CN1594353A - Process for preparing periplocin - Google Patents
Process for preparing periplocin Download PDFInfo
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- CN1594353A CN1594353A CN 200410019967 CN200410019967A CN1594353A CN 1594353 A CN1594353 A CN 1594353A CN 200410019967 CN200410019967 CN 200410019967 CN 200410019967 A CN200410019967 A CN 200410019967A CN 1594353 A CN1594353 A CN 1594353A
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- periplocoside
- preparation
- macroporous resin
- bark
- root
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- KWBPKUMWVXUSCA-AXQDKOMKSA-N 3-[(3s,5s,8r,9s,10r,13r,14s,17r)-5,14-dihydroxy-3-[(2r,4s,5r,6r)-4-methoxy-6-methyl-5-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-10,13-dimethyl-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl] Chemical compound C1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C)CC[C@@H](C[C@@]5(O)CC[C@H]4[C@@]3(O)CC2)O[C@H]2C[C@@H]([C@@H]([C@@H](C)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)OC)=CC(=O)OC1 KWBPKUMWVXUSCA-AXQDKOMKSA-N 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- LMZSCLAHNSQLBW-UHFFFAOYSA-N periplocin Natural products COC1CC(OC2CCC3(C)C(CCC4C3CCC5(C)C(CCC45C)C6=CC(=O)OC6)C2)OC(C)C1OC7OC(CO)C(O)C(O)C7O LMZSCLAHNSQLBW-UHFFFAOYSA-N 0.000 title abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 235000002470 Asclepias syriaca Nutrition 0.000 claims abstract description 48
- 241000893851 Periploca graeca Species 0.000 claims abstract description 48
- 238000002360 preparation method Methods 0.000 claims abstract description 37
- 229940097217 cardiac glycoside Drugs 0.000 claims abstract description 35
- 239000002368 cardiac glycoside Substances 0.000 claims abstract description 35
- LPMXVESGRSUGHW-HBYQJFLCSA-N ouabain Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1C[C@@]2(O)CC[C@H]3[C@@]4(O)CC[C@H](C=5COC(=O)C=5)[C@@]4(C)C[C@@H](O)[C@@H]3[C@@]2(CO)[C@H](O)C1 LPMXVESGRSUGHW-HBYQJFLCSA-N 0.000 claims abstract description 35
- 229920005989 resin Polymers 0.000 claims abstract description 35
- 239000011347 resin Substances 0.000 claims abstract description 35
- 229930002534 steroid glycoside Natural products 0.000 claims abstract description 35
- 238000010828 elution Methods 0.000 claims abstract description 29
- 239000007864 aqueous solution Substances 0.000 claims abstract description 20
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 15
- 239000007791 liquid phase Substances 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- 229930186642 periplocoside Natural products 0.000 claims description 52
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000000605 extraction Methods 0.000 claims description 29
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- 239000012071 phase Substances 0.000 claims description 19
- 239000002904 solvent Substances 0.000 claims description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 16
- 238000001179 sorption measurement Methods 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 7
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 239000012916 chromogenic reagent Substances 0.000 claims description 5
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000013078 crystal Substances 0.000 claims description 4
- 238000001953 recrystallisation Methods 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 3
- XYLMUPLGERFSHI-UHFFFAOYSA-N alpha-Methylstyrene Chemical compound CC(=C)C1=CC=CC=C1 XYLMUPLGERFSHI-UHFFFAOYSA-N 0.000 claims description 2
- MPMBRWOOISTHJV-UHFFFAOYSA-N but-1-enylbenzene Chemical compound CCC=CC1=CC=CC=C1 MPMBRWOOISTHJV-UHFFFAOYSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 238000004040 coloring Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 238000010992 reflux Methods 0.000 description 9
- 241000753128 Periploca <moth> Species 0.000 description 8
- VYWYYJYRVSBHJQ-UHFFFAOYSA-N 3,5-dinitrobenzoic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1 VYWYYJYRVSBHJQ-UHFFFAOYSA-N 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 7
- 239000012445 acidic reagent Substances 0.000 description 7
- 238000002203 pretreatment Methods 0.000 description 7
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- WZUODJNEIXSNEU-UHFFFAOYSA-N 2-Hydroxy-4-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C(O)=C1 WZUODJNEIXSNEU-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 206010007559 Cardiac failure congestive Diseases 0.000 description 4
- 206010019280 Heart failures Diseases 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000208327 Apocynaceae Species 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- OKQKFNTWEWCEEK-UHFFFAOYSA-N 6-[[17-hydroxy-17-[1-[5'-(5-hydroxy-4-methoxy-6-methyloxan-2-yl)oxy-4'-methoxy-6',9-dimethylspiro[4,5a,6,7,9,9a-hexahydropyrano[3,4-c][1,2,5]trioxepine-3,2'-oxane]-7-yl]oxyethyl]-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanth Chemical compound O1C(C)C(O)C(OC)CC1OC1C(OC)CC2(OOC3C(C)OC(OC(C)C4(O)C5(CCC6C7(C)CCC(CC7=CCC6C5CC4)OC4C(C(OC)=CC(C)O4)=O)C)CC3OC2)OC1C OKQKFNTWEWCEEK-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 229910052799 carbon Inorganic materials 0.000 description 2
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- 239000004615 ingredient Substances 0.000 description 2
- 239000002917 insecticide Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
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- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000007225 Limnophila aromatica Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 244000236146 brown strophanthus Species 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
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- 239000000039 congener Substances 0.000 description 1
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- 150000008131 glucosides Chemical group 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- -1 glycosyl carbon Chemical compound 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a process for preparing periplocin which consists the steps of, (1) extracting medicinal material of Chinese silkvine bark, obtaining aqueous solution of Chinese silkvine bark, (2) carrying out macroporous resin absorption to the aqueous solution, eluting using 10-95% ethanol, collecting the elution position, concentrating and proceeding thin-layer chromatography, coloring with color-developing agent, collecting and merging positions with cardiac glycoside, (3) preparation and separation of liquid phase.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of preparation method who treats the cardiac glycoside of acute and chronic congestive heart failure.
Background technology
Root-bark of Chinese Silkvine is the dry root skin of asclepiadaceae plant periploca spium, and Root-bark of Chinese Silkvine is usually used in treating acute and chronic congestive heart failure (CHF) in recent years.Root-bark of Chinese Silkvine is poisonous, and excessive use can produce untoward reaction, and the report of causing death is also arranged clinically.The assay item of Root-bark of Chinese Silkvine adopts 4-methoxysalicylaldehyde to measure for the HPLC method down and uses reference substance in the Pharmacopoeia of the People's Republic of China of version in 2000, but up to the present there is no the report that 4-methoxysalicylaldehyde has relevant pharmacological action.Periplocoside (periplocin) is that periplocoside G (structural formula as follows) is a kind of cardiac glycoside composition in the Root-bark of Chinese Silkvine, and is closely related with the clinical efficacy of Root-bark of Chinese Silkvine, therefore comparatively reasonable as the quality control index of Root-bark of Chinese Silkvine treatment CHF with periplocoside.But can't buy the periplocoside reference substance from the domestic and international market at present.
Periplocoside C
36H
56O
13
Japanese scholar isolated periplocoside and had determined structure (Seiichi Sakama et al.On theStructure of Glycoside G and K of Bei-Wujiapi.Chem.Pharm.Bull from Root-bark of Chinese Silkvine in 1969,1969,17 (10): 2183.), the method that he adopts is methyl alcohol or extraction using alcohol, extracting solution concentrates the back and extracts with benzene, the water saturated n-butanol extraction of water layer after the benzene collection, after concentrating, n-butanol portion, obtains periplocoside monomer (productive rate 0.02%) at last repeatedly through silica gel column chromatography.Hu Yingjie (Hu Yingjie in 1989, the full chapter of wood. the chemical ingredients of Yunnan periploca spium. Yunnan plant grinds [J] .1989,11 (4): 465～479) also isolate periplocoside from the congener Yunnan periploca spium (black keel) of periploca spium, the employing method is 95% alcohol reflux, alcohol extract is behind petroleum ether degreasing, use the acetone refluxing extraction, the solvable position of acetone obtains periplocoside (productive rate 0.023%) through silica gel column chromatography and RP-8 silica gel reversed phase column chromatography.
Up to the present the preparation method of the periplocoside that is adopted has plenty of raw material 95% alcohol reflux, and is incomplete to the extraction of medicinal material, and the low polar impurity that extracts is many, is unfavorable for the monomeric separation of periplocoside; What have will be through silica gel column chromatography repeatedly, and it is time-consuming again promptly to take solvent, and productive rate is low, and cost is higher.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of preparation method of periplocoside is provided.
Technical scheme of the present invention is summarized as follows:
(1) extraction of Root-bark of Chinese Silkvine medicinal material: with concentration of volume percent is that 40%～80% ethanol or 40%～100% methyl alcohol serve as to extract solvent, and Root-bark of Chinese Silkvine is extracted, and boils off solvent after the extraction, adds water filtration or centrifugal, the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: the Root-bark of Chinese Silkvine aqueous solution is through macroporous resin adsorption, with 10%～95% ethanol gradient elution, collect each wash-out position, carry out thin-layer chromatography respectively after concentrating, with the special-purpose chromogenic reagent of cardiac glycoside, collect the position that merging contains cardiac glycoside;
(3) the high performance liquid phase preparation separates: the high performance liquid phase preparation is carried out with reverse-phase chromatographic column in the cardiac glycoside position, with volume ratio 30～60: 70～40 methyl alcohol: water, or 15～40: 85～60 acetonitrile: water is moving phase, adopt UV-detector to collect with guide product synchronously, collect liquid and behind concentrate drying, obtain periplocoside in 217～220nm supervision elution curve.
The preferred concentration of extraction etoh solvent of Root-bark of Chinese Silkvine medicinal material is 50%～70%, and optimum concn is 60%.
Described macroporous resin is non-polar macroporous resin or low-pole macroporous resin.
Described non-polar macroporous resin is to be the macroporous resin of skeleton with vinylbenzene or ethyl styrene or alpha-methyl styrene.
Described low-pole macroporous resin is to be the macroporous resin of skeleton with vinylbenzene.
Also can carry out silica gel column chromatography to the cardiac glycoside position between step (2) and step (3) separates: on the cardiac glycoside position behind the sample, with volume ratio 11: 1～6: 1 water saturation ethyl acetate: methyl alcohol, or 65: 3～65: 20 chloroform: methyl alcohol gradient elution, each flow point of elutriant carries out thin-layer chromatography respectively, with the special-purpose chromogenic reagent of cardiac glycoside, collect the flow point that merging contains cardiac glycoside.
Also can carry out recrystallization to periplocoside afterwards in step (3), solvent for use is methyl alcohol or ethanol or ethyl acetate, gets the periplocoside crystal.
With " a kind of preparation method of periplocoside " of the present invention prepared periplocoside through UV, IR,
13C-NMR, MS analysis confirmation are periplocoside; Detect this product purity through TLC, HPLC (PDA detector) and reach 98.8%, products obtained therefrom can reach the requirement of assay with the Chemistry for Chinese Traditional Medicine reference substance.And preparing periplocoside with the inventive method, medicinal material extract is complete, the productive rate height; Consumption of organic solvent is few.
Description of drawings
Fig. 1 is the IR collection of illustrative plates of the periplocoside of the present invention's preparation.
Fig. 2 is the C of the periplocoside of the present invention's preparation
13-NMR collection of illustrative plates, table 1 are C
13-NMR data.
Fig. 3 is the MS collection of illustrative plates of the periplocoside of the present invention's preparation.
Fig. 4 is the three dimensional chromatogram of the periplocoside of the present invention's preparation.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated:
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is 40% ethanolic soln that the 500g Root-bark of Chinese Silkvine adds the 4000mL concentration of volume percent, refluxing extraction 3 times, each 2 hours, united extraction liquid, be evaporated to about 500mL, add 500mL water dilution after-filtration, get the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: the Root-bark of Chinese Silkvine aqueous solution is adsorbed with the good AB-8 macroporous resin (Chemical Plant of Nankai Univ.'s production) of pre-treatment, with 10%～95% ethanol gradient elution, collect each wash-out position, the evaporated under reduced pressure solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) silica gel column chromatography separates: 160～200 order chromatographic silica gels, 300 grams are adorned post with the ethyl acetate wet method, sample on the dry method of cardiac glycoside position, it with volume ratio 11: 1～6: 1 ethyl acetate: the methyl alcohol gradient elution, every 200mL elutriant is a, each flow point carries out thin-layer chromatography after the evaporated under reduced pressure solvent adds dissolve with ethanol respectively, with the special-purpose chromogenic reagent of cardiac glycoside, collect the flow point that merging contains cardiac glycoside, the result shows the cardiac glycoside composition in the flow point 2～6;
(4) the high performance liquid phase preparation separates: carry out the high performance liquid phase preparation after 2～6 flow points are filtered with millipore filtration (0.45 μ m) and separate chromatographic column: C
18Reverse-phase chromatographic column (Hypersil, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is methyl alcohol: water=30: 70; Flow velocity: 2.5mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 220nm supervision elution curve.
(5) recrystallization: will collect the liquid concentrating under reduced pressure, and volatilize solvent, and add 2mL methyl alcohol, and be transferred in the crystallizing dish, and put into moisture eliminator (CaCl
2Siccative), separate out white, needle-shaped crystals gradually, crystallization filtered out, 3 times so repeatedly, dry back altogether crystallization 412mg, productive rate is 0.08%.
(6) structure and purity detecting:
Structural confirmation:
A. UV scanning: UVmax (EtOH)=218nm (has Δ
α, βThe characteristic absorbance of lactone ring five membered cardiac glycoside).
B.IR analyzes: mainly be absorbed as 3600～3200 (OH), 1782,1741,1631 (beta substitution-Δs
α, β-gamma lactone), 1077,1044cm
-1Collection of illustrative plates is seen accompanying drawing 1.
C.C
13-NMR: data and Hu Yingjie, the full chapter of wood. the chemical ingredients of Yunnan periploca spium. Yunnan plant grinds [J] .1989, and 11 (4): 465～479 unanimities of being announced.Data see Table 1, and collection of illustrative plates is seen Fig. 2.
D.MS analyzes: m/z:697.4 (M
+), 534.1 (M
+-C
6H
10O
5).Collection of illustrative plates is seen accompanying drawing 3.
Confirm as periplocoside by analysis.
Purity check:
A.TLC: point sample 0.3 μ g, 3 μ g, 30 μ g on the high-efficient silica gel plate, chloroform: methyl alcohol; Water=lower floor launched in 65: 30: 10, used 3 respectively, 5-dinitrobenzoic acid and 10%H
2SO
4Single redness and black splotch are all only seen in colour developing.
B. detect through HPLC (PDA), purity reaches 98.8%.See accompanying drawing 4, in the drawings, 12 minutes peak of retention time is the periplocoside peak.
The prepared periplocoside of table 1
13The C-NMR data
Glucoside unit carbon number periplocoside own product glycosyl carbon number periplocoside 1
1 25.9 25.96 1’ 97.3 97.32
2 26.3 26.33 2’ 36.6 36.56
3 73.6 73.64 3’ 77.8 77.9
4 35.3 35.35 4’ 82.6 82.88
5 75.2 75.32 5’ 69.4 69.48
6 35.4 35.42 6’ 18.5 18.61
7 24.2 24.28 OMe 58.5 58.5
8 40.9 40.91 1” 106.3 106.48
9 39.2 39.17 2” 75.9 75.97
10 41.1 41.13 3” 78.2 78.38
11 21.9 21.95 4” 71.9 71.76
12 39.9 39.85 5” 78.2 78.32
13 49.9 49.91 6” 62.8 62.96
14 84.6 84.58
15 33 33.06
16 27.2 27.2
17 51.2 51.25
18 16 16.1
19 17.1 17.17
20 175.9 175.86
21 73.6 73.66
22 117.6 117.64
23 174.4 174.45
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is 80% ethanolic soln that the 500g Root-bark of Chinese Silkvine adds the 3000mL concentration of volume percent, refluxing extraction 3 times, each 2 hours, united extraction liquid, be evaporated to about 500mL, add 800mL water dilution after-filtration, get the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: with the Root-bark of Chinese Silkvine aqueous solution good D of pre-treatment
101(production of Tianjin insecticide factory) macroporous resin adsorption with 10%～95% ethanol gradient elution, is collected each wash-out position, the evaporated under reduced pressure solvent, and residue adds dissolve with ethanol, carries out thin-layer chromatography respectively.Use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development has the red-purple spot in display area 3～5 (40%～60% ethanol elution position) as a result, with its merging, the cardiac glycoside position;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate.Chromatographic column: C
18Reverse-phase chromatographic column (Hypersil, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is methyl alcohol: water=40: 60, flow velocity: 2.5mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 217nm supervision elution curve;
To collect the liquid concentrating under reduced pressure, and volatilize solvent, and get periplocoside 495mg after the drying, productive rate is 0.10%.
Embodiment 3
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is 50% ethanolic soln that the 500g Root-bark of Chinese Silkvine adds the 3500mL concentration of volume percent, refluxing extraction 2 times, and each 3 hours, united extraction liquid was evaporated to about 400mL, adds the 1000mL water filtration, got the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: with the Root-bark of Chinese Silkvine aqueous solution good Dm2 (production of the Tianjin Chemical Plant of Nankai Univ.) macroporous resin adsorption of pre-treatment, with 10%～95% ethanol gradient elution, collect each wash-out position, the evaporated under reduced pressure solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate chromatographic column: C
18Reverse-phase chromatographic column (Hypersil ODS, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is methyl alcohol: water=50: 50, flow velocity: 2.5mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 220nm supervision elution curve;
To collect the liquid concentrating under reduced pressure, wave, get periplocoside after the drying in solvent.
Embodiment 4
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is 70% ethanolic soln that the 500g Root-bark of Chinese Silkvine adds the 2500mL concentration of volume percent, and refluxing extraction 1 hour is extracted 4 times, and united extraction liquid is evaporated to 800mL, adds the 300mL water filtration, gets the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: the Root-bark of Chinese Silkvine aqueous solution is adsorbed with the good HPD100 macroporous resin of pre-treatment (the precious grace chemical industry in Cangzhou company limited produces), with 10%～95% ethanol gradient elution, collect each wash-out position, decompression is steamed in solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate chromatographic column: C
18Reverse-phase chromatographic column (Hypersil ODS, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is acetonitrile: water=15: 85, flow velocity: 2.5mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside in 217nm supervision elution curve.
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is that 40% methanol solution carries out the diacolation extraction that the 500g Root-bark of Chinese Silkvine adds the 8000mL concentration of volume percent, and extracting solution is evaporated to about 400mL, adds 700mL water dilution after-filtration, gets the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: with the Root-bark of Chinese Silkvine aqueous solution good D2 (Chemical Plant of Nankai Univ.'s production) macroporous resin adsorption of pre-treatment, with 10%～95% ethanol gradient elution, collect each wash-out position, the evaporated under reduced pressure solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate chromatographic column: C
8Reverse-phase chromatographic column (Hypersil ODS, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is acetonitrile: water=40: 60, flow velocity: 2.0mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 217nm supervision elution curve;
(4) recrystallization: will collect the liquid concentrating under reduced pressure, and volatilize solvent, and add 2mL methyl alcohol, and be transferred in the crystallizing dish, and put into moisture eliminator (CaCl
2Siccative), separates out white, needle-shaped crystals gradually, crystallization is leached, the washing after drying.
Embodiment 6
(1) extraction of Root-bark of Chinese Silkvine medicinal material: the 500g Root-bark of Chinese Silkvine adds the 3500mL methanol solution, and refluxing extraction 2 hours is extracted 4 times, and united extraction liquid is evaporated to about 500mL, adds the 700mL water filtration, gets the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: with the Root-bark of Chinese Silkvine aqueous solution good GDX-104 (production of the Tianjin reagent two factories) macroporous resin adsorption of pre-treatment, with 10%～95% ethanol gradient elution, collect each wash-out position, the evaporated under reduced pressure solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate chromatographic column: (C
18Reverse-phase chromatographic column Hypersil ODS, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is acetonitrile: water=27: 73, flow velocity: 2.0mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 217nm supervision elution curve;
To collect the liquid concentrating under reduced pressure, get periplocoside after the drying.
Embodiment 7
(1) extraction of Root-bark of Chinese Silkvine medicinal material: it is 60% ethanolic soln that the 500g Root-bark of Chinese Silkvine adds the 3500mL concentration of volume percent, and refluxing extraction 2 hours is extracted 4 times, and united extraction liquid is evaporated to about 500mL, adds the 700mL water filtration, gets the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: with the Root-bark of Chinese Silkvine aqueous solution good GDX-104 (production of the Tianjin reagent two factories) macroporous resin adsorption of pre-treatment, with 10%～95% ethanol gradient elution, collect each wash-out position, the evaporated under reduced pressure solvent, residue adds dissolve with ethanol, carry out thin-layer chromatography respectively, use chloroform: methyl alcohol: the lower floor of water=65: 30: 10 is a developping agent, 3,5-dinitrobenzoic acid reagent colour development, in display area 3～5 (40%～60% ethanol elution position) the red-purple spot is arranged as a result,, get the cardiac glycoside position its merging;
(3) the high performance liquid phase preparation separates: carry out the HPLC preparation after the cardiac glycoside position is filtered with millipore filtration (0.45 μ m) and separate chromatographic column: (C
18Reverse-phase chromatographic column Hypersil ODS, 5 μ m, 10 * 250mm); Chromatographic condition: moving phase is methyl alcohol: water=38: 62, flow velocity: 2.0mL/min; Adopt UV-detector to collect with guide product synchronously, obtain periplocoside and collect liquid in 217nm supervision elution curve;
To collect the liquid concentrating under reduced pressure, get periplocoside after the drying.
When actually operating, can also select for use other model non-polar macroporous resin as HPD 100 or HPD 300 (Cangzhou precious grace chemical industry company limited); GDX-104 (Tianjin reagent two factories); SIP-1100 or SIP-1200 or SIP-1300 or SIP-1400 (Shanghai Institute of Pharmaceutical Industry); Last examination 101 or go up examination 102 or go up examination 401 or go up examination 402 (Shanghai chemical reagent works); Nanjing University 3520 or D1 or D2, or Dm2, or Dm5 (Chemical Plant of Nankai Univ.), or the like.The model of the low-pole macroporous resin that can select for use is: AB-8 (Chemical Plant of Nankai Univ.); HPD 500, HPD 400 (the precious grace chemical industry in Cangzhou company limited); D201, D301 (Tianjin insecticide factory), external Diaion (HP10, HP20, HP21), Amberlite (XAD-1, XAD-2), or the like.
Except Root-bark of Chinese Silkvine, also can asclepiadaceae plant Yunnan periploca spium or Greece's periploca spium or U.S. leaf periploca spium, or the seed of apocynaceae plant strophanthus hispidus is that raw material extracts periplocoside.
The available preparative column of industrial preparation periplocoside has: Waters C18 (20mm * 250mm), Zorbax SB-C18 (9.4 * 250mm) or the like.
Claims (8)
1. the preparation method of a periplocoside is characterized in that comprising the steps:
(1) extraction of Root-bark of Chinese Silkvine medicinal material: with concentration of volume percent is that the methyl alcohol of 40%～80% ethanol or 40%～100% serves as to extract solvent, and Root-bark of Chinese Silkvine is extracted, and boils off solvent after the extraction, adds water filtration or centrifugal, the clarifying Root-bark of Chinese Silkvine aqueous solution;
(2) macroporous resin adsorption is separated: the Root-bark of Chinese Silkvine aqueous solution is through macroporous resin adsorption, with 10%～95% ethanol gradient elution, collect each wash-out position, carry out thin-layer chromatography respectively after concentrating, with the special-purpose chromogenic reagent of cardiac glycoside, collect the position that merging contains cardiac glycoside;
(3) the high performance liquid phase preparation separates: the high performance liquid phase preparation is carried out with reverse-phase chromatographic column in the cardiac glycoside position, with volume ratio 30～60: 70～40 methyl alcohol: water, or 15～40: 85～60 acetonitrile: water is moving phase, adopt UV-detector to collect with guide product synchronously, collect liquid and behind concentrate drying, obtain periplocoside in 217～220nm supervision elution curve.
2. the preparation method of a kind of periplocoside according to claim 1 is characterized in that described concentration of ethanol is 50%～70%.
3. the preparation method of a kind of periplocoside according to claim 2 is characterized in that described concentration of ethanol is 60%.
4. the preparation method of a kind of periplocoside according to claim 1 is characterized in that described macroporous resin is non-polar macroporous resin or low-pole macroporous resin.
5. the preparation method of a kind of periplocoside according to claim 4 is characterized in that described non-polar macroporous resin is is the macroporous resin of skeleton with vinylbenzene or ethyl styrene or alpha-methyl styrene.
6. the preparation method of a kind of periplocoside according to claim 4 is characterized in that described low-pole macroporous resin is is the macroporous resin of skeleton with vinylbenzene.
7. the preparation method of a kind of periplocoside according to claim 1, it is characterized in that, between step (2) and step (3) silica gel column chromatography being carried out at the cardiac glycoside position separates: on the cardiac glycoside position behind the sample, with volume ratio 11: 1～6: 1 water saturation ethyl acetate: methyl alcohol, or 65: 3～65: 20 chloroform: methyl alcohol gradient elution, each flow point of elutriant carries out thin-layer chromatography respectively, with the special-purpose chromogenic reagent of cardiac glycoside, collects the flow point that merging contains cardiac glycoside.
8. according to the preparation method of claim 1 or 7 described a kind of periplocosides, it is characterized in that periplocoside is carried out recrystallization with methyl alcohol or ethanol or ethyl acetate, make the periplocoside crystal.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305480C (en) * | 2005-06-03 | 2007-03-21 | 中国科学院昆明植物研究所 | Antineoplastic medicinal composition, its preparation method and application |
| CN103027932A (en) * | 2012-11-29 | 2013-04-10 | 江苏大学 | Cortex periplocae anti-tumor effective component extractive as well as preparation method and application thereof |
| CN105777514A (en) * | 2014-12-24 | 2016-07-20 | 河北以岭医药研究院有限公司 | Extraction and separation method for chemical components in cortex periplocae |
| WO2019179465A1 (en) * | 2018-03-22 | 2019-09-26 | 石家庄以岭药业股份有限公司 | Application of cortex periplocae extract in preparation of medicament for treating heart failure complications |
-
2004
- 2004-07-12 CN CN 200410019967 patent/CN1253467C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305480C (en) * | 2005-06-03 | 2007-03-21 | 中国科学院昆明植物研究所 | Antineoplastic medicinal composition, its preparation method and application |
| CN103027932A (en) * | 2012-11-29 | 2013-04-10 | 江苏大学 | Cortex periplocae anti-tumor effective component extractive as well as preparation method and application thereof |
| CN105777514A (en) * | 2014-12-24 | 2016-07-20 | 河北以岭医药研究院有限公司 | Extraction and separation method for chemical components in cortex periplocae |
| CN105777514B (en) * | 2014-12-24 | 2020-08-14 | 河北以岭医药研究院有限公司 | Extraction and separation method of chemical components of cortex periplocae |
| WO2019179465A1 (en) * | 2018-03-22 | 2019-09-26 | 石家庄以岭药业股份有限公司 | Application of cortex periplocae extract in preparation of medicament for treating heart failure complications |
| CN110292589A (en) * | 2018-03-22 | 2019-10-01 | 石家庄以岭药业股份有限公司 | Application of the Cortex Periplocae Radicis extract in treatment heart failure complication |
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