CN101683353B - 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 antihypoxic drugs, antihypoxic health products and other functional foods.
Description
Technical field:
The present invention relates to the method for preparing 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 of tissue and cell not enough; Like some disease or be in special low-oxygen environment (like the plateau) etc.; And anoxia can cause cell, tissue injury and even various serious disease conversely, hangs down inferior like serious respiratory system disease and concurrent cardiovascular and cerebrovascular disease, immune function.Severe depletion of oxygen becomes the dead immediate cause of many relevant critical patients 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 Chinese medicine voluminous dictionary, the first volume for its bark and fruit medicine; Shanghai, the Shanghai People's Press,, 397-398 page or leaf and the 1564th page in 1977); Dry fruit is as Chinese medicine " Fructus Choerospondiatis " income China version pharmacopeia (Chinese Pharmacopoeia Commission, Pharmacopoeia of People's Republic of China, one one, Beijing in 2005; 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 southern wild jujube fruit general flavone also once had document record (Li Zengxi etc.; The fructus choerospondiatis general flavone is to the protective effect of anti-anoxic of animal 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-anoxic active component.The flavone that from Fructus Choerospondiatis, separates preparation that the present invention relates to and anti-hypoxia activity of Flavane compound and uses thereof are not appeared in the newspapers so far.
Summary of the invention:
The present invention aims to provide the method for preparing 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 that the aqueous alcohol extractum of medicinal plants Fructus Choerospondiatis, acetic acid ethyl ester extract, n-butyl alcohol extract and the column chromatography component etc. of aqueous alcohol extractum 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; Below be called compound I again), Quercetin-7-O-β-D-glucopyranoside (formula II chemical compound; Below be called compound I I again), 3 '-O-galloyl anthocyanidin B-3 (formula III chemical compound; Below be called compound III 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
3The formula chemical compound below is called compound VI again), gambiriinan A
1Seven flavone such as (formula VII compound, below be called compound VI I again) and flavan derivatives, wherein compound I is a noval chemical compound.
Formula I formula II
Formula III formula IV
Formula V formula VI formula VII
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 method for preparing 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; Said 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 aspect further 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 method for preparing of above-claimed cpd I, II, III, IV, V, VI and VII is following: with aqueous alcohol soaking and extracting medicinal plants Fructus Choerospondiatis material; Acquisition contains the CE of above-claimed cpd, and the conventional separation means purification of reuse obtains the above-claimed cpd in the CE.
The aquiferous ethanol that said aqueous alcohol is, the preferably aquiferous ethanol of 60%～95% (v/v).Described conventional separation means comprises the liquid-liquid extraction that the professional person knew, 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 the mtt assay test evaluation anti-hypoxia of above-claimed cpd I, II, III, IV, V, VI, VII active.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 property diseases or improvement and alleviate various malaise symptoms relevant with anoxia under the low-oxygen environment.
The compounds of this invention can use or make up use separately; Also can with pharmaceutically acceptable various carriers, excipient or supplementary product compatibility; Process anti-anoxic medicine and be used to prevent and treat the various diseases that anoxia causes; Maybe can process health product or food additive, be used to prevent, improve or alleviate the 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; The buccal medication, local application, non-enterally administer is 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, PPOX, 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, like monoglyceride or two glyceride.
It may be noted that in addition; The using dosage of The compounds of this invention and method for using depend on many 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 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 ".
Formula I formula II
Formula III formula IV
Formula V formula VI formula VII
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 the 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,, concentrating under reduced pressure is dry, 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 respectively to extract respectively 4 times with 3 liters, merges 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 an amount of dissolve with methanol, add an amount of polyamide absorption, drying; (ethyl acetate center pillar bed 7.0cm * 50cm) with the ethyl acetate-methanol gradient elution of different volumes ratio, collects eluent and merges according to the thin layer testing result last polyamide column; Concentrating under reduced pressure is dry; Obtain four the thick component of chromatography: E-1 (4g, eluent ethyl acetate part), E-2 (35g, ethyl acetate-methanol 9:1 eluting part), E-3 (25g; Ethyl acetate-methanol 1:1 eluting part) 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; (post bed 3.8cm * 50cm) with chloroform-methanol (4:1) solvent system eluting, collects eluent and merges according to the thin layer testing result last polyamide column; Concentrating under reduced pressure is dry, 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 bullion, and recrystallizing and refining gets the pure article 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
The Fructus Choerospondiatis n-butyl alcohol extract 300g that obtains among the embodiment 1 is used an amount of dissolve with methanol; Add an amount of macroporous resin AB-8 absorption, drying; Last macroporous resin AB-8 post (water center pillar bed 8.5cm * 48cm); (gradient elution of 100:0 → 0:100) is collected according to the thin layer testing result and to be merged eluent, and concentrating under reduced pressure is dry with the water-ethanol of different volumes ratio; Get two washing B component-1 (20g), B-2 (38g) and some follow-up outflow aquiferous ethanol elution fraction B-3 (amounting to 230g, water-ethanol 90:10 → 0:100 eluting part) according to the eluting elution order.
B component-2 (38g) is used an amount of dissolve with methanol, adds an amount of polyamide absorption, drying, and last polyamide column (water center pillar bed 7.5cm * 18.5cm); With the water-acetone gradient elution of different volumes ratio, merge collected eluent, concentrating under reduced pressure according to the thin layer testing result; Get eight component: B-2-1 (10g, water elution part), B-2-2 (2g, water elution part), B-2-3 (1.5g; Water-acetone 9:1 eluting part), B-2-4 (1.7g, water-acetone 9:1 eluting part), B-2-5 (2g, water-acetone 9:1 eluting part), B-2-6 (5g; Water-acetone 7:3 eluting part), B-2-7 (5g, water-acetone 5:5 eluting part) 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 obtains 3 B component-2-2-1 (350mg, water-methanol 7:3 eluting part), B-2-2-2 (1.4g; Water-methanol 4:6 methanol-eluted fractions partly) 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 water-methanol 2:8 eluting, 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, brs, 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 water-ethanol 4:6 eluant solution, 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%; Soaked 7 days for 25 liters with aquiferous ethanol at every turn, carry merge extractive liquid, altogether 3 times; Concentrating under reduced pressure is dry, 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, concentrating under reduced pressure is dry, obtains five components: WE-1 (10g; The water elution part), WE-2 (17g, water elution part), WE-3 (2g, water-ethanol volume ratio 75:25 solvent elution part), WE-4 (15g; Ethanol elution partly) and WE-5 (20g, acetone eluting part).
WE-4 (15g) uses an amount of dissolve with methanol, adds an amount of polyamide absorption, drying, and last polyamide column (post bed 4.5cm * 50cm); With the ethyl acetate-methanol solvate gradient elution of different volumes ratio, collect the merging eluent according to the thin layer testing result, concentrating under reduced pressure is dry; Obtain seven components: WE-4-1 (2.2g; The eluent ethyl acetate part), WE-4-2 (300mg, ethyl acetate-methanol 20:1 eluting part), WE-4-3 (520mg, ethyl acetate-methanol 20:1 eluting part), WE-4-4 (1.5g; Ethyl acetate-methanol 20:1 eluting part), WE-4-5 (2.8g; Ethyl acetate-methanol 15:1 eluting part), WE-4-6 (0.7g, ethyl acetate-methanol 15:1 eluting part), 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, polyamide, Sephadex LH-20 column chromatography obtain compound IV (105mg) to a main eluting stream part warp 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-MSm/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) or6 (t)), 100.5 (C-4a (t)); (100.1 C-4a (u)), 96.1 (C-8 (u) or6 (u)), 95.7 (C-6 (t) or8 (t)), 95.2 (C-6 (u) or8 (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-MSm/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 the polyamide column chromatography that obtains among the embodiment 1 (15g) is used an amount of dissolve with methanol, add the polyamide absorption of about 2 times of amounts, 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, concentrating under reduced pressure is dry, obtains 2 component E-4-1 (2.0g; Chloroform-methanol volume ratio 1:1 eluting partly) and E-4-2 (12g, methanol-eluted fractions part).E-4-2 (12g) is with methanol-water dissolving the carrying out Sephadex LH-20 gel filtration chromatography of an amount of percentage by volume 90%; Methanol-water eluting with equal volume percent 90%; Collect eluent and merge according to the 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); Sephadex LH-20 through repeatedly separates with polyamide column chromatography; Collect compound VI and VII bullion respectively according to the thin layer chromatography testing result, recrystallizing and refining from methanol respectively again, pure article compound VI (102mg) and VII (113mg).
Compound VI is a light brown crystal type powder (methanol), and fusing point is 172-174 ℃.ESI-MSm/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-MSm/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, supplies to survey active.
Activity test method: the PC12 cell of the trophophase of taking the logarithm, use contain 10% newborn calf serum and penicillin, streptomycin respectively the fresh DMEM culture medium of 100 μ g/mL to be made into cell density be 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 normal cultured 24h 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, and mixing is hatched 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 ELIASA.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 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 (5)
1. formula I chemical compound or its pharmaceutically acceptable salt are used to prepare the purposes of anti-anoxic medicine and health products of anti-anoxia,
Formula I.
2. formula I chemical compound or its pharmaceutically acceptable salt with oxygen lack resistant function, said formula I chemical compound is shown below,
Formula I.
3. pharmaceutical composition with oxygen lack resistant function, it comprises the described formula I chemical compound of claim 2, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carrier or excipient.
4. health food with oxygen lack resistant function, it comprises the described formula I chemical compound of claim 2, or its pharmaceutically acceptable salt.
5. the method for preparing of the said formula I chemical compound of claim 1, this method may further comprise the steps:
The dry bark 3.2kg that gets Fructus Choerospondiatis pulverizes the back with the lixiviate of commercially available medical ethanol room temperature, uses the aquiferous ethanol of 95% (V/V) to soak 7 days for 25 liters at every turn, carries altogether 4 times; Merge extractive liquid,, concentrating under reduced pressure is dry, gets ethanol extraction 750g; This ethanol extraction 750g is suspended in 3 premium on currency, uses chloroform, ethyl acetate, n-butanol extraction successively, and all kinds of solvents is used 3 liters at every turn; Respectively extract respectively 4 times; 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 an amount of dissolve with methanol, add an amount of polyamide absorption, drying, last polyamide column; Wherein ethyl acetate center pillar bed 7.0cm * 50cm with the ethyl acetate-methanol gradient elution of different volumes ratio, collects eluent and merges the concentrating under reduced pressure drying according to the thin layer testing result; Obtain four the thick component of chromatography: E-1,4g is eluent ethyl acetate part, E-2; 35g is 9: 1 eluting parts of ethyl acetate-methanol, E-3,25g; Be 1: 1 eluting part of ethyl acetate-methanol, and E-4,15g is the methanol-eluted fractions part;
Get E-3,25g uses an amount of dissolve with methanol, adds an amount of polyamide absorption, drying; Last polyamide column, post bed 3.8cm * 50cm with 4: 1 chloroform-methanol solvent system eluting, collects eluent and merges according to the thin layer testing result; Concentrating under reduced pressure is dry, gets three component E-3-1, E-3-2 and E-3-3, wherein E-3-2; 1.5g, carry out Sephadex LH-20 column chromatography behind the ethanol water dissolution with an amount of percentage by volume 90%, post bed 2.8cm * 27cm; Ethanol water elution with equal volume percent 90% gets the compound I bullion, and recrystallizing and refining gets the pure article 517mg of compound I in the methanol aqueous solution of percentage by volume 30% again, promptly obtains formula I chemical compound.
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| CN101224218A (en) * | 2008-01-07 | 2008-07-23 | 苏州大学 | Uses of 8-beta-D glucopyranosyl-4',7-dihydroxy isoflavone |
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| CN101224218A (en) * | 2008-01-07 | 2008-07-23 | 苏州大学 | Uses of 8-beta-D glucopyranosyl-4',7-dihydroxy isoflavone |
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