new drug development.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention It encloses.

Claims (4)

1. the triterpene compound having the following structure or the triterpene compound having the following structure can pharmaceutically receive Salt preparing the purposes in anti-influenza virus medicament as sole active agent,
2. the triterpene compound having the following structure or the triterpene compound having the following structure can pharmaceutically receive Salt preparing the purposes in anti-influenza virus medicament as sole active agent,
3. purposes according to claim 1 or 2, which is characterized in that the triterpene compound or the triterpenes chemical combination The pharmaceutically acceptable salt of object and other substances with resisiting influenza virus effect are collectively as active constituent, and pharmaceutically The anti-influenza virus medicament is prepared by conventional method in acceptable auxiliary material；It is described other with resisiting influenza virus work Substance is selected from one of amantadine, Rimantadine, Oseltamivir, zanamivir and Peramivir or a variety of.
4. purposes according to claim 1 or 2, which is characterized in that the anti-influenza virus medicament include oral preparation and Non-oral formulation；
The oral preparation be selected from one of tablet, capsule, pill, suspension, syrup, oral solution and granule or It is a variety of；The non-oral formulation is selected from one of injection, spray, nasal drop and suppository or a variety of.
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