CN103550215B - Applications of peptides compound in rhizome sparganii - Google Patents
Applications of peptides compound in rhizome sparganii Download PDFInfo
- Publication number
- CN103550215B CN103550215B CN201310486948.8A CN201310486948A CN103550215B CN 103550215 B CN103550215 B CN 103550215B CN 201310486948 A CN201310486948 A CN 201310486948A CN 103550215 B CN103550215 B CN 103550215B
- Authority
- CN
- China
- Prior art keywords
- compound
- peptides
- time
- phe
- tyr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- JAYLGCXPHQNDIB-UHFFFAOYSA-N Cc1cc(CC(C(NC2Cc3ccccc3)=O)NC2=O)ccc1N Chemical compound Cc1cc(CC(C(NC2Cc3ccccc3)=O)NC2=O)ccc1N JAYLGCXPHQNDIB-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses peptides in a kind of trigone to prepare the application in anticoagulation and/or antithrombotic reagent, shown in the peptides structural formula such as formula (I). The present invention provides for the first time to be isolated and purified to obtain the peptides from trigone and provides its application, the compound has the effect of extension trend to prothrombin time (PT), active partial thromboplastin time (APTT) and thrombin time (TT), with good anticoagulant active, provided a strong basis for the exploitation of antithrombotic natural drug.
(I).
Description
Technical field
The present invention relates to medical art, more specifically, relate to one
threethe application of peptides in rib.
Background technology
Sparganium stoloniferum is Sparganiaceae plant rhizoma scirpi
sparganium stoloniferumthe dry tuber of Buch.-Ham, head is loaded in supplement to the Herbal, and existing is that " Chinese Pharmacopoeia " records, and its bitter in the mouth, property are flat, enter liver, spleen channel, have the function such as removing blood stasis circulation of qi promoting, removing food stagnancy pain relieving.The traditional Chinese medical science is except alone raw product, vinegar goods, and Chang Peiwu Rhizoma Curcumae, the Radix Astragali etc. heighten the effect of a treatment.
Modern pharmacological research shows, in the middle of 35 kinds of conventional drug for invigorating blood circulation and eliminating stasis, the action intensity of Rhizoma Sparganii occupies first 10, and is widely used in cardiovascular and cerebrovascular disease, the treatment of tumor and gynecological's various diseases.Particularly for the Gynecological stubborn diseases such as hysteromyoma, endometriosis, Rhizoma Sparganii shows stronger dispelling the stagnated QI of invigorating blood circulation, dissipate tired lame therapeutical effect, shows its extensive exploitation Utilization prospects in gynecological, tumor, hepatopathy and cardiovascular and cerebrovascular disease etc.
But, the complex chemical composition of Rhizoma Sparganii, containing flavone and flavonoid glycoside, Phenylpropanoid Glycosides glycosides, steroidal and the compound such as glycosides, organic acid thereof, although modern chemistry and pharmacological research part illustrate the contact between chemical composition and clinical practice, but also have a lot of active component not found, a lot of pharmacological research also only rests on the extract stage, limits the further genralrlization application of Rhizoma Sparganii compound.Therefore disclose further chemical composition and clinical medicine in Rhizoma Sparganii apply between contact, have very important meaning for instructing clinical application and new drug development.
Summary of the invention
The technical problem to be solved in the present invention fills up the deficiency of existing Rhizoma Sparganii compound application technology, provides
threethe application of peptides in rib.
Goal of the invention of the present invention is achieved by the following technical programs:
There is provided peptides in a kind of Rhizoma Sparganii preparing the application in anticoagulation and/or antithrombotic reagent, the structural formula of described compound as shown in the formula (I):
（Ⅰ）。
In described Rhizoma Sparganii, peptides comprises the enantiomer of compound shown in formula I, tautomer, physiology functional derivatives or pharmaceutically acceptable salt.
Further preferably, described compound is ring-(phenylalanine-tyrosine) [cyclo-(Phe-Tyr)].
Described compound can adopt the method for chemosynthesis to obtain.Invention also provides the compound that a kind of method for extraction and purification prepares structure shown in formula I, comprise the following steps:
S1. take Rhizoma Sparganii medical material, pulverize, add ethanol extraction, after extracting solution decompression and solvent recovery, obtain ethanol extract; Obtain through the continuous experimental summary of the present invention, adopt 60%(v/v) the extraction ratio of ethanol to Rhizoma Sparganii medicinal material extract the highest, preferably adopt 60%(v/v) ethanol;
S2. by ethanol extract aqueous dispersion, be extracted with ethyl acetate, combined ethyl acetate part, after decompression and solvent recovery, obtain ethyl acetate extract;
S3. adopt silica gel chromatographic column to be separated ethyl acetate extract, carry out gradient elution with chloroform-methanol solvent system, Fr38-40 merges, with dissolve with methanol after recycling design, room temperature is placed, and obtains white, needle-shaped crystals, obtains compound ring-(phenylalanine-tyrosine);
Fr41-43 merges, and through silica gel column chromatography segmentation, carry out gradient elution with chloroform-methanol solvent system (25:1,20:1,10:1), Fr2-4 merges, and room temperature separates out white, needle-shaped crystals after placing, and obtains compound ring-(phenylalanine-tyrosine).
The compound that the present invention adopts said extracted purification process to prepare has good anticoagulant active, can be advantageously applied to and prepare antithrombotic reagent aspect.
The present invention has following beneficial effect:
The present invention summarizes the process conditions of science through great many of experiments, adopt suitable method to be separated first from Sparganium stoloniferum and obtain a kind of CYCLIC DIPEPTIDES compounds, and confirm that it is to prothrombin time (prothrombin time through great many of experiments, PT), activated partial thromboplastin time (activated partial thrombop lastintime, and thrombin time (thrombin time APTT), TT) prolongation trend is had, there is good anticoagulant active, fill up prior art blank, exploitation for antithrombotic natural drug provides new foundation.
Accompanying drawing explanation
Fig. 1 is the extraction route map of Rhizoma Sparganii effective site.
Fig. 2 is the rough segmentation flow chart of ethyl acetate extract.
Fig. 3 is the segmentation flow chart of Fr41-43 section.
Fig. 4 the compounds of this invention
13c-NMR spectrogram.
Fig. 5 the compounds of this invention
1h-NMR spectrogram.
Fig. 6 the compounds of this invention affects result figure to prothrombin time.
Fig. 7 the compounds of this invention affects result figure to active partial thromboplastin time.
Fig. 8 the compounds of this invention affects result figure to thrombin time.
Detailed description of the invention
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, the reagent that the embodiment of the present invention adopts unless stated otherwise is conventional commercial reagent, and unless stated otherwise, the equipment of employing and method are equipment and the method for the art routine.But therefore do not limit the present invention.
embodiment 1the extracting and developing purification of compound shown in formula I and Structural Identification
1, experiment material:
(1) medical material
Rhizoma Sparganii medical material writes pharmaceutcal corporation, Ltd purchased from Guangzhou, and lot number is 090301, is accredited as Sparganiaceae Sparganium plant rhizoma scirpi through TCD identification teaching and research room of Chinese medicine institute of Guangdong Pharmaceutical University professor Li Shuyuan
sparganium stoloniferumbuch.-Ham prunes the dry tuber of crust.
(2) reagent and filler
Chloroform (analytical pure, Tianjin Fu Yu Fine Chemical Co., Ltd);
Methanol (analytical pure, Tianjin Fu Yu Fine Chemical Co., Ltd);
Vanillin (Guang Lianjin chemical plant, Guangzhou);
Column chromatography is with silica gel (80-100 order, 100-200 order, 200-300 order, Haiyang Chemical Plant, Qingdao);
Tlc silica gel (silica gel G, Qingdao Marine Chemical Co., Ltd.);
Sulphuric acid (analytical pure, Shanghai Shen Xiang chemical reagent company limited);
Sodium carboxymethyl cellulose CMC-Na(Shanghai Condar Chemical Engineering Technology service department).
(3) experimental apparatus
6202 miniature high-speed pulverizers (Beijing Huan Ya Tianyuan Mechanical Technology Inc.);
EC-1500/VC-600 type extracts thickener (Taiwan unit becomes mechanical limited company) automatically;
AGX1-2001-P type Ai Kepu (Aquapro) ultrapure water system (Chongqing Yi Yang enterprise development company limited);
RE-52A type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant);
SHZ-D type circulating water type vacuum pump (Ying Yu instrument plant of Henan Gongyi City);
GZX digital display air dry oven (Shanghai Bo Xun Medical Equipment Plant);
DLSB type low-temperature cooling fluid circulating pump (Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd.);
KQ-100 type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.);
Japan Yanaco MP-S3 type micro melting point apparatus;
Electric-heated thermostatic water bath (HHS type, Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd.);
Thin layer automatic coating device (YOKO-BF, Wuhan Yao Ke new technology development company);
Superconducting pulse Fourier-transform nuclear magnetic resonance spectrogrph: Bruker AVANCE AV 500;
Superconducting pulse Fourier-transform nuclear magnetic resonance spectrogrph: Varian UNITY INOVA 500.
2, the extraction of medical material:
Extraction process route map as shown in Figure 1.Get the dry tuber 14kg of Sparganium stoloniferum, be ground into coarse powder, put in multi-function extractor, with 60%(v/v) alcohol heating reflux extracts three times, each two hours, filters, and merges subsequent filtrate, reclaims ethanol, dry, obtains total extractum 752g.Total extractum is suspended in water, ultrasonic 20min, uses petroleum ether, chloroform, ethyl acetate, n-butanol extraction three times successively, merge each extract, reclaim under reduced pressure, obtain petroleum ether part extractum 26g, chloroform extract extractum 34g, ethyl acetate extract extractum 11.5g, n-butanol portion extractum 42g.
3, the separation and purification of composition:
By gained ethyl acetate extract 11.5g extractum, use about 50ml acetic acid ethyl dissolution sample, dry method mixes sample, through silica gel (200 ~ 300 order) column chromatography rough segmentation, to carry out gradient (100:1 with chloroform-methanol solvent system, 20:1,10:1,5:1, volume ratio) eluting, TLC inspects, and merges thin layer behavior phase homogeneous turbulence part.The attached rough segmentation flow chart that Figure 2 shows that ethyl acetate extract.In accompanying drawing 2, in square frame, alphabetical A represents the compounds of this invention.
(1) Fr38-40 merges, and use Rotary Evaporators to concentrate, then use dissolve with methanol, room temperature is placed, and obtains white, needle-shaped crystals, obtains described compound.
(2) by stream part of Fr41-43 merging, concentrating under reduced pressure, dissolve with appropriate chloroform, dry method mixes sample, segment through silica gel (200 ~ 300 order) column chromatography, carry out gradient elution with chloroform-methanol solvent system (25:1,20:1,10:1, volume ratio), TLC inspects, merge phase homogeneous turbulence part, merged by Fr2-4, room temperature separates out white, needle-shaped crystals after placing, and obtains described compound.The attached segmentation flow chart that Figure 3 shows that Fr41-43 section, in square frame, alphabetical A represents the compounds of this invention.
Said method is separated the Structural Identification of the compound obtained:
Colorless needle crystals (methanol), m.p.301 ~ 303 DEG C,
1in H-NMR (500MHz, DMSO) spectrum, δ 7.27 (2H, m), δ 7.20 (1H, m), δ 7.05 (2H, t, J=8.28Hz), there is monosubstituted phenyl, according to its chemical shift in display molecule, substituent group can only be alkane etc., and can not be remarkable electron-donating group or electron withdraw group; , there is para-orientation phenyl structure fragment in display molecule in δ 6.84 (2H, d, J=8.40Hz), δ 6.67 (2H, d, J=8.4Hz), its chemical shift prompting may have hydroxyl coupled; High field region also shows δ 3.94 (1H, brt.), and δ 3.88 (1H, brd.), δ 2.58 (2H, m) and δ 2.21 (2H, m), is the H signal in saturated carbon.
13c-NMR (500MHz, DMSO) spectrum provides 18 carbon signals.
By right
13the careful analysis of C-NMR can find, have 3 in molecule to signal extremely close in number, be respectively δ 166.2 and δ 166.1, δ 55.7 and δ 55.4, δ 39.9 and δ 39.3, corresponding group is 2 amide groups, 2 CH, 2 CH2; Infer thus, these former molecular structure fragments should be symmetrical structure, its data difference come from phenyl that 2 ends connect and p-hydroxybenzene on its chemical shift affect different.
This compound structure attribution data is as follows:
1 h-NMR (500MHz, DMSO):δ 9.22 (1H, s, OH), 7.86 (2H, s, CONH), 7.27 (2H, t,
j=7.0Hz, Phe-3 ', 5 '), 7.20 (1H, t,
j=7.5Hz, Phe-4 '), 7.05 (2H, t, Phe-2 ', 6 '), 6.84 (2H, d, J=8.4Hz, Tyr-2 ', 6 '), 6.67 (2H, d, J=8.40Hz, Tyr-3 ', 5 '), 3.94 (1H, brt, Tyr-α), 3.88 (1H, brd, Phe-α), 2.58 (2H, m, Phe-β) and 2.21 (2H, m, Tyr-β).
13 ，DMSO)：δ166.2(Tyr-co)，166.1(Phe-co)，156.1(Tyr-4′)， 136.6(Phe-1′)，130.7(Tyr-2′，6′)，129.7(Phe-3′，5′)，128.2(Phe-2′，6′)，126.4(Phe-4′ and Tyr-1′)，115.0(Tyr-3′，5′)，55.7(Tyr-α)，55.4(Phe-α)，39.9(Tyr-β)，39.3(Phe-β)。
As shown in figures 4 and 5.According to above-mentioned spectral data, and contrast the nuclear magnetic data reported, thus the structure determining this compound is ring-(phenylalanine-tyrosine).
The structural formula of described compound is as shown in the formula (I):
（Ⅰ）。
embodiment 2compound anticoagulant functions test of the present invention
1, experiment material
(1) medicine
Embodiment 1 gained compound: Cyclic dipeptides A [ring-(phenylalanine-tyrosine)].
(2) laboratory animal
SD rat, body weight 180 ~ 250g, is provided by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.
(3) reagent
Heparin (sigma, lot number: 20120509);
Chloral hydrate (Guangzhou Si Le biochemical reagents company limited);
(Bioengineering Research Institute is built up in Nanjing to prothrombin time PT test kit, lot number: 20130314);
(Bioengineering Research Institute is built up in Nanjing to activated partial thromboplastin time APTT test kit, lot number: 20130201);
(Bioengineering Research Institute is built up in Nanjing to thrombin time TT test kit, lot number: 20130314);
Normal saline (world, Guizhou pharmaceutcal corporation, Ltd, lot number A13011209);
Dimethyl sulfoxide (DMSO) (analytical pure, Guangzhou Chemical Reagent Factory);
Sodium citrate (analytical pure, Tianjin Chemical Co., Ltd.'s all generations, lot number: 0110).
(4) experimental apparatus
Desk centrifuge (Beijing Medical Centrifugal Machine Factory);
Electric-heated thermostatic water bath (HHS type, Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd.);
FA1104 type electronic analytical balance (Shanghai balance equipment factory);
Stopwatch (upper Asterias amurensis Lutken bores stopwatch company limited);
EP manages (Shanghai Shen Xiang chemical reagent company limited);
Liquid-transfering gun (Correspondent development in science and technology company limited of Beijing China);
Syringe (setting analysis Instrument Ltd. of Shanghai Chu).
2, experimental technique:
(1) blood plasma preparation
Rat 10% chloral hydrate (350mg/kg) intraperitoneal injection of anesthesia, abdominal aortic blood, is sub-packed in the plastic centrifuge tube containing 0.4ml, 109mmol/L sodium citrate anti-freezing liquid, and often pipe 3.6mL, puts upside down mixing immediately gently.The centrifugal 15min of 3000rpm, collects supernatant, obtain platelet poor plasma (PPP), tested in 2 hours, or-20 DEG C frozen for subsequent use.
(2) anticoagulating active measures
Embodiment 1 gained compound DMSO is dissolved into the solution that concentration is 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0mg/mL, and manual method measures its APTT, PT and TT value.
Carry out compound on prothrombin time, activated partial thromboplastin time and affect thrombin time impact experiment.Specific experiment operation illustrates according to prothrombin time PT test kit, activated partial thromboplastin time APTT test kit and thrombin time TT test kit respectively and operates.
(3) outcome record
Add corresponding reagent mix immediately and start stopwatch, test tube is still dipped in water-bath, takes out to during about 10s,
Upwards provoke every 2s and stop timing to generation solid filament, repeat above operation 3 times, get average and namely obtain PT, APTT or TT time.
3, experimental result and evaluation:
The results are shown in Table shown in 1 and accompanying drawing 6 ~ 8.
Table 1
Result shows, and in coagulation function, more blank group of PT, APTT and TT value of test-compound group all has prolongation trend, illustrates that the compounds of this invention has anticoagulant effect.
The present invention is separated the compound obtained from Sparganium stoloniferum has prolongation trend to prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), have good anticoagulant active, the exploitation for antithrombotic natural drug provides strong foundation.
Claims (1)
1. in Rhizoma Sparganii, peptides is preparing the application in anticoagulation and/or antithrombotic reagent, and described compound is ring-(phenylalanine-tyrosine), its structural formula as shown in the formula (I):
（Ⅰ）。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310486948.8A CN103550215B (en) | 2013-10-17 | 2013-10-17 | Applications of peptides compound in rhizome sparganii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310486948.8A CN103550215B (en) | 2013-10-17 | 2013-10-17 | Applications of peptides compound in rhizome sparganii |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103550215A CN103550215A (en) | 2014-02-05 |
| CN103550215B true CN103550215B (en) | 2015-06-03 |
Family
ID=50004692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310486948.8A Active CN103550215B (en) | 2013-10-17 | 2013-10-17 | Applications of peptides compound in rhizome sparganii |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103550215B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108904502B (en) * | 2018-08-24 | 2021-03-16 | 广东药科大学 | Application of burreed tuber component B in rhizoma sparganii in preparation of anti-blood stasis medicine |
| CN109172577B (en) * | 2018-08-24 | 2021-03-16 | 广东药科大学 | Application of burreed tuber component C in rhizoma sparganii in preparation of anti-blood stasis medicine |
| CN108926566B (en) * | 2018-08-24 | 2021-03-16 | 广东药科大学 | Application of burreed tuber component A in rhizoma sparganii in preparation of medicine for resisting blood stasis |
| CN113694066B (en) * | 2021-08-13 | 2023-02-17 | 广东药科大学 | Application of scirpusin C in preparation of medicine for treating cerebral ischemic stroke |
-
2013
- 2013-10-17 CN CN201310486948.8A patent/CN103550215B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 三棱的化学成分;董学等;《药学学报》;20081231;第43卷(第1期);第63-66页 * |
| 不同三棱提取物药理活性的比较研究;胡旭光等;《陕西中医》;20091231;第30卷(第8期);第1091-1093页 * |
| 中药三棱的化学成分及药理研究进展;董学等;《齐鲁药事》;20051231;第24卷(第10期);第612-614页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103550215A (en) | 2014-02-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103550215B (en) | Applications of peptides compound in rhizome sparganii | |
| CN103145677B (en) | Method for separating active ingredients from aquilaria sinensis lamina by utilizing high-speed countercurrent chromatography | |
| CN103536899B (en) | The application of peptide class reactive compound in Rhizoma Sparganii | |
| CN103520160B (en) | Application of peptide compound in rhizoma sparganii | |
| CN111187365B (en) | Chinese rose polysaccharide, extraction method and application thereof in preparation of anticoagulant drugs | |
| CN110294673A (en) | Caffeoylquinic acids butyl ester class isomer and its preparation method and application | |
| CN111689965B (en) | Alkaloid compound with antitumor activity and preparation method and application thereof | |
| CN105434482A (en) | Enrichment method of total flavonoids in pine needles of Cedrus deodara and application of total flavonoids in pine needles of Cedrus deodara in tumor suppression | |
| CN107936069B (en) | Coagulation-promoting apple flower effective component and extraction and separation method and application thereof | |
| CN103610762B (en) | Extract of corydalis impatiens total alkaloids and extraction method thereof | |
| CN103340916B (en) | Lindley eupatorium extract as well as preparation method and application thereof | |
| CN108578461B (en) | Cercis chinensis extract, its extraction method and application in preparing antithrombotic medicines | |
| CN114790222B (en) | Flavonoids based on epimedium and preparation method thereof | |
| CN103524576A (en) | Separation and purification process of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside | |
| CN105601693A (en) | Preparation method and antitumor effect of ginsenoside F1 | |
| CN110305079A (en) | A kind of anticancer drug supercritical fluid extraction method of purification | |
| CN101851266A (en) | Novel cycloartane triterpenoidsaponins and preparation method and application thereof | |
| CN103623066B (en) | Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof | |
| CN108164574B (en) | Compound in caulis Sinomenii, and preparation method and application thereof | |
| CN112538016B (en) | Method for extracting chlorogenic acid n-butyl ester from peach blossom | |
| CN108623645B (en) | Flavonoid compound and preparation method and application thereof | |
| CN112125937A (en) | Macrocyclic phenolic glycoside compound and preparation method and application thereof | |
| CN114933522B (en) | Preparation method and application of shikonin compound | |
| CN112624928B (en) | Anticoagulant peach blossom effective component and application thereof | |
| CN110066306A (en) | A kind of Isorhamnetin-3-O-neohespeidoside preparative liquid chromatography separation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 510240 40 Haizhuqu District, Guangdong, Guangzhou. Patentee after: Guangdong Pharmaceutical University Address before: 510006 No. 280 East Ring Road, Guangzhou City University, Guangdong Patentee before: Guangdong Pharmaceutical University |