CN110452281A - The method that Qingyangshenglycoside A is extracted using Cynanchum otophyllum Schneid - Google Patents
The method that Qingyangshenglycoside A is extracted using Cynanchum otophyllum Schneid Download PDFInfo
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- CN110452281A CN110452281A CN201910773666.3A CN201910773666A CN110452281A CN 110452281 A CN110452281 A CN 110452281A CN 201910773666 A CN201910773666 A CN 201910773666A CN 110452281 A CN110452281 A CN 110452281A
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- qingyangshenglycoside
- silica gel
- methanol
- alcoholic solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
- C07J53/002—Carbocyclic rings fused
- C07J53/004—3 membered carbocyclic rings
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of methods for extracting Qingyangshenglycoside A using Cynanchum otophyllum Schneid; four steps are purified including raw material extraction, macroreticular resin chromatography, silica gel column chromatography, preparative HPLC; the present invention utilizes acyl group stable feature under mildly acidic conditions; medicinal material is extracted using acidic aqueous solution; reduce the percent hydrolysis of para hydroxybenzene formyl group; the recovery rate for improving Qingyangshenglycoside A is conducive to further purify；It is enriched with using macroreticular resin, silica gel, eliminates impurity, Qingyangshenglycoside A has been further purified, also the filler of preparative efficient liquid phase, related accessory are protected；Qingyangshenglycoside A is isolated and purified using UV detector on-line monitoring, the synchronous progress of separation detection is realized, avoids the waste of resource, wrong receipts is also avoided, leaks the mistake for receiving sample, increase yield.
Description
Technical field
The invention belongs to Chinese medicine extractive technique fields, and in particular to a kind of to extract Qingyangshenglycoside A using Cynanchum otophyllum Schneid
Method.
Background technique
Auricledleaf swallowwort root (Cynanchum otophyllum Schneid) English name RADIX CYNANCHI OTOPHYLLI.This product is Asclepiadaceae plant blueness sheep
The dry root of Cynanchum otophyllum C.K.Schneider.Summer, autumn excavation, impurity is removed, is dried.Function master
Control: wind-damp dispelling, qi-restoratives is empty, antispastic, detoxification.For treating rheumatic ostealgia, lumbar muscle strain, physically weak mind declines, infantile malnutrition, slow frightened, epilepsy,
Dog bite.Nature and flavor: it is sweet, pungent, warm, it is slightly poisonous.The Chinese patent drug of Cynanchum otophyllum Schneid piece is developed, main component is that Cynanchum otophyllum Schneid is total
Glycosides.In recent years to each side such as the chemical component of Cynanchum otophyllum Schneid, pharmacological toxicology activity, assay, pharmacognostical study and reproduction techniques
Face makes further research.But there are following deficiencies for research:
1. Cynanchum otophyllum Schneid chemical component separation identification research is more, fail to provide what a large amount of standard substances were studied as other
Material base.
2. assay only resides within the level of measurement aglycon and total glycosides due to lacking standard substance.
3. show that Cynanchum otophyllum Schneid has the effects of wind-damp dispelling, qi-restoratives, antispastic, detoxification according to theory of traditional Chinese medical science and clinical research, but
Specific material base and mechanism of action not yet thoroughly research, existing Cynanchum otophyllum Schneid one-component research is not complete, and clinical effectiveness leaves a question open.
4. clinical research proves that Cynanchum otophyllum Schneid is long-term or largely takes with toxicity, but wherein specific toxicant with
Toxic dose research is less.
5. the total glycosides research of Cynanchum otophyllum Schneid is more, single saponin(e or structural modification etc. research are less.
Above 5 points insufficient, is to be studied due to failing to extract the one-component of auricledleaf swallowwort root at present comprehensively, in Cynanchum otophyllum Schneid
Main component be Qingyangshenglycoside A, also study at present it is very few, to find out its cause, be that Qingyangshenglycoside A preparative separation is difficult, point
It is few from amount.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for extracting Qingyangshenglycoside A using Cynanchum otophyllum Schneid, by Cynanchum otophyllum Schneid
A large amount of preparations of main component Qingyangshenglycoside A provide a large amount of substance for the subsequent pharmacology of Cynanchum otophyllum Schneid, toxicity, structural modification research
Basis.
To achieve the above object, The technical solution adopted by the invention is as follows:
The method for extracting Qingyangshenglycoside A using Cynanchum otophyllum Schneid, includes the following steps:
(1) raw material extracts: by Cynanchum otophyllum Schneid pulverizing medicinal materials at coarse powder, be added alcoholic solution, then plus organic acid adjust PH to 4-5,
It is 60 DEG C of -80 DEG C of refluxing extractions at least 2 times, 2-3 hours each, merge alcoholic solution and is concentrated into no alcohol taste；
(2) macroreticular resin chromatographs: the extracting solution after taking step (1) to be concentrated staticly settles, through macroporous resin enrichment, successively
It is eluted with 0%-90% alcoholic solution, collects target product section, be concentrated spare；
(3) silicagel column silica gel column chromatography: is added after addition silica gel uniformly mixes sample, drying in concentrate obtained by step (2)
It in chromatography, with methanol-ethyl acetate system gradient elution, is monitored by thin-layer chromatography, collects object fraction, merged dense
It contracts spare；
(4) preparative HPLC purifies: the concentrate medicinal extract that step (3) is obtained is dissolved, miillpore filter mistake with methanol solution
Sample introduction after filter, on-line ultraviolet monitoring, wavelength select 210nm, and chromatographic column filler selects ODS-2C18 filler, and mobile phase selects volume ratio
The acetonitrile-aqueous solution of 25:75 recycles acetonitrile, common vetch NM-200 polymer filler of receiving dehydration, concentration, dry white powder.Micropore
In membrane filtration, optional 0.23 μm, 0.45 μm or 0.8 μm of miillpore filter.
The white powder that the present invention is prepared is Qingyangshenglycoside A, and structural formula is as follows:
Preferably, in step (1), the additional amount of alcoholic solution is 6-10L/ kilograms of medicinal material.
Preferably, in step (1), the alcoholic solution is the methanol or ethanol solution of 40%-70%.
Preferably, in step (2), the alcoholic solution is methanol or ethanol solution.
Preferably, it in step (2), is successively eluted with 30%, 50%, 90% alcoholic solution.
Preferably, in step (3), in the silica gel column chromatography, mixing sample silica gel and dress column silica gel ratio is 1:3~1:
5。
Preferably, in step (3), the thin-layer developing agent of thin-layer chromatography is n-butanol/ice second that volume ratio is 5:1:4
Acid/water upper solution, spray is heated to clear spot with ethanol solution of sulfuric acid after expansion.
Compared with prior art, the invention has the following advantages:
1, the present invention mentions medicinal material using acidic aqueous solution using acyl group stable feature under mildly acidic conditions
It takes, reduces the percent hydrolysis of para hydroxybenzene formyl group, improve the recovery rate of Qingyangshenglycoside A, be conducive to further pure
Change；
2, present invention employs macroreticular resin, silica gel to be enriched with, and eliminates impurity, Qingyangshenglycoside A has been further purified, also
The filler of preparative efficient liquid phase, related accessory are protected；
3, present invention employs UV detector on-line monitorings to isolate and purify Qingyangshenglycoside A, realizes separation detection
It is synchronous to carry out, the waste of resource is avoided, wrong receipts is also avoided, leaks the mistake for receiving sample, increase yield.
Detailed description of the invention
1 embodiment of the present invention of attached drawing, 1 gained object Qingyangshenglycoside A HPLC liquid phase detects spectrogram；
2 embodiment of the present invention of attached drawing, 1 gained object Qingyangshenglycoside A ESI-MS m/z:931.4747, mass spectrogram；
3 embodiment of the present invention of attached drawing 1 gained object Qingyangshenglycoside A 1H-NMR (400MHz, CD3OD) nuclear magnetic spectrogram；
4 embodiment of the present invention of attached drawing 1 gained object Qingyangshenglycoside A 13C-NMR (400MHz, CD3OD) nuclear magnetic spectrogram.
Specific embodiment
Below with reference to each embodiment, the invention will be further described, and mode of the invention includes but are not limited to following reality
Apply example.
Embodiment
The present embodiment provides a kind of methods for extracting Qingyangshenglycoside A using Cynanchum otophyllum Schneid, include the following steps:
1) it extracts: Cynanchum otophyllum Schneid 100kg is ground into coarse powder, 70% methanol solution of 10 times of amounts is added, glacial acetic acid tune is added
PH to 5 is saved, refluxing extraction 3 times, 2 hours every time, combined extract was concentrated into no alcohol taste, obtained extracting solution 250L；
2) macroporous resin enrichment: the extracting solution in step 1) after concentration is taken, is staticly settled；Through macroporous resin enrichment, successively
It is eluted with 30%, 50%, 90% ethanol solution, collects target product section.It is concentrated into a small amount of 15L；
3) silica gel column chromatography: gained concentrate 60-80 mesh silica gel 7.5kg in step 2) is mixed into sample, drying, fills column silica gel
100-200 mesh 22.5kg successively uses methanol-ethyl acetate gradient elution, collects the eluent containing object, and concentration and recovery is molten
Agent；It is monitored by thin-layer chromatography, the thin-layer developing agent of thin-layer chromatography is n-butanol/glacial acetic acid/water that volume ratio is 5:1:4
Upper solution, after expansion spray with 5% ethanol solution of sulfuric acid, be heated to clear spot；
4) preparative HPLC purifies: medicinal extract 220g in step 3) is dissolved, 0.45 μm of miillpore filter mistake with 50% methanol
Filter, it is spare.
Chromatographic column filler: ODS-2C18 filler
Mobile phase: acetonitrile-aqueous solution V/V=25:75
Take above-mentioned filtering with microporous membrane filtrate sample introduction, each sample introduction 20g, on-line ultraviolet monitoring, wavelength: 210nm, 500ml
The mobile phase of every bottle of collection target compound.Acetonitrile is recycled, common vetch NM-200 polymer filler of receiving dehydration is concentrated, whitely dry
Powder 12g.
HPLC detects 98% or more purity (attached drawing 1), MS map such as Fig. 2, and the molecular weight of product is 932, molecular weight symbol
It closes；Its hydrogen spectrum such as Fig. 3, carbon spectrum such as Fig. 4, it was demonstrated that the present embodiment has obtained the higher Qingyangshenglycoside A of purity really.
Above-described embodiment is only one of the preferred embodiments of the invention, should not be taken to limit protection model of the invention
It encloses, as long as in subject of the present invention design philosophy and mentally making skimble-skamble change or polishing, the technology solved
Problem is still consistent with the present invention, should all be included within protection scope of the present invention.
Claims (7)
1. the method for extracting Qingyangshenglycoside A using Cynanchum otophyllum Schneid, which comprises the steps of:
(1) raw material extracts: by Cynanchum otophyllum Schneid pulverizing medicinal materials at coarse powder, be added alcoholic solution, then plus organic acid adjust PH to 4-5,60 DEG C-
It is 80 DEG C of refluxing extractions at least 2 times, 2-3 hours each, merge alcoholic solution and is concentrated into no alcohol taste；
(2) macroreticular resin chromatographs: the extracting solution after taking step (1) to be concentrated is staticly settled, through macroporous resin enrichment, is successively used
The elution of 0%-90% alcoholic solution, collects target product section, is concentrated spare；
(3) silica gel column chromatography silica gel column chromatography: is added after addition silica gel uniformly mixes sample, drying in concentrate obtained by step (2)
In, it with methanol-ethyl acetate system gradient elution, is monitored by thin-layer chromatography, collects object fraction, it is standby to merge concentration
With；
(4) preparative HPLC purifies: the concentrate medicinal extract that step (3) is obtained is dissolved with methanol solution, after filtering with microporous membrane
Sample introduction, on-line ultraviolet monitoring, wavelength select 210nm, and chromatographic column filler selects ODS-2C18 filler, and mobile phase selects volume ratio 25:
75 acetonitrile-aqueous solution recycles acetonitrile, common vetch NM-200 polymer filler of receiving dehydration, concentration, dry white powder.
2. the method as described in claim 1, which is characterized in that in step (1), the additional amount of alcoholic solution is 6-10L/ kilograms of medicine
Material.
3. the method as described in claim 1, which is characterized in that in step (1), the alcoholic solution is the methanol of 40%-70%
Or ethanol solution.
4. the method as described in claim 1, which is characterized in that in step (2), the alcoholic solution is methanol or ethanol solution.
5. method as described in claim 1 or 4, which is characterized in that in step (2), successively with 30%, 50%, 90% alcohol
Solution is eluted.
6. the method as described in claim 1, which is characterized in that in step (3), in the silica gel column chromatography, mix sample silica gel with
Dress column silica gel ratio is 1:3~1:5.
7. method as described in claim 1 or 6, which is characterized in that in step (3), the thin-layer developing agent of thin-layer chromatography is body
Product is than the n-butanol/glacial acetic acid/water upper solution for being 5:1:4, and it is clear to be heated to spot with ethanol solution of sulfuric acid for spray after expansion
It is clear.
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| CN201910773666.3A CN110452281A (en) | 2019-08-21 | 2019-08-21 | The method that Qingyangshenglycoside A is extracted using Cynanchum otophyllum Schneid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114195750A (en) * | 2021-12-23 | 2022-03-18 | 新疆维吾尔自治区人民医院 | Camel thorn treatment process |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148958A (en) * | 1996-08-29 | 1997-05-07 | 中国科学院昆明植物研究所 | Pharmaceutical application of glucoside A and B of auricledleaf swallowort |
| CN1634097A (en) * | 2004-11-09 | 2005-07-06 | 云南云药实验室有限公司 | Application of C21 steroid glycoside in pharmacy |
-
2019
- 2019-08-21 CN CN201910773666.3A patent/CN110452281A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148958A (en) * | 1996-08-29 | 1997-05-07 | 中国科学院昆明植物研究所 | Pharmaceutical application of glucoside A and B of auricledleaf swallowort |
| CN1634097A (en) * | 2004-11-09 | 2005-07-06 | 云南云药实验室有限公司 | Application of C21 steroid glycoside in pharmacy |
Non-Patent Citations (2)
| Title |
|---|
| XIN LI 等: "Pregnane glycosides from the antidepressant active fraction of cultivated Cynanchum otophyllum", 《FITOTERAPIA》 * |
| 殷敏敏 等: "青阳参乙酸乙酯萃取部位的化学成分", 《中国药科大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114195750A (en) * | 2021-12-23 | 2022-03-18 | 新疆维吾尔自治区人民医院 | Camel thorn treatment process |
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