CN102247354B - Application of flavane derivative - Google Patents

Application of flavane derivative Download PDF

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CN102247354B
CN102247354B CN201110247285.5A CN201110247285A CN102247354B CN 102247354 B CN102247354 B CN 102247354B CN 201110247285 A CN201110247285 A CN 201110247285A CN 102247354 B CN102247354 B CN 102247354B
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methanol
anoxia
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崔承彬
李长伟
蔡兵
韩冰
李明明
范明
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Institute of Pharmacology and Toxicology of AMMS
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Abstract

The invention relates to flavone and a flavane derivative which are contained in choerospondias axillaries serving as a medicinal plant, a preparation method of the flavone and the flavane derivative, and application of the compounds to the preparation of functional food such as anti-anoxia medicaments, anti-anoxia health-care products and the like.

Description

A kind of purposes of flavan derivatives
The application is to be 200810211765.4 based on application number, and the applying date is JIUYUE in 2008 24 days, and denomination of invention is the divisional application of the Chinese patent application of " preparation method and its usage of flavone and flavan derivatives ".
Technical field:
The present invention relates to the preparation method of the contained flavone of medicinal plants Fructus Choerospondiatis and flavan derivatives, and these compounds are for the preparation of the purposes of the functional food such as anti-anoxic medicine, health products of anti-anoxia.
Background technology:
Oxygen is indispensable in people's vital movement, and in the energy metabolism of tissue and cell, oxygen molecule plays an important role in vivo.Many factors can cause the confession hypoxgia of tissue and cell, as some disease or be in special low-oxygen environment (as plateau) etc., and anoxia can cause cell, tissue injury and even various serious disease conversely, as serious respiratory system disease and concurrent cardiovascular and cerebrovascular disease, immune function low inferior.Severe depletion of oxygen becomes the immediate cause of many relevant critical patient's death toward contact.Therefore, the research and development of anti-hypoxia agent receive publicity.
Fructus Choerospondiatis Choerospon dias axillaries (Roxb.) Burtt.et Hill. is Anacardiaceae Fructus Choerospondiatis platymiscium, (the new medical college in Jiangsu is compiled for its bark and fruit medicine, Chinese medicine voluminous dictionary, the first volume, Shanghai, Shanghai People's publishing house, 1977,397-398 page and the 1564th page), dry fruit is as version pharmacopeia (Chinese Pharmacopoeia Commission in 2005 of Chinese medicine " Fructus Choerospondiatis " income China, Pharmacopoeia of People's Republic of China, one, Beijing, Chemical Industry Press, 2005,29-30 page).The chemical composition of Fructus Choerospondiatis has been reported a bit, and antibacterial, anticoagulant, the antitumor isoreactivity of its crude extract, total flavones or some monomeric compound also have report (Li Changwei etc.; The progress of Fructus Choerospondiatis: PLA's Acta Pharmaceutica Sinica, the 24th the 3rd phase of volume in 2008,231-234 page).In addition, the hypoxia protection effect of Fructus Choerospondiatis fruit total flavones also once had document record (Lee increases Xi etc.; The protective effect of Flavones of Choerospondias Axillaris Fructus to animal anoxia enduring and acute myocardial ischemia: Chinese herbal medicine, the 15th the 6th phase of volume in 1984,25-27 page), but do not illustrate wherein anti-hypoxia active component.The flavone that separates preparation from Fructus Choerospondiatis the present invention relates to and anti-hypoxia activity of Flavane compound and uses thereof so far there are no report.
Summary of the invention:
The present invention aims to provide the preparation method of flavone and the flavan derivatives with anti-hypoxia activity, and these compounds are as the purposes of anti-hypoxia agent.
The inventor finds the aqueous alcohol extractum of medicinal plants Fructus Choerospondiatis, the acetic acid ethyl ester extract of aqueous alcohol extractum, n-butyl alcohol extract and column chromatography component etc. have good anti-hypoxia activity, and therefrom separate first naringenin-4 '-O-(6 " O-galloyl-β-D-glucopyanosyl) glycosides (the formula I compound that has obtained having anti-hypoxia activity, below be called again Compound I), Quercetin-7-O-β-D-glucopyranoside (formula II compound, below be called again Compound I I), 3 '-O-galloyl anthocyanidin B-3 (formula III compound, below be called again compound III), (+)-catechin (6 '-8) (+)-catechin (formula IV compound, below be called again compound IV), dihydrodicatechin A (formula V compound, below be called again compound V), gambiriinA 3(formula VI compound, below be called again compound VI), gambiriin A 1seven flavone such as (formula VII compound, below be called again compound VI I) and flavan derivatives, wherein Compound I is noval chemical compound.
Formula I
Figure BDA0000086155960000022
Formula II
Figure BDA0000086155960000031
Formula III formula IV
Figure BDA0000086155960000032
Formula V formula VI formula VII
The compounds of this invention I, II, III, IV, V, VI, VII can form salt by the acidic-groups such as institute's phenolic hydroxy group and alkali metal or alkaline-earth metal, or ammonium salt, also can with polyvalent metal ion formation complex or the chelate such as magnesium, calcium, ferrum.
Therefore, first aspect of the present invention relates to flavanone kind composition I (formula I compound) or its pharmaceutically acceptable salt, complex or chelate and for the preparation of the purposes in the functional food such as anti-anoxic medicine, health products of anti-anoxia.
Second aspect of the present invention relates to above-mentioned flavone compound II (formula II compound) and flavan derivatives compounds III (formula III compound), IV (formula IV compound), V (formula V compound), VI (formula VI compound), VII (formula VII compound) or its pharmaceutically acceptable salt, complex or chelate for the preparation of the purposes in the functional food such as anti-anoxic medicine, health products of anti-anoxia.
The 3rd aspect of the present invention relates to the preparation method of above-mentioned flavanone kind composition I (formula I compound), flavone compound II (formula II compound), flavan derivatives compounds III (formula III compound), IV (formula IV compound), V (formula V compound), VI (formula VI compound) and VII (formula VII compound) or its pharmaceutically acceptable salt, complex or chelate class, described method comprises by alcohol or aqueous alcohol to be extracted as Fructus Choerospondiatis plant material, then obtains target compound through separation and purification.
The further aspect of the present invention relates to Compound I (formula I compound).
The present invention further aspect relates to anti-anoxia medicinal composition, it comprises one or more above-claimed cpds I, II, III, IV, V, VI, VII, or its pharmaceutically acceptable salt, complex or chelate, and one or more pharmaceutically acceptable carrier or excipient.
The 5th aspect of the present invention relates to the functional foods such as health products of anti-anoxia, and it contains above-mentioned one or more Compound I, II, III, IV, V, VI, VII, or its pharmaceutically acceptable salt, complex or chelate.
Specifically, the preparation method of above-claimed cpd I, II, III, IV, V, VI and VII is as follows: soak and extract medicinal plants Fructus Choerospondiatis material by aqueous alcohol, the crude extract that acquisition contains above-claimed cpd, then purify and obtain the above-claimed cpd in crude extract by conventional separation means.
The aquiferous ethanol that described aqueous alcohol is, the preferably aquiferous ethanol of 60%～95% (v/v).Described conventional separation means comprises the known liquid-liquid extraction of professional person, column chromatography, thin layer chromatography, high performance liquid chroma-tography and the recrystallization etc. in natural product chemistry field.
The present invention adopts Rat pheochromocytoma PC12 cell, with mtt assay test evaluation the anti-hypoxia activity of above-claimed cpd I, II, III, IV, V, VI, VII.Confirm through experiment, above-claimed cpd all has fine protective effect to the anoxia-induced apoptosis of PC12 cell.
Therefore, above-claimed cpd I of the present invention, II, III, IV, V, VI, VII all can be used as anti-hypoxia agent for the various malaise symptoms relevant to anoxia under preventing and treating various Hypoxic diseases or improvement and alleviating low-oxygen environment.
The compounds of this invention can be used singly or in combination, also can with pharmaceutically acceptable various carriers, excipient or supplementary product compatibility, make the various diseases that anti-anoxic medicine causes for preventing and treating anoxia, maybe can make health product or the food additive with hypoxia tolerance, for preventing, improve or alleviating the various symptoms under anoxia condition.
Term " pharmaceutically acceptable salt " in the present invention refers to medicinal inorganic or organic salt.In Compound I, II, III, IV, V, VI, VII, the acidic-group such as institute's phenolic hydroxy group can make compound and alkali metal or alkaline-earth metal form pharmaceutical salts, also can form pharmaceutically acceptable ammonium salt, preferably but be not limited to ammonium (or amine) salt, sodium salt, potassium salt, magnesium salt or calcium salt.Term " pharmaceutically acceptable complex or chelate " in the present invention refers to medicinal metal complex or chelate, preferably but be not limited to metal complex or the chelates such as magnesium, calcium, ferrum.
Compound of the present invention can be separately or with the form administration of pharmaceutical composition.Route of administration can be oral, non-intestinal or topical.Pharmaceutical composition can be made into various suitable dosage forms according to route of administration.
The pharmaceutical composition of the compounds of this invention can be used with any-mode below: oral, spraying sucks, rectal application, nasal cavity applied medicine, buccal medication, local application, non-enterally administer, as subcutaneous, vein, intramuscular, intraperitoneal, in sheath, in ventricle, in breastbone and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Wherein preferred oral, intraperitoneal or intravenous administration mode.
In the time of oral medication, the compounds of this invention can be made into oral acceptable dosage form arbitrarily, includes but not limited to tablet, capsule, aqueous solution or water slurry.Wherein, the carrier that tablet uses generally comprises lactose and corn starch, also can add in addition lubricant as magnesium stearate.The diluent that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation normally mixes use by active component with suitable emulsifying agent and suspending agent.Optionally, in above oral formulations form, also can add some sweeting agents, aromatic or coloring agent.
In the time of topical application, the compounds of this invention can be made into suitable ointment, lotion or cream dosage form, wherein active component is suspended or is dissolved in one or more carriers.The spendable carrier of ointment formulation includes but not limited to: mineral oil, Albolene, white vaseline, propylene glycol, polyethylene glycol oxide, polypropylene oxide, emulsifing wax and water; The spendable carrier of lotion or cream includes but not limited to: mineral oil, sorbitan monostearate, polysorbate60, hexadecane ester type waxes, hexadecene are fragrant and mellow, 2-octyldodecanol, benzyl alcohol and water.
The all right aseptic injection preparation form medication of the compounds of this invention, comprises aseptic injection water or oil suspension or aseptic injectable solution.Wherein, spendable carrier and solvent comprise water, Ringer's mixture and isotonic sodium chlorrde solution.In addition, the fixed oil of sterilizing also can be used as solvent or suspension media, as monoglyceride or two glyceride.
It may be noted that in addition, using dosage and the using method of the compounds of this invention depend on factors, comprise activity intensity, Time of Administration, metabolic rate, the order of severity of disease and diagnosis and treatment doctor's the subjective judgment of patient's age, body weight, sex, natural health situation, nutriture, various extract or various compounds.
The specific embodiment:
The following example will further illustrate the present invention, but the present invention not formed to any restriction.
In following examples, what be called Compound I is flavanone kind composition " naringenin-4 '-O-(6 " O-galloyl-β-D-glucopyanosyl) glycosides, i.e. formula I compound "; What be called Compound I I is flavonoid glycoside compound " Quercetin-7-O-β-D-glucopyranoside, i.e. formula II compound "; What be called compound III is galloyl anthocyan compound " 3 '-O-galloyl anthocyanidin B-3, i.e. formula III compound "; What be called compound IV is anthocyan compound " (+)-catechin (6 '-8) (+)-catechin, i.e. formula IV compound "; What be called compound V is dimeric-catechin compounds " dihydrodicatechin A, i.e. formula V compound "; That be called compound VI is Cha Er-catechin compounds " gambiriin A 3, i.e. formula VI compound ", that be called compound VI I is another Cha Er-catechin compounds " gambiriin A 1, i.e. formula VII compound ".
Figure BDA0000086155960000061
Formula I
Figure BDA0000086155960000062
Formula II
Figure BDA0000086155960000071
Formula III formula IV
Figure BDA0000086155960000072
Formula V formula VI formula VII
The separation preparation of embodiment 1. Compound I
The dry bark 3.2kg (picking up from area, Menla, Yunnan in August, 1999) of Fructus Choerospondiatis Choerospon dias axillaries (Roxb.) Burtt.et Hill. pulverizes rear with the lixiviate of commercially available medical ethanol room temperature, each 25 liters of immersions of aquiferous ethanol with 95% (V/V) 7 days, carry altogether 4 times, merge extractive liquid,, concentrating under reduced pressure is dry, obtains ethanol extraction 750g.This ethanol extraction 750g is suspended in 3 premium on currency, use successively chloroform, ethyl acetate, n-butanol extraction, various solvents are each with 3 liters, each extraction 4 times respectively, merge identical extract, concentrating under reduced pressure is dry, obtains chloroform extract 60g, acetic acid ethyl ester extract 310g, n-butyl alcohol extract 300g and water layer residue 80g.
Get acetic acid ethyl ester extract 100g, use appropriate dissolve with methanol, add appropriate polycaprolactam, dry, upper polyamide column (ethyl acetate center pillar bed 7.0cm × 50cm), with ethyl acetate-methanol gradient elution of different volumes ratio, collect eluent and merge according to thin layer testing result, concentrating under reduced pressure is dry, obtain four thick components of chromatography: E-1 (4g, eluent ethyl acetate part), E-2 (35g, 9: 1 eluting parts of ethyl acetate-methanol), E-3 (25g, 1: 1 eluting part of ethyl acetate-methanol) and E-4 (15g, methanol-eluted fractions part).
E-3 (25g) uses appropriate dissolve with methanol, add appropriate polycaprolactam, dry, upper polyamide column (post bed 3.8cm × 50cm), with chloroform-methanol (4: 1) solvent system eluting, collect eluent and merge according to thin layer testing result, concentrating under reduced pressure is dry, obtains three component E-3-1, E-3-2 and E-3-3.To wherein after the ethanol water dissolution of E-3-2 (1.5g) by appropriate percentage by volume 90%, carry out Sephadex LH-20 column chromatography (post bed 2.8cm × 27cm), obtain Compound I crude product with the ethanol water elution of same volume percent 90%, then recrystallizing and refining obtains Compound I sterling (517mg) in the methanol aqueous solution of percentage by volume 30%.
Compound I is white crystals type powder (methanol), and fusing point is 160-162 ℃.Cation ESI-MS m/z:587[M+H] +, 609[M+Na] +, 625[M+K] +, with its molecular composition C 28h 26o 4corresponding molecular weight 586 is consistent. 1H-NMR(400MHz，MeOH-d 4)δ：7.29(1H，d，J＝8.4Hz，2′，6′-H)，7.10(2H，s，2″″，6″″-H)，7.06(1H，d，J＝8.4Hz，3′，5′-H)，5.90(1H，d，J＝2.0Hz，6-H)，5.88(1H，d，J＝2.0Hz，8-H)，5.31(1H，dd，J＝2.8，12.8Hz，2-H)，4.91(1H，d，J＝7.6Hz，1″-H)，4.61(1H，dd，J＝2.0，12.0Hz，6″-Ha)，4.38(1H，dd，J＝8.4，12.0Hz，6″-Hb)，3.78(1H，m，5″-H)，3.38～3.55(3H，m，2″-4″-H)，3.07(1H，dd，J＝12.8，16.8Hz，3-H-transe)，2.66(1H，dd，J＝2.8，16.8Hz，3-H-cis). 13C-NMR(100MHz，MeOH-d 4)δ：197.3(C-4)，167.9(C-1′″)，167.6(C-7)，164.9(C-5)，164.3(C-8a)，158.5(C-4′)，146.0(C-3″″，5″″)，139.4(C-4″″)，133.5(C-1′)，128.4(C-2′，6′)，120.8(C-1″″)，117.1(C-3′，5′)，109.8(C-2″″，6″″)，102.9(C-4a)，101.5(C-1″)，96.6(C-6)，95.8(C-8)，77.5(C-3″)，75.1(C-5″)，74.3(C-2″)，71.5(C-4″)，64.4(C-6″)，43.3(C-3).
Embodiment 2. Compound I I and III separate preparation
By the appropriate dissolve with methanol of Fructus Choerospondiatis n-butyl alcohol extract 300g obtaining in embodiment 1, add appropriate macroporous resin AB-8 absorption, dry, upper macroporous resin AB-8 post (water center pillar bed 8.5cm × 48cm), with water-ethanol (100: 0 → 0: the 100) gradient elution of different volumes ratio, collect and merge eluent according to thin layer testing result, concentrating under reduced pressure is dry, obtain two washing B component-1 (20g) according to eluting elution order, B-2 (38g) and some follow-up outflow aquiferous ethanol elution fraction B-3 (amount to 230g, 90: 10 → 0: 100 eluting part of water-ethanol).
(38g) uses appropriate dissolve with methanol in B component-2, add appropriate polycaprolactam, dry, upper polyamide column (water center pillar bed 7.5cm × 18.5cm), with the boiling gradient elution of different volumes ratio, merge collected eluent according to thin layer testing result, concentrating under reduced pressure, obtain eight components: B-2-1 (10g, water elution part), B-2-2 (2g, water elution part), B-2-3 (1.5g, 9: 1 eluting parts of boiling), B-2-4 (1.7g, 9: 1 eluting parts of boiling), B-2-5 (2g, 9: 1 eluting parts of boiling), B-2-6 (5g, 7: 3 eluting parts of boiling), B-2-7 (5g, 5: 5 eluting parts of boiling) and B-2-8 (5g, acetone eluting part).B-2-2 (2g) dissolves by suitable quantity of water, upper Sephadex LH-20 chromatographic column (post bed 2.8cm × 27cm), water-methanol solvate system gradient elution, obtain 3 B component-2-2-1 (350mg, 7: 3 eluting parts of water-methanol), B-2-2-2 (1.4g, 4: 6 methanol-eluted fractions parts of water-methanol) and B-2-2-3 (200mg, methanol-eluted fractions part).B-2-2-3 (200mg) is again through Sephadex LH-20 column chromatography, with 2: 8 eluting of water-methanol, collects the eluting part containing Compound I I, concentrating under reduced pressure and from methanol recrystallizing and refining, Compound I I (26mg).
Compound I I is yellow crystal type powder (methanol), and fusing point is 173-175 ℃.ESI-MSm/z:465[M+H] +, 463[M-H] -, with its molecular composition C 21h 20o 12corresponding molecular weight 464 is consistent, 1h-NMR (400MHz, MeOH-d 4) δ: 7.65 (1H, br s, 2 '-H), 7.56 (1H, d, J=8.2Hz, 6 '-H), 6.78 (1H, d, J=8.2Hz, 5 '-H), 6.35 (1H, d, J=2.0Hz, 6-H), 6.62 (1H, d, J=2.0Hz, 8-H), 4.95 (1H, d, J=7.2Hz, 1 " H), 3.84 (1H, dd, J=12.2,1.6Hz, 6 "-Ha), 3.63 (1H, dd, J=12.2,5.6Hz, 6 " Hb), 3.30-3.50 (4H, m, 2 " H～5 "-H). 13c-NMR (100M Hz, MeOH-d 4) δ: 177.4 (C-4), 164.4 (C-7), 162.1 (C-5), 157.6 (C-8a), 148.9 (C-2), 148.7 (C-3 '), 146.2 (C-4 '), 137.9 (C-3), 123.9 (C-1 '), 121.8 (C-6 '), 116.2 (C-2 '), 116.1 (C-5 '), 102.3 (C-4a), 101.6 (C-1 "), 100.1 (C-6), 95.5 (C-8), 78.3 (C-3 "), 77.8 (C-5 "), 74.7 (C-2 "), , 71.2 (C-4 "), 62.4 (C-6 ").
B component-3 (230g) are with repeatedly add absorption in appropriate polyamide, dry after dissolve with methanol in batches, carry out polyamide column chromatography (post bed 7.0cm × 50cm), with ethanol-acetone system eluting, mainly be divided into ethanol elution part (most of material) and acetone eluting part (on a small quantity), its acetone eluting part continues repeatedly to carry out Sephadex LH-20 column chromatography, with 4: 6 eluant solutions of water-ethanol, obtain compound III (84mg).
Compound III is brown amorphous powder.Cation ESI-MS m/z:731[M+H] +, 753[M+Na] +, 769[M+K] +, with its molecular composition C 37h 30o 16corresponding molecular weight 730 is consistent.
1H-NMR(400MHz，MeOH-d 4)δ：6.92(2H，s，2″，6″-H)，6.61～6.86(6H，m，2′(u)，5′(u)，6′(u)，2′(t)，5′(t)，6′(t)-H)，6.01(1H，br?s，8(u)-H)，5.84(2H，br?s，6(u)，6(t)-H)，5.38(1H，br?s，3(t)-H)，5.29(1H，br?s，2(t)-H)，4.54(1H，br?s，2(u)-H)，3.98(2H，br?s，3(u)，4(u)-H)，2.89(1H，br?s，4(t)-Ha)，2.52(1H，br?s，4(t)-Hb). 13C-NMR(100MHz，MeOH-d 4)δ：167.0(C-1′)，155～157(C-5(u)，7(u)，8a(u)，5(t)，7(t)，8a(t))，146.1(C-3′(t)，4′(t))，145.7(C-4′(u))，145.5(C-3′(u))，145.5(C-3″，5″)，139.7(C-4″)，131.8(C-1′(u)，1′(t))，121.3(C-1″)，120.5(C-6′(t))，119.3(C-6′(u))，115.9(C-5′(u))，115.8(C-2′(u)，2′(t))，114.9(C-5′(t))，110.1(C-8(t)，2″，6″)，102.5(C-4a(t))，101.2(C-4a(u))，96.8(C-6(t))，96.0(C-6(u))，95.8(C-8(u))，82.6(C-2(u))，75.9(C-2(t))，74.8(C-3(u))，68.5(C-3(t))，34.6(C-4(u))，30.3(C-4(t)).
Embodiment 3. compound IV and V separate preparation
Medicinal residues after percentage by volume 95% ethanol extraction in embodiment 1 are further carried with the ethanol hydroecium warm macerating of percentage by volume 60%, each with 25 liters of immersions of aquiferous ethanol 7 days, carry altogether merge extractive liquid, 3 times, concentrating under reduced pressure is dry, obtains aquiferous ethanol and extracts extractum 90g.
Get this aquiferous ethanol and extract extractum 80g, use appropriate dissolve with methanol, add polyamide 150g absorption, dry, upper polyamide column (loads 250 grams of polyamide in water, post bed 8.5cm × 22cm), water-ethanol-acetone system eluting, collect and merge eluent according to thin layer testing result, concentrating under reduced pressure is dry, obtain five components: WE-1 (10g, water elution part), WE-2 (17g, water elution part), WE-3 (2g, 75: 25 solvent elution parts of water-ethanol volume ratio), WE-4 (15g, ethanol elution part) and WE-5 (20g, acetone eluting part).
WE-4 (15g) uses appropriate dissolve with methanol, add appropriate polycaprolactam, dry, upper polyamide column (post bed 4.5cm × 50cm), with ethyl acetate-methanol solvate gradient elution of different volumes ratio, collect and merge eluent according to thin layer testing result, concentrating under reduced pressure is dry, obtain seven components: WE-4-1 (2.2g, eluent ethyl acetate part), WE-4-2 (300mg, 20: 1 eluting parts of ethyl acetate-methanol), WE-4-3 (520mg, 20: 1 eluting parts of ethyl acetate-methanol), WE-4-4 (1.5g, 20: 1 eluting parts of ethyl acetate-methanol), WE-4-5 (2.8g, 15: 1 eluting parts of ethyl acetate-methanol), WE-4-6 (0.7g, 15: 1 eluting parts of ethyl acetate-methanol), WE-4-7 (4.5g, methanol-eluted fractions part).
Appropriate chloroform-methanol for WE-4-6 (0.7g) (volume ratio 1: 1) solution dissolves upper Sephadex LH-20 chromatographic column, with chloroform-methanol (1: 1) solvent system eluting, main eluting stream part is through polyamide, Sephadex LH-20 column chromatography obtain compound IV (105mg) repeatedly.WE-4-7 (4.5g) carries out polyamide column chromatography (post bed 2.8cm × 30cm), with ethyl acetate-methanol (3: 1) solvent system eluting, merge flow point according to thin layer chromatography result and obtain 4 component WE-4-7-1 (1.2g), WE-4-7-2 (1.4g), WE-4-7-3 (1.1g), WE-4-7-4 (0.5g), wherein WE-4-7-4 is through polyamide, Sephadex LH-20 column chromatography obtain compound V (37mg) repeatedly.
Compound IV is brown unformed powder.ESI-MS m/z:579[M+H] +, 596[M+NH 4] +, 577[M-H] -, with its molecular composition C 30h 26o 12corresponding molecular weight 578 is consistent. 1h-NMR (400MHz, MeOH-d 4) δ: 6.71 (1H, d, J=2.0Hz, 2 ' (t)-H), 6.70 (1H, d, J=8.0Hz, 5 ' (t)-H), 6.82 (1H, s, 2 ' (u)-H), 6.64 (1H, s, 5 ' (u)-H), 6.59 (1H, dd, J=2.0, 8.0Hz, 6 ' (t)-H), 6.08 (1H, s, 6 (t)-H), 5.90 (1H, d, J=2.0Hz, 8 (u)-H), 5.82 (1H, d, J=2.0Hz, 6 (u)-H), 4.78 (1H, d, J=6.0Hz, 2 (u)-H), 4.73 (1H, d, J=6.0Hz, 2 (t)-H), 4.01 (1H, m, 3 (u)-H), 3.97 (1H, m, 3 (t)-H), 2.73 (1H, dd, J=5.2, 16.0Hz, 4 (t)-Ha), 2.66 (1H, dd, J=4.8, 16.0Hz, 4 (u)-Ha), 2.60 (1H, dd, J=5.8, 16.0Hz, 4 (t)-Hb), 2.42 (1H, dd, J=5.8, 16.0Hz, 4 (u)-Hb). 13c-NMR (400MHz, MeOH-d 4) δ: 157.3 (C-7 (u), 8a (u)), 156.7 (C-5 (u)), 156.4 (C-8a (t)), 154.6 (C-5 (t)), 153.0 (C-7 (t)), 145.6 (C-3 ' (t), 4 ' (t)), 145.5 (C-3 ' (u), 4 ' (u)), 132.2 (C-1 ' (t)), 131.6 (C-1 ' (u)), 126.2 (C-6 ' (u)), 119.6 (C-5 ' (u)), 119.1 (C-6 ' (t)), 115.7 (C-5 ' (t)), 114.3 (C-2 ' (t)), 114.0 (C-2 ' (u)), 108.0 (C-8 (t) or 6 (t)), 100.5 (C-4a (t)), 100.1 (C-4a (u)), 96.1 (C-8 (u) or 6 (u)), 95.7 (C-6 (t) or 8 (t)), 95.2 (C-6 (u) or 8 (u)), 81.7 (C-2 (t)), 79.9 (C-2 (u)), 68.1 (C-3 (t)), 67.6 (C-3 (u)), 27.0 (C-4 (t)), 26.3 (C-4 (u)).
Compound V yellow crystal type powder (methanol), fusing point is 196-198 ℃.ESI-MS m/z:577[M+H] +, 599[M+Na] +, 575[M-H] -, with its molecular composition C 30h 24o 12corresponding molecular weight 576 is consistent. 1h-NMR (400MHz, MeOH-d 4) δ: 6.84 (1H, d, J=2.2Hz, 2 ' (t)-H), 6.78 (1H, d, J=8.0Hz, 5 ' (t)-H), 6.73 (1H, dd, J=2.2, 8.0Hz, 6 ' (t)-H), 6.41 (1H, s, 5 ' (u)-H), 6.11 (1H, s, 6 (t)-H), 5.89 (1H, d, J=2.6Hz, 8 (u)-H), 5.52 (1H, d, J=2.6Hz, 6 (u)-H), 4.90 (1H, d, covered by water peak, 2 (t)-H), 4.10 (1H, m, 3 (t)-H), 3.96 (2H, m, 2 (u), 3 (u)-H), 2.92 (1H, dd, J=5.8, 14.4Hz, 4 (u)-Ha), 2.85 (1H, dd, J=5.2, 16.4Hz, 4 (t)-Ha), 2.67 (1H, d, J=11.6Hz, 2 ' (u)-Ha), 2.52 (1H, dd, J=9.0, 14.4Hz, 4 (u)-Hb), 2.48 (1H, d, J=11.6Hz, 2 ' (u)-Hb). 13c-NMR (400MHz, MeOH-d 4) δ: 192.8 (C-4 ' (u)), 166.7 (C-7 (t)), 164.9 (C-5 (t)), 162.8 (C-6 ' (u)), 156.7 (C-8a (u)), 156.4 (C-7 (u)), 153.8 (C-8a (t)), 155.0 (C-5 (u)), 145.2 (C-4 ' (t)), 145.0 (C-3 ' (t)), 129.9 (C-1 ' (t)), 118.4 (C-6 ' (t)), 115.0 (C-5 ' (t)), 113.5 (C-2 ' (t)), 111.5 (C-5 ' (u)), 104.3 (C-8 (t)), 102.6 (C-4a (t)), 99.1 (C-4a (u)), 95.7 (C-8 (u)), 94.4 (C-6 (u)), 94.0 (C-3 ' (u)), 89.6 (C-6 (t)), 88.5 (C-1 ' (u)), 82.1 (C-2 (t)), 78.1 (C-2 (u)), 66.5 (C-3 (t)), 65.5 (C-3 (u)), 44.3 (C-2 ' (u)), 27.0 (C-4 (u)), 26.5 (C-4 (t)).
Embodiment 4. compound VI and VII separate preparation
Thick the polyamide column chromatography obtaining in embodiment 1 component E-4 (15g) is used to appropriate dissolve with methanol, add approximately 2 times of amount polycaprolactams, dry, carry out polyamide column chromatography (post bed 3.8cm × 50cm), with the chloroform-methanol solvent system gradient elution of different volumes ratio, collect eluent and merge according to thin layer testing result, concentrating under reduced pressure is dry, obtain 2 component E-4-1 (2.0g, 1: 1 eluting part of chloroform-methanol volume ratio) and E-4-2 (12g, methanol-eluted fractions part).E-4-2 (12g) dissolves and carries out Sephadex LH-20 gel filtration chromatography with the methanol-water of appropriate percentage by volume 90%, with the methanol-water eluting of same volume percent 90%, collect eluent and merge according to thin layer testing result, concentrating under reduced pressure is dry, obtains 3 component: E-4-2-1 (7.2g), E-4-2-2 (2.3g) and E-4-2-3 (2.5g).Get E-4-2-3 (2.5g), separate with polyamide column chromatography through Sephadex LH-20 repeatedly, collect respectively compound VI and VII crude product according to thin layer chromatography testing result, recrystallizing and refining from methanol respectively again, obtains sterling compound VI (102mg) and VII (113mg).
Compound VI is light brown crystal type powder (methanol), and fusing point is 172-174 ℃.ESI-MS m/z:581[M+H] +, 603[M+Na] +, 579[M-H] -, with its molecular composition C 30h 28o 12corresponding molecular weight 580 is consistent, 1h-NMR (400MHz, MeOH-d 4) δ: 6.87 (1H, d, J=1.2Hz, 2 ' (t)-H), 6.78 (1H, d, J=8.4Hz, 5 ' (u)-H), 6.75 (2H, dd, J=1.6, 6.8Hz, 6 ' (u), 6 ' (t)-H), 6.67 (1H, d, J=2.0Hz, 2 ' (u)-H), 6.65 (1H, d, J=8.0Hz, 5 ' (t)-H), 6.02 (1H, s, 8 (t)-H), 5.90 (2H, s, 3 (u), 5 (u)-H), 4.83 (1H, d, J=3.2Hz, α-H), 4.62 (1H, d, J=7.6Hz, 2 (t)-H), 4.58 (1H, br s, β-H), 4.03 (1H, m, 3 (t)-H), 2.90 (1H, dd, J=4.8, 16.4Hz, 4 (t)-Ha), 2.90 (1H, dd, J=4.8, 14.8Hz, γ-Ha), 2.62 (1H, dd, J=8.4, 16.4Hz, 4 (t)-Hb), 2.50 (1H, dd, J=10.0, 14.8Hz, γ-Hb). 13c-NMR (400MHz, MeOH-d 4) δ: 158.2 (C-2 (u), 6 (u)), 158.0 (C-4 (u)), 156.8 (C-7 (t)), 156.2 (C-5 (t)), 155.4 (C-8a (t)), 146.5 (C-4 ' (u), 4 ' (t)), 146.0 (C-3 ' (t)), 144.3 (C-3 ' (u)), 135.5 (C-1 ' (u)), 132.6 (C-1 ' (t)), 120.8 (C-6 ' (u)), 120.4 (C-6 ' (t)), 117.0 (C-2 ' (u)), 116.4 (C-5 ' (t)), 116.2 (C-5 ' (u)), 115.6 (C-2 ' (t)), 108.5 (C-6 (t)), 106.6 (C-1 (u)), 102.1 (C-4a (t)), 96.2 (C-3 (u), 5 (u)), 95.6 (C-8 (t)), 83.0 (C-2 (t)), 78.1 (C-β), 69.4 (C-3 (t)), 46.6 (C-α), 30.6 (C-4 (t)), 29.5 (C-γ).
Compound VI I is light brown crystal type powder (methanol), and fusing point is 167-169 ℃.ESI-MS m/z:581[M+H] +, 603[M+Na] +, 579[M-H] -, with its molecular composition C 30h 28o 12corresponding molecular weight 580 is consistent, 1h-NMR (400MHz, MeOH-d 4) δ: 6.78 (1H, d, J=1.6Hz, 2 ' (t)-H), 6.75 (1H, br s, 2 ' (u)-H), 6.68 (1H, d, J=8.0Hz, 5 ' (t)-H), 6.63 (3H, br s, 5 ' (u), 6 ' (u), 6 ' (t)-H), 6.04 (1H, s, 6 (t)-H), 5.84 (2H, s, 3 (u), 5 (u)-H), 4.70 (1H, br s, α, 2 (t)-H), 4.59 (1H, br s, β-H), 3.93 (1H, m, 3 (t)-H), 2.90 (2H, m, γ, 4 (t)-H), 2.56 (1H, m, 4 (t)-H), 2.49 (1H, m, γ-H). 1h-NMR (400MHz, MeOH-d 4) δ: 158.5 (C-2 (u), 6 (u)), 158.0 (C-4 (u)), 156.3 (C-7 (t)), 156.2 (C-5 (t)), 155.5 (C-8a (t)), 146.5 (C-3 ' (t)), 146.4 (C-4 ' (t)), 146.0 (C-3 ' (u)), 144.5 (C-4 ' (u)), 136.2 (C-1 ' (u)), 132.8 (C-1 ' (t)), 121.4 (C-6 ' (u)), 120.8 (C-6 ' (t)), 117.4 (C-2 ' (u)), 116.5 (C-5 ' (t)), 116.3 (C-5 ' (u)), 115.6 (C-2 ' (t)), 107.9 (C-8 (t)), 106.6 (C-1 (u)), 101.4 (C-4a (t)), 97.9 (C-6 (t)), 96.4 (C-3 (u), 5 (u)), 83.2 (C-2 (t)), 77.6 (C-β), 69.3 (C-3 (t)), 46.9 (C-α), 30.8 (C-γ), 29.6 (C-4 (t)).
Embodiment 5. anti-hypoxia active testings
Cell line and cell culture: active testing adopts Rat pheochromocytoma PC12 cell strain, the DMEM culture medium that contains 10% newborn calf serum and penicillin, the each 100 μ g/mL of streptomycin for cell, passes into 5%CO in 37 ℃ 2safeguard with cellar culture and subculture in the incubator of 95% air.
Sample and sample solution preparation: Compound I, II, III, IV, V, VI and VII in embodiment 1～embodiment 4.Precision weighing sample is appropriate, dissolves by the DMEM culture medium containing 10% newborn calf serum and penicillin and the each 100 μ g/mL of streptomycin, is mixed with the sample solution of 50 μ g/mL, 100 μ g/mL and 200 μ g/mL, active for surveying.
Activity test method: the PC12 cell of the trophophase of taking the logarithm, being made into cell density by the fresh DMEM culture medium that contains 10% newborn calf serum and penicillin, the each 100 μ g/mL of streptomycin is every milliliter 1 × 10 5individual cell suspension, is inoculated in 96 orifice plates, and every hole 150 μ L, pass into 5%CO in 37 ℃ 2with in the incubator of 95% air, cultivate 18～24h, attract to discard culture fluid, be divided into Normal group, anoxia matched group and anoxia administration group, respectively organize each sample and all establish three holes.The each 150 μ L of fresh DMEM culture medium of every Kong Jiahan 10% newborn calf serum of Normal group and anoxia matched group, penicillin and the each 100 μ g/mL of streptomycin, the every hole of anoxia administration group adds the each 150 μ L of sample solution.Matched group and administration group are cultivated 1h in incubator, and anoxia matched group and anoxia administration group are with 31.25 μ M CoCl 2process 2h and cause the anoxia-induced apoptosis to cell, then the normal 24h that cultivates in incubator.After cultivation finishes, every hole adds the each 15 μ L of MTT solution (with the PBS liquid preparation of 0.01M) of 5mg/mL, mix, hatch 4h for 37 ℃, turnover panel method discards culture fluid in hole, every hole adds the each 150 μ L of DMSO, and vibration 10min fully dissolves MTT purple product, measures every hole in the OD at 490nm place value by microplate reader.Data are take Normal group as 100%, get each group of OD meansigma methods and be calculated as follows the cell survival rate of anoxia matched group and anoxia administration group: cell survival rate (%)=anoxia matched group or anoxia administration group OD meansigma methods/Normal group OD meansigma methods × 100%.Get independent experiment data 8 times, calculate corresponding cell survival rate (%), and use Student-t method of inspection, the significant difference of cell survival rate between two groups of statistical test anoxia matched group and anoxia administration groups.
Experimental result refers to table 1.Shown in table 1, test result shows, the compounds of this invention I～VII has remarkable protective effect to the anoxia-induced apoptosis of PC12 cell in trial concentration range.
The protective effect of table 1. Compound I～VII to the damage of PC12 cell hypoxia
Figure BDA0000086155960000151
*asterisk shows there is significant difference compared with corresponding anoxia matched group, P < 0.01
Conclusion:
Compound I of the present invention, II, III, IV, V, VI, VII have significant protective effect to the anoxia-induced apoptosis of PC12 cell.Therefore, above-claimed cpd of the present invention can be used as anti-hypoxia active component for the preparation of the functional food such as anti-anoxic medicine and health products of anti-anoxia.

Claims (2)

1. formula V compound or its pharmaceutically acceptable salt be for the preparation of the purposes of anti-anoxic medicine and health products of anti-anoxia,
Formula V.
2. formula V compound or its pharmaceutically acceptable salt be for the preparation of the purposes of the medicine of various malaise symptoms relevant to anoxia under preventing and/or treating various Hypoxic diseases or improvement and alleviating low-oxygen environment,
Formula V.
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Publication number Priority date Publication date Assignee Title
CN1980654A (en) * 2004-07-05 2007-06-13 三得利株式会社 Lipase inhibitors

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Title
Cobalt Chloride Induces PC12 Cells Apoptosis Through Reactive Oxygen Species and Accompanied by AP-1 Activation.;Weiguo Zou等;《Journal of Neuroscience Research》;20011231;第64卷;第646–653页 *
Evaluation of Cytotoxic Compounds from Calligonum comosum L. Growing in Egypt.;Farid A. Badria等;《Z. Naturforsch》;20071231;第62C卷;第656-660页 *
Farid A. Badria等.Evaluation of Cytotoxic Compounds from Calligonum comosum L. Growing in Egypt..《Z. Naturforsch》.2007,第62C卷第656-660页.
Weiguo Zou等.Cobalt Chloride Induces PC12 Cells Apoptosis Through Reactive Oxygen Species and Accompanied by AP-1 Activation..《Journal of Neuroscience Research》.2001,第64卷第646–653页.

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