CN105037124B - A kind of preparation method of selaginellin N - Google Patents
A kind of preparation method of selaginellin N Download PDFInfo
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- CN105037124B CN105037124B CN201510104832.2A CN201510104832A CN105037124B CN 105037124 B CN105037124 B CN 105037124B CN 201510104832 A CN201510104832 A CN 201510104832A CN 105037124 B CN105037124 B CN 105037124B
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Abstract
The invention belongs to pharmaceutical technology field, it is provided that a kind of therefrom powder stick cypress is prepared the preparation method of content selaginellin N more than 95%.This compound has the structure of following formula (I).Heretofore described selaginellin N is that to use the dry herb raw material of Spikemoss plant Herba Selaginellae Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata Selaginella pulvinata (Hook.et Grev.) Maxim. be initiation material, and the separation method carrying out extracting and use macroporous resin adsorption and Sephadex LH 20 absorption to combine with organic solvent and/or water carries out isolated.It is simple, reproducible that the present invention has technological operation, and productivity is high, the advantage being suitable for industrialized production, and the purity of this product is higher, and its content uses HPLC method to detect more than 95%.
Description
Technical field:
The present invention relates to the preparation method of selaginellin N in Herba Selaginellae, its content uses HPLC method to detect should be 95%
Above.Belong to Chinese medicine extraction separation, purification art.
Background technology:
In China's coastal port, regulation Herba Selaginellae is Spikemoss plant Herba Selaginellae Selaginella tamariscina
(Beauv.) Spring or the dry herb of Selaginella pulvinata Selaginella pulvinata (Hook.et Grev.) Maxim..
The whole year all can gather, and removes fibrous root and silt, dries.Pungent, flat.Return liver, heart channel.There is promoting blood circulation to restore menstrual flow.For amenorrhea dysmenorrhea,
Abdominal mass mass in the abdomen, injury from falling down.Herba Selaginellae charcoal removing stasis to stop bleeding.For spitting blood, metrorrhagia, have blood in stool, proctoptosis.Herba Selaginellae becomes containing multiple chemistry
Point, such as flavonoid, it has now been found that containing apigenin (apigenin), amentoflavone (amentoflavone), flat in Herba Selaginellae
Cypress bisflavone (hinokiflavone), isocryptomerin (isocryptomerin) and Oman's torr yellow (amentoflavone)
Deng；The compounds such as Phenylpropanoid Glycosides class, alkaloids, phenols, organic acid.Pharmacological action shows have: immunity and antitumor action,
To the effect of blood system, antiviral, antibacterial act on antiinflammatory action, antalgic and sedative etc..It is a kind of convenient, simple to the invention provides
Just the method, quickly preparing high-purity selaginellin N.Currently available technology has no this selaginellin N's
Extracting method.
Summary of the invention:
The technical problem to be solved is to provide in one therefrom powder stick cypress a kind of content of extraction more than 95%
The preparation method of selaginellin N, it is therefore an objective to obtain content higher (content is more than 95%) by simple technical process
Selaginellin N.
The technical scheme is that and therefrom powder stick cypress extracts a kind of content selaginellin N more than 95%
Preparation method, its concrete grammar step is:
A) by Spikemoss plant Herba Selaginellae Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata
The dry herb raw material of Selaginella pulvinata (Hook.et Grev.) Maxim. is cut into a cun section；
B) with the one in ethanol, methanol, water or the backflow of methanol-water, alcohol-water (1:9～9:1) mixed solvent in proportion
Extract, add and be equivalent to 2-16 times of solvent of medical material weight, reflux, extract, 1-5 time, each 0.5 to 6 hour, united extraction liquid,
Filter, obtain extracting solution；
C) by extracting solution recycling design and be concentrated into the clear paste or thick paste that relative density is 1.05-1.40 (60 DEG C), by than
Example (1:10～10:1) adds water, suspendible, obtains suspension.
D) suspension is adsorbed in D101 or various polarity, low pole macroporous adsorptive resins, then by alcohol-water or first
The mixed solvent eluting of alcohol-water (10:1～1:1 in proportion), respectively with every part 1/100～the volume of 1/20 total amount eluant
Collect eluting liquid stream part, with chloroform: methanol (10:1～1:1) is that developing solvent uses TLC to check speckle, merges identical palm fibre
Brown spot washing out method stream part.
E) the identical sepia spot washing out method stream part recycling design that will merge, obtains sepia pressed powder.
F) by the sepia pressed powder petroleum ether of 2-100 times amount, ether, ethyl acetate, acetone, chloroform, second
The mixed organic solvents that one or both (1:10～10:1) in alcohol, methanol mix dissolves.
G) organic solvent or the mixed organic solvents of sepia pressed powder are added with Sephadex LH-20 for absorption
Adsorb on the chromatographic column of agent.
H) by absorption the Sephadex LH-20 chromatographic column petroleum ether of sepia pressed powder, ether, ethyl acetate, third
The mixed solvent that one or both (1:10～10:1) in ketone, chloroform mix respectively with ethanol or methanol (10:1～1:
1) mixed solvent eluting, collects Sephadex LH-20 layer with the volume of every part 1/100～1/20 total amount eluant respectively
The yellow colour band of analysis post, until completely.With chloroform: methanol (10:1～1:1) is that developing solvent uses TLC follow-up investigations speckle
Point, merges identical sepia spot washing out method stream part.
I) the identical sepia spot washing out method stream part recycling design that will merge, obtains sepia pressed powder, i.e.
Selaginellin N highly finished product, its content uses HPLC method to detect should be more than 95%, and the rate of transform is up to more than 75%.
Described methanol-water, the concentration of alcohol-water are 30-75%；The preferred D101 of described macroporous resin column.
Chloroform in step (d): the preferred 3:1 of methanol.
In step (h), chloroform: methanol=3:1-4:1.
In step (f), described organic solvent is the 10-30 times amount of pressed powder.
Selaginellin N structure elucidation
Red powder, is soluble in methanol and DMSO, water insoluble.The aobvious pink of 10% sulphuric acid, iodine is smoked there is pale brown mottle
Point, pointing out this compound is selaginellin compounds.
UVλmax(MeOH) nm:299,274 and 264nm.
ESI-TOF-MS collection of illustrative plates is given m/z:495.1607 [M-H]-, value of calculation 495.1602.In conjunction with1H-NMR and13C-
NMR speculates that molecular formula is C34H24O4。
1H-NMR (600MHz, DMSO-d6) 24 proton signals of display, compare with compound 1, lacked the hydrogen of methylol
Signal；13C-NMR (150MHz, DMSO-d6) compare with compound 1, methylol carbon signal disappears, it could therefore be concluded that this chemical combination
Thing is to lack a methylol on the parent nucleus of compound 1.
1H-NMR (600MHz, DMSO-d6δ 6.79 (2H, d, J=8.5Hz) in), δ 6.56 (2H, d, J=8.5Hz)；δ
6.96 (2H, d, J=8.7Hz), δ 6.64 (2H, d, J=8.7Hz)；δ 6.82 (4H, d, J=8.4Hz) shows three phenyl ring pair
The AA'BB' Coupling System formed when position replaces.Speculate that compound 2 exists two identical structure fragments according to HSQC Yu HMBC
A。
1H-NMR,13C-NMR by signal δ 3.71 (3H, s), δ 55.4 is speculated as a methoxyl group signal, in conjunction with HSQC with
HMBC speculates that compound 2 exists a structure fragment B.
By signal δ 7.62 (1H, d, J=7.7Hz), δ 7.55 (1H, t, J=7.8Hz), δ 7.33 (1H, d, J=7.7Hz)
Judge that compound 2 exists the neighbour three replacement structure fragment C on a phenyl ring.
13C-NMR (150MHz, DMSO-d6) display, δ 185.8 is the carbon signal of C=O,1H-NMR (600MHz, DMSO-d6)
Display, δ 7.42 (1H, dd, J=10.0,2.6Hz), δ 7.23 (1H, dd, J=10.0,2.6Hz), δ 6.36 (1H, dd, J=
10.0,2.1Hz), δ 6.32 (1H, dd, J=10.0,2.1Hz) combines HSQC and HMBC, and document[19]Comparison, thus it is speculated that compound
2 structures there may be structure fragment D.
It addition,13C-NMR (150MHz, DMSO-d6) signal shows: the carbon signal of δ 86.6, δ 93.7 should be the letter of C ≡ C
Number；HMBC shows, δ 6.96 (H-28) and δ 93.7 (C-27) has relevant peaks, releases C-27 with C-33 and is connected；δ142.4(C-18)
Be correlated with δ 6.79 (H-20, H-24), then C-18 with C-25 is connected；δ 93.7 (C-27) and δ 6.96 (H-28,32) has relevant peaks,
Therefore speculate that compound 2 exists structure fragment E.
Comprehensively1H-NMR,13C-NMR, HSQC and HMBC data analysis, connects above fragment combination, determines that compound is
(R,S)-4-[[4'-hydroxy-3-[2-(4-hydroxyphenyl)ethynyl][1,1'-biphenyl]-2-
Yl] (4-methoxyphenyl) methyl ene]-2,5-cyclohexadien-1-one, this compound is for having no document report
The noval chemical compound in road, named selaginellin N.The important the most relevant of this compound is seen1H-NMR,13C-NMR, HSQC
(Table) is seen with HMBC attribution data.
Table NMR spectral data and HMBC Correlation of selaginellin N(600
MHz, DMSO-d6)
The economic benefit of the present invention is:
(1) technological operation is simple, reproducible, and productivity is high, is suitable for industrialized production.
(2) purity of this product is higher, and its content uses HPLC method to detect more than 95%.
The application of selaginellin N:
(1) the selaginellin N of the present invention is suitable for preparing the a kind new medicine preparation used clinically.
(2) the selaginellin N of the present invention can also be prepared clinically with other compound or Drug combination
The new drug preparation used.
(3) the selaginellin N of the present invention can make the various pharmaceutical dosage form used clinically.
(3) the selaginellin N of the present invention can also use as reference substance.
(4) the selaginellin N of the present invention can also make the various health food used clinically and functional
Cosmetics.
Detailed description of the invention:
Carry out the more detailed description present invention below in conjunction with embodiment, but the present invention is not limited to these embodiments restrictions.
Embodiment 1:
By dry Herba Selaginellae herb 100g, it is cut into a cun section, adds 30% alcohol reflux three times, add 11 times amount, often every time
Secondary 2 hours, united extraction liquid, filter, obtain extracting solution.Extracting solution is reclaimed ethanol and to be concentrated into relative density be 1.20 (60 DEG C)
Clear paste, add 5 times amount water suspendibles, obtain suspension.Just suspension adds the layer with D101 type macroporous adsorbent resin as adsorbent
Adsorb on analysis post.Then adsorption column ethanol and the mixed solvent gradient elution of water (10:1～1:1), wash with every part of 1/40 total amount
The volume of de-agent collects eluting liquid stream part, uses TLC to check speckle (with chloroform: methanol 3:1 is as developing solvent), merges phase
Same sepia spot washing out method stream part.The identical sepia spot washing out method stream part recycling design that will merge, obtains sepia solid powder
End.After the sepia pressed powder acetone solution by 30 times amount, add the chromatographic column with Sephadex LH-20 as adsorbent
Upper absorption.By the mixed solvent eluting of Sephadex LH-20 chromatographic column chloroform Yu methanol (3:1), respectively with every part 1/
The volume of 30 total amount eluant collects the sepia colour band of Sephadex LH-20 chromatographic column, until completely.TLC is used to follow the tracks of
Check speckle (with chloroform: methanol 4:1 is as developing solvent), merge identical sepia spot washing out method stream part.The phase that will merge
With sepia spot washing out method stream part recycling design, obtain sepia pressed powder 0.67g, i.e. selaginellinN highly finished product, yield
Being 0.67%, its content uses HPLC method to be detected as on 98.9%.
Embodiment 2:
By dry Herba Selaginellae herb 2kg, it is cut into a cun section, adds 75% alcohol reflux three times, add 10 times amount, every time every time
1.5 hours, united extraction liquid, filter, obtain extracting solution.Extracting solution is reclaimed ethanol and to be concentrated into relative density be 1.10 (60 DEG C)
Clear paste, add 7 times amount water suspendibles, obtain suspension.Suspension is added the chromatography with D101 type macroporous adsorbent resin as adsorbent
Adsorb on post.Then adsorption column ethanol and the mixed solvent gradient elution of water (10:1～1:1), with every part of 1/30 total amount eluting
The volume of agent collects eluting liquid stream part, uses TLC to check speckle (with chloroform: methanol 2:1 is as developing solvent), merges identical
Sepia spot washing out method stream part.The identical sepia spot washing out method stream part recycling design that will merge, obtains sepia pressed powder.
After the sepia pressed powder acetone solution by 15 times amount, add and inhale on the chromatographic column with Sephadex LH-20 as adsorbent
Attached.By the mixed solvent eluting of Sephadex LH-20 chromatographic column chloroform Yu methanol (3:1), 1/20 total with every part respectively
The volume of amount eluant collects the sepia colour band of Sephadex LH-20 chromatographic column, until completely.Use TLC follow-up investigations
Speckle (with chloroform: methanol 3:1 is as developing solvent), merges identical sepia spot washing out method stream part.The identical palm fibre that will merge
Brown spot washing out method stream part recycling design, obtains sepia pressed powder 11.4g, i.e. selaginellin N highly finished product, and yield is
0.57%, its content uses HPLC method to be detected as 97.5%.
Embodiment 3:
By dry Herba Selaginellae herb 30kg, it is cut into a cun section, adds 50% alcohol reflux three times, add 9 times amount, every time every time
1.5 hours, united extraction liquid, filter, obtain extracting solution.Extracting solution is reclaimed ethanol and to be concentrated into relative density be 1.25 (60 DEG C)
Clear paste, add 10 times amount water suspendibles, obtain suspension.Suspension is added the chromatography with D101 type macroporous adsorbent resin as adsorbent
Adsorb on post.Then adsorption column ethanol and the mixed solvent gradient elution of water (10:1～1:1), with every part of 1/20 total amount eluting
The volume of agent collects eluting liquid stream part, uses TLC to check speckle (with chloroform: methanol 3:1 is as developing solvent), merges identical
Sepia spot washing out method stream part.The identical sepia spot washing out method stream part recycling design that will merge, obtains sepia pressed powder.
After the sepia pressed powder acetone solution by 10 times amount, add and inhale on the chromatographic column with Sephadex LH-20 as adsorbent
Attached.By the mixed solvent eluting of Sephadex LH-20 chromatographic column chloroform Yu methanol (3:1), 1/30 total with every part respectively
The volume of amount eluant collects the sepia colour band of Sephadex LH-20 chromatographic column, until completely.Use TLC follow-up investigations
Speckle (with chloroform: methanol 3:1 is as developing solvent), merges identical sepia spot washing out method stream part.The identical palm fibre that will merge
Brown spot washing out method stream part recycling design, obtains sepia pressed powder 150g, i.e. selaginellin N highly finished product, and yield is
0.5%, its content uses HPLC method to be detected as 95.2%.
Claims (7)
1. the preparation method of the selaginellin N shown in a formula (I), it is characterised in that:
A) the dry herb raw material of Chinese medicine Herba Selaginellae is cut into a cun section；
B) by the one in ethanol, methanol, water or methanol-water, alcohol-water 1:9～9:1 mixed solvent reflux, extract, in proportion；
United extraction liquid, filters, obtains extracting solution；
C) by extracting solution recycling design and concentrate, 1:10～10:1 adds water, suspendible in proportion, obtains suspension;
D) suspension is adsorbed in D101 macroporous adsorptive resins, then with alcohol-water or methanol-water 10:1～1:1 in proportion
Mixed solvent eluting, collect eluting liquid stream part, with three chloromethanes with the volume of every part 1/100～1/20 total amount eluant respectively
Alkane: methanol 10:1～1:1 is that developing solvent uses TLC to check speckle, merges identical eluting stream part;
E) the identical eluting stream part recycling design that will merge, obtains pressed powder;
F) by the pressed powder petroleum ether of 2-100 times amount, ether, ethyl acetate, acetone, chloroform, ethanol, methanol
One or both mixing mixed organic solvents dissolve, two kinds mixing ratios be 1:10～10:1;
G) organic solvent or the mixed organic solvents of pressed powder are added the chromatographic column with Sephadex LH-20 as adsorbent
Upper absorption;
H) by absorption the Sephadex LH-20 chromatographic column petroleum ether of sepia pressed powder, ether, ethyl acetate, acetone,
Mixed solvents of one or both mixing in chloroform respectively with the mixed solvent eluting of ethanol or methanol 10:1～1:1,
The ratio of two kinds of mixing is 1:10～10:1, collects with the volume of every part 1/100～1/20 total amount eluant respectively
The yellow colour band of Sephadex LH-20 chromatographic column, until completely, with chloroform: methanol 10:1～1:1 is that developing solvent uses
TLC follow-up investigations speckle, merges identical eluting stream part;
I) the identical eluting stream part recycling design that will merge, obtains pressed powder, i.e. selaginellin N highly finished product;
。
Preparation method the most according to claim 1, it is characterised in that add the 2-being equivalent to medical material weight in step (b)
16 times of solvents, reflux, extract, 1-5 time, each 0.5 to 6 hour.
The preparation method of selaginellinN the most according to claim 1, it is characterised in that: described Chinese medicine Herba Selaginellae is:
Spikemoss plant Herba Selaginellae Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata Selaginella
The dry herb raw material of pulvinata (Hook.et Grev.) Maxim..
Preparation method the most according to claim 1, it is characterised in that methanol-water or the concentration of alcohol-water in step b)
For 30-75%.
Preparation method the most according to claim 1, it is characterised in that the macroporous resin in step d) is D101.
Preparation method the most according to claim 1, it is characterised in that in step h), chloroform: methanol=3:1-4:1.
Preparation method the most according to claim 1, it is characterised in that in step f), described organic solvent is solid powder
The 10-30 times amount at end.
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