CN101683353A - Preparation method and application of flavone and flavan derivatives - Google Patents
Preparation method and application of flavone and flavan derivatives Download PDFInfo
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Abstract
The invention relates to flavone and flavan derivatives contained in medical plant choerospondias axillaries, a preparation method thereof, and an application of the compounds in preparing antihypoxicdrugs, antihypoxic health products and other functional foods.
Description
Technical field:
The present invention relates to the preparation method of contained flavone of medicinal plants Fructus Choerospondiatis and flavan derivatives, and these chemical compounds are used to prepare the purposes of functional foods such as anti-anoxic medicine, health products of anti-anoxia.
Background technology:
Oxygen is indispensable in people's vital movement, and oxygen molecule is being brought into play important effect in the energy metabolism of tissue and cell in vivo.Multiple factor can cause the oxygen supply deficiency of tissue and cell, as some disease or be in special low-oxygen environment (as the plateau) etc., and anoxia can cause cell, tissue injury and even various serious disease conversely, hangs down inferior as serious respiratory system disease and concurrent cardiovascular and cerebrovascular disease, immune function.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 Choerospondias axillaries (Roxb.) Burtt.et Hill. is an Anacardiaceae Fructus Choerospondiatis platymiscium, and (the new medical college in Jiangsu is compiled the Chinese medicine voluminous dictionary for its bark and fruit medicine, the first volume, Shanghai, the Shanghai People's Press, 1977,397-398 page or leaf and the 1564th page), dry fruit is as Chinese medicine " Fructus Choerospondiatis " income China version pharmacopeia in 2005 (Chinese Pharmacopoeia Commission, Pharmacopoeia of People's Republic of China, one one, Beijing, Chemical Industry Press, 2005, the 29-30 page or leaf).Some existing report of the chemical constituent of Fructus Choerospondiatis, antibiotic, 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, 2008 the 24th the 3rd phases of volume, 231-234 page or leaf).In addition, the hypoxia protection effect of Fructus Choerospondiatis fruit total flavones also once had document record (Lee increased Xi etc.; The Fructus Choerospondiatis total flavones is to the protective effect of animal anoxia enduring and acute myocardial ischemia: Chinese herbal medicine, 1984 the 15th the 6th phases of volume, 25-27 page or leaf), but do not illustrate wherein anti-hypoxia active component.Separate from the Fructus Choerospondiatis flavone of preparation and the anti-hypoxia activity of Flavane compound and uses thereof that the present invention relates to are not appeared in the newspapers so far.
Summary of the invention:
The present invention aims to provide the preparation method with the active flavone of anti-hypoxia and flavan derivatives, and these chemical 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 obtained having anti-hypoxia active naringenin-4 '-O-(6 " O-galloyl-β-D-glucopyanosyl) glycosides (formula I chemical compound; the following Compound I that is called again); Quercetin-7-O-β-D-glucopyranoside (formula II chemical compound; the following Compound I I that is called again); 3 '-O-galloyl anthocyanidin B-3 (formula III chemical compound; the following compound III that is called again); (+)-catechin (6 '-8) (+)-catechin (formula IV chemical compound; below be called compound IV again); dihydrodicatechin A (formula V chemical compound, below be called chemical compound V again); gambiriinA
3(formula VI chemical compound, below be called compound VI again), gambiriin A
1Seven flavone such as (formula VII chemical compound, below be called compound VI I again) and flavan derivatives, wherein Compound I is a noval chemical compound.
The compounds of this invention I, II, III, IV, V, VI, VII can pass through acidic-group and alkali metal or alkaline-earth metal formation salt such as institute's phenolic hydroxy group, or ammonium salt, also can form complex or chelates with polyvalent metal ions such as magnesium, calcium, ferrum.
Therefore, first aspect of the present invention relates to flavanone kind composition I (formula I chemical compound) or its pharmaceutically acceptable salt, complex or chelate and is used for preparing the purposes of functional foods such as anti-anoxic medicine, health products of anti-anoxia.
Second aspect of the present invention relates to the purposes that above-mentioned flavone compound II (formula II chemical compound) and flavan derivatives compounds III (formula III chemical compound), IV (formula IV chemical compound), V (formula V chemical compound), VI (formula VI chemical compound), VII (formula VII chemical compound) or its pharmaceutically acceptable salt, complex or chelate are used for preparing functional foods 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 chemical compound), flavone compound II (formula II chemical compound), flavan derivatives compounds III (formula III chemical compound), IV (formula IV chemical compound), V (formula V chemical compound), VI (formula VI chemical compound) and VII (formula VII chemical compound) or its pharmaceutically acceptable salt, complex or chelate class, described method comprises with alcohol or aqueous alcohol to be extracted plant material such as Fructus Choerospondiatis, obtains target compound through separation and purification again.
The further aspect of the present invention relates to Compound I (formula I chemical 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 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 with aqueous alcohol and extract medicinal plants Fructus Choerospondiatis material, acquisition contains the crude extract of above-claimed cpd, and the conventional separation means purification of reuse obtains the above-claimed cpd in the crude extract.
The aquiferous ethanol that described aqueous alcohol is, the preferably aquiferous ethanol of 60%～95% (v/v).The known liquid-liquid extraction of professional person, column chromatography, thin layer chromatography, high performance liquid chroma-tography and recrystallization etc. that described conventional separation means comprises the natural product chemistry field.
The present invention adopts rat pheochromocytoma PC12 cell, with the mtt assay test evaluation anti-hypoxia activity of above-claimed cpd I, II, III, IV, V, VI, VII.Through experiment confirm, 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 the anti-hypoxia agent and are used to prevent and treat various anoxia diseases or improvement and alleviate various malaise symptoms relevant with anoxia under the 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 anti-anoxic medicine and be used to prevent and treat the various diseases that anoxia causes, maybe can make health product or food additive, be used to prevent, improve or alleviate various symptoms under the anoxia condition with anti-hypoxia function.
Term among the present invention " pharmaceutically acceptable salt " is meant medicinal inorganic or organic salt.Acidic-group such as institute's phenolic hydroxy group can make chemical compound and alkali metal or alkaline-earth metal form pharmaceutical salts among Compound I, II, III, IV, V, VI, the VII, also can form pharmaceutically acceptable ammonium salt, preferred but be not limited to ammonium (or amine) salt, sodium salt, potassium salt, magnesium salt or calcium salt.Term among the present invention " pharmaceutically acceptable complex or chelate " is meant medicinal metal complex or chelate, and is preferred but be not limited to metal complex or chelates such as magnesium, calcium, ferrum.
Chemical 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 following any-mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, buccal medication, local application, non-enterally administer, as subcutaneous, vein, intramuscular, intraperitoneal is in the sheath, in the ventricle, breastbone interior and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Wherein preferred oral, intraperitoneal or intravenous administration mode.
When medicine for oral use, 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 lubricant such as magnesium stearate in addition.The diluent that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation then normally mixes use with active component with examples of suitable emulsifiers and suspending agent.Randomly, also can add some sweeting agents, aromatic or coloring agent in the above oral formulations form.
When 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 sterilization also can be used as solvent or suspension media, as monoglyceride or two glyceride.
It may be noted that in addition, the using dosage of The compounds of this invention and using method depend on all multifactor, 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 all cpds.
The specific embodiment:
The following example will further specify the present invention, but the present invention not constituted 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 chemical compound "; What be called Compound I I is flavonoid glycoside compound " Quercetin-7-O-β-D-glucopyranoside, i.e. formula II chemical compound "; What be called compound III is galloyl anthocyan chemical compound " 3 '-O-galloyl anthocyanidin B-3, i.e. formula III chemical compound "; What be called compound IV is anthocyan chemical compound " (+)-catechin (6 '-8) (+)-catechin, i.e. formula IV chemical compound "; What be called chemical compound V is dimeric-catechin compounds " dihydrodicatechin A, i.e. formula V chemical compound "; That be called compound VI is Cha Er-catechin compounds " gambiriin A
3, i.e. formula VI chemical compound ", that be called compound VI I is another Cha Er-catechin compounds " gambiriin A
1, i.e. formula VII chemical compound ".
The separation preparation of embodiment 1. Compound I
The dry bark 3.2kg of Fructus Choerospondiatis Choerospondias axillaries (Roxb.) Burtt.et Hill. (picking up from area, Menla, Yunnan in August, 1999) pulverizes back with the lixiviate of commercially available medical ethanol room temperature, aquiferous ethanol with 95% (V/V) soaked 7 days for 25 liters at every turn, carry altogether 4 times, merge extractive liquid,, the concentrating under reduced pressure drying gets ethanol extraction 750g.This ethanol extraction 750g is suspended in 3 premium on currency, use chloroform, ethyl acetate, n-butanol extraction successively, all kinds of solvents is each with 3 liters, respectively extract respectively 4 times, merge identical extract, the concentrating under reduced pressure drying 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 an amount of dissolve with methanol, add an amount of polyamide absorption, drying, last polyamide column (ethyl acetate center pillar bed 7.0cm * 50cm), ethyl acetate-methanol gradient elution with the different volumes ratio, collect eluent and merge according to the thin layer testing result, the concentrating under reduced pressure drying, obtain four thick components of chromatography: E-1 (4g, the 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 an amount of dissolve with methanol, add an amount of polyamide absorption, drying, last polyamide column (post bed 3.8cm * 50cm), with chloroform-methanol (4: 1) solvent system eluting, collect eluent and merge according to the thin layer testing result, the concentrating under reduced pressure drying gets three component E-3-1, E-3-2 and E-3-3.Will be wherein carry out Sephadex LH-20 column chromatography (post bed 2.8cm * 27cm) behind the ethanol water dissolution of E-3-2 (1.5g) with an amount of percentage by volume 90%, ethanol water elution with equal volume percent 90% gets the Compound I crude product, and recrystallizing and refining gets the pure product of Compound I (517mg) in the methanol aqueous solution of percentage by volume 30% again.
Compound I is a 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 unanimities.
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
With an amount of dissolve with methanol of Fructus Choerospondiatis n-butyl alcohol extract 300g that obtains among the embodiment 1, add an amount of macroporous resin AB-8 absorption, drying, last macroporous resin AB-8 post (water center pillar bed 8.5cm * 48cm), water-ethanol (100: 0 → 0: 100) gradient elution with the different volumes ratio, collect the merging eluent according to the thin layer testing result, the concentrating under reduced pressure drying, get two washing B component-1 (20g), B-2 (38g) and some follow-up outflow aquiferous ethanol elution fraction B-3 (amounting to 230g, 90: 10 → 0: 100 eluting part of water-ethanol) according to the eluting elution order.
B component-2 (38g) is used an amount of dissolve with methanol, add an amount of polyamide absorption, dry, last polyamide column (water center pillar bed 7.5cm * 18.5cm), water-acetone gradient elution with the different volumes ratio, merge collected eluent according to the thin layer testing result, concentrating under reduced pressure, get eight components: B-2-1 (10g, the water elution part), B-2-2 (2g, the water elution part), B-2-3 (1.5g, 9: 1 eluting parts of water-acetone), B-2-4 (1.7g, 9: 1 eluting parts of water-acetone), B-2-5 (2g, 9: 1 eluting parts of water-acetone), B-2-6 (5g, 7: 3 eluting parts of water-acetone), B-2-7 (5g, 5: 5 eluting partly of water-acetone) and B-2-8 (5g, acetone eluting part).B-2-2 (2g) dissolves with suitable quantity of water, last 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 partly of water-methanol) and B-2-2-3 (200mg, methanol-eluted fractions part).B-2-2-3 (200mg) is once more through Sephadex LH-20 column chromatography, with 2: 8 eluting of water-methanol, collects the eluting part that contains Compound I I, concentrating under reduced pressure and from methanol recrystallizing and refining, Compound I I (26mg).
Compound I I is a 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 unanimities;
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) is with repeatedly add absorption, drying in an amount of polyamide behind the 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 partly continues to carry out repeatedly 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 unanimities.
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 behind percentage by volume 95% ethanol extraction among the 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 merge extractive liquid, altogether 3 times, the concentrating under reduced pressure drying gets aquiferous ethanol and extracts extractum 90g.
Get this aquiferous ethanol and extract extractum 80g, use an amount of dissolve with methanol, add polyamide 150g absorption, drying, last polyamide column (filling 250 gram polyamide in the water, post bed 8.5cm * 22cm), with water-ethanol-acetone system eluting, collect the merging eluent according to the thin layer testing result, the concentrating under reduced pressure drying obtains five component: WE-1 (10g, water elution part), WE-2 (17g, the 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 an amount of dissolve with methanol, add an amount of polyamide absorption, dry, last polyamide column (post bed 4.5cm * 50cm), ethyl acetate-methanol solvate gradient elution with the different volumes ratio, collect the merging eluent according to the thin layer testing result, the concentrating under reduced pressure drying, obtain seven components: WE-4-1 (2.2g, the 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).
WE-4-6 (0.7g) goes up Sephadex LH-20 chromatographic column with the dissolving of an amount of chloroform-methanol (volume ratio 1: 1) solution, 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 the 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 chemical 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 unanimities.
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)).
Chemical compound V yellow crystal type powder (methanol), fusing point are 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 unanimities.
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 are covered by the 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
The thick component E-4 of polyamide column chromatography (15g) that obtains among the embodiment 1 is used an amount of dissolve with methanol, add the absorption of about 2 times of amount polyamide, drying, carry out polyamide column chromatography (post bed 3.8cm * 50cm), chloroform-methanol solvent system gradient elution with the different volumes ratio, collect eluent and merge according to the thin layer testing result, the concentrating under reduced pressure drying obtains 2 component E-4-1 (2.0g, 1: 1 eluting partly of chloroform-methanol volume ratio) and E-4-2 (12g, methanol-eluted fractions part).E-4-2 (12g) is with the methanol-water of an amount of percentage by volume 90% dissolving carrying out Sephadex LH-20 gel filtration chromatography, methanol-water eluting with equal volume percent 90%, collect eluent and merge according to the thin layer testing result, the concentrating under reduced pressure drying 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 repeatedly Sephadex LH-20, collect compound VI and VII crude product respectively according to the thin layer chromatography testing result, recrystallizing and refining from methanol respectively again, pure product compound VI (102mg) and VII (113mg).
Compound VI is a 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 unanimities;
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 a 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 unanimities;
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, and cell feeds 5%CO with the DMEM culture medium that contains 10% newborn calf serum and penicillin, each 100 μ g/mL of streptomycin in 37 ℃
2With conventional the cultivation and the subculture maintenance in the incubator of 95% air.
Sample and sample solution preparation: Compound I, II, III, IV, V, VI and VII among embodiment 1～embodiment 4.The precision weighing sample is an amount of, with the DMEM culture medium dissolving that contains each 100 μ g/mL of 10% newborn calf serum and penicillin and streptomycin, is mixed with the sample solution of 50 μ g/mL, 100 μ g/mL and 200 μ g/mL, and is active for surveying.
Activity test method: the PC12 cell of the trophophase of taking the logarithm, being made into cell density with the fresh DMEM culture medium that contains 10% newborn calf serum and penicillin, 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 feed 5%CO in 37 ℃
2With cultivate 18～24h in the incubator of 95% air, attract to discard culture fluid, be divided into normal control group, anoxia matched group and anoxia administration group, each is organized each sample and all establishes three holes.Normal control group and the every hole of anoxia matched group add each the 150 μ L of fresh DMEM culture medium that contain each 100 μ g/mL of 10% newborn calf serum, penicillin and streptomycin, and the every hole of anoxia administration group adds each 150 μ L of sample solution.Matched group and administration group are cultivated 1h in incubator, anoxia matched group and anoxia administration group are with 31.25 μ M CoCl
2Handle the anoxia-induced apoptosis that 2h causes pair cell, then the normal 24h that cultivates in incubator.After cultivating end, every hole adds each 15 μ L of MTT solution (with the PBS liquid preparation of 0.01M) of 5mg/mL, mixing, hatch 4h for 37 ℃, the turnover panel method discards culture fluid in the hole, every hole adds each 150 μ L of DMSO, and vibration 10min fully dissolves MTT purple product, measures every hole in the OD at 490nm place value with microplate reader.Data are 100% with the normal control group, get and respectively organize the cell survival rate that the OD meansigma methods is calculated as follows anoxia matched group and anoxia administration group: cell survival rate (%)=anoxia matched group or anoxia administration group OD meansigma methods/normal control group OD meansigma methods * 100%.Get the independent experiment data 8 times, calculate corresponding cell survival rate (%), and use the 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 sees table 1 for details.Test result shows shown in the table 1, and The compounds of this invention I～VII anoxia-induced apoptosis to the PC12 cell in the examination concentration range has remarkable protective effect.
Table 1. Compound I～VII is to the protective effect of PC12 cell hypoxia damage
* asterisk shows with corresponding anoxia matched group and has compared significant difference, 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 the anti-hypoxia active component and is used to prepare functional foods such as anti-anoxic medicine and health products of anti-anoxia.
Claims (7)
1. formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII chemical compound, or its pharmaceutically acceptable salt, complex or chelate are used to prepare the purposes of functional foods such as anti-anoxic medicine and health products of anti-anoxia,
Formula I
Formula II formula III formula IV
Formula V formula VI formula VII.
2. formula I chemical compound or its pharmaceutically acceptable salt, complex or chelate:
Formula I.
3. pharmaceutical composition, it comprises the described formula I chemical compound of claim 2, or its pharmaceutically acceptable salt, complex or chelate, and one or more pharmaceutically acceptable carrier or excipient.
4. health food, it comprises the described formula I chemical compound of claim 2, or its pharmaceutically acceptable salt, complex or chelate.
5. the preparation method of the described formula I of claim 1～formula VII chemical compound, this method comprises with the Fructus Choerospondiatis being raw material, soaks through aqueous alcohol and extracts, and obtains to contain the crude extract of above-claimed cpd, the conventional separation means of reuse, separation and purification obtains the above-claimed cpd in the crude extract.
6. the described preparation method of claim 5, wherein said aqueous alcohol is the aquiferous ethanol of 60%～95% (v/v).
7. the described preparation method of claim 5, described conventional separation means comprises liquid-liquid extraction, column chromatography, thin layer chromatography, high performance liquid chroma-tography and recrystallization etc.
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| WO2015169121A1 (en) * | 2014-05-05 | 2015-11-12 | 南京睿鹰润泽生物医药科技有限公司 | Use of quercetin-o-glycoside derivatives in treating lipid metabolism disorders |
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| CN101224218B (en) * | 2008-01-07 | 2011-02-09 | 苏州大学 | Uses of 8-beta-D glucopyranosyl-4',7-dihydroxy isoflavone |
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| CN104208202A (en) * | 2014-08-08 | 2014-12-17 | 西北农林科技大学 | Cortex choerospondias proanthocyanidins and preparation method and application thereof |
| CN104208202B (en) * | 2014-08-08 | 2016-08-24 | 西北农林科技大学 | A kind of Fructus Choerospondiatis procyanidin and its preparation method and application |
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