CN101817852B - Containing primeverin and methyl phenyl ketone glycoside extracts and application thereof - Google Patents
Containing primeverin and methyl phenyl ketone glycoside extracts and application thereof Download PDFInfo
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Abstract
The invention provides a kind of from anaesthetic bulk drug containing primeverin and methyl phenyl ketone glycoside extracts.Invention particularly provides from primeverin in root of Maximowicz Primrose bulk drug and methyl phenyl ketone glycoside extracts, and the extracting method of this extract.Present invention also offers the pharmaceutical composition utilizing this extract.Extracting method of the present invention is simple and practical, and refining effect is good.
Description
Technical field
The present invention relates to a kind of primeverin from anaesthetic bulk drug and methyl phenyl ketone glycoside extracts, particularly relate to the methyl phenyl ketone glycoside extracts containing primeverin from root of Maximowicz Primrose bulk drug.
Background technology
Anaesthetic root of Maximowicz Primrose (section of having another name called is heralded spring, kermes is heralded spring) is the herb (anaesthetic name: Sa is Christiane Knacke good fortune all) of Primulaceae primula root of Maximowicz Primrose Primula maximowiczii Regel, be born in the place that subalpine meadow is upper or mountain region sylvan life, border and moist soil ulmin are abundant, the ground such as northeast, the Inner Mongol, Hebei, Shanxi, Shaanxi, Gansu, Qinghai are distributed in China, there is pain relieving, wind action of dispelling, cure mainly the disease such as epilepsy, headache.
Summary of the invention
The present inventor is in studying the primeverin in root of Maximowicz Primrose and methyl phenyl ketone glycoside extracts, and what discovery obtained contains primeverin and serial methyl phenyl ketone glycoside extracts, thus completes content of the present invention.
That extracts in anaesthetic primeverin provided by the invention and methyl phenyl ketone glycoside extracts, especially anaesthetic root of Maximowicz Primrose contains primeverin and methyl phenyl ketone glycoside extracts.
The invention provides a kind of primeverin from anaesthetic bulk drug and methyl phenyl ketone glycoside extracts, wherein at least containing primeverin composition, in this extract, the content of primeverin is at least not less than 5% by weight percentage, and preferably 30%, but be no more than 95%.
Present invention also offers the extracting method of the said extracted thing from root of Maximowicz Primrose bulk drug, to obtain primeverin and the higher extract of methyl phenyl ketone glycosides content.
According to anaesthetic primeverin provided by the invention and methyl phenyl ketone glycoside extracts, wherein at least containing primeverin, the primeverin contained in every gram of extract and methyl phenyl ketone methods of glycosides are no less than 500 milligrams in primeverin.Especially, 50 milligrams are no less than containing primeverin in every gram of extract.
Extract of the present invention can from any bulk drug containing described primeverin and methyl phenyl ketone glycosides composition, preferably from primeverin in root of Maximowicz Primrose bulk drug and methyl phenyl ketone methods of glycosides.
By groping the test of composition, invention particularly provides the root of Maximowicz Primrose extract that application macroporous adsorbent resin and the extracting method refined of polycaprolactam obtain, there is higher primeverin and methyl phenyl ketone glycoside active constituent content, and maintain the composition of natural matter.
It is the different sugar of parent nucleus is substituent compound that the present invention's alleged " methyl phenyl ketone glycoside " refers to methyl phenyl ketone.
According to noted earlier, extract of the present invention is from anaesthetic root of Maximowicz Primrose, except containing except primeverin, can also comprise any methyl phenyl ketone glycoside position, such as its composition also comprises one or several that be selected from 2-primrose glycosyl-4-methoxy-acetophenone, 4-O-β-D-Glucose base-methyl phenyl ketone, 4-hydroxy-acetophenone, 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters, 4-primrose glycosyl-methyl phenyl ketone, 2-primrose glycosyl-5-methoxy-acetophenone etc.According to preferred version of the present invention, this extract is from primeverin in root of Maximowicz Primrose bulk drug and methyl phenyl ketone methods of glycosides.
Extract of the present invention can be obtained by any feasible method, can be preferably to adopt macroporous adsorbent resin and polycaprolactam to refine to obtain, and makes product have higher purity and yield.
The present invention can adopt raw material crude drug, also can adopt the medicine materical crude slice after processing.According to the preferred method of the present invention, adopt root of Maximowicz Primrose medicine materical crude slice or unprocessed root of Maximowicz Primrose crude drug to be raw material, if unprocessed root of Maximowicz Primrose crude drug, can carry out cleaning, the processing such as airing and section as process in early stage, extracting method is as follows:
Root of Maximowicz Primrose pulverizing medicinal materials, organic solvent (such as ethanol) extracts, recycling design, concentrating and precipitating, filter, macroporous adsorbent resin on filtrate, with organic solvent (such as alcohol, preferred alcohol) gradient elution, collect the composition that 10%-90% concentration elutes, the composition that preferred 20%-50% concentration elutes, more preferably the composition that elutes of 20%-40% concentration, upper polyamide column, with 20%-35% ethanol elution, obtaining compound primeverin and methyl phenyl ketone methods of glycosides is main component.
Structural Identification
Compound 1:2-primrose glycosyl-4-methoxy-acetophenone
Brownish-yellow powder,
224,262.
1aBX Coupling System is there is: δ 7.65 (1H, d, J=8.5Hz in this compound structure of H-NMR data presentation, H-6), 6.78 (1H, d, J=2.0Hz, H-3), 6.66 (1H, dd, J=8.5,2.0Hz, H-5) and 1 methoxyl group δ 3.83 (3H, s, H-9), 1 methyl δ 2.57 (3H, s, H-8) and 2 sugared terminal hydrogen δ 5.03 (1H, d, J=7.5Hz, H-1 '), 4.15 (1H, d, J=7.5Hz, H-1 "), show that 2 monose are beta comfiguration.
13c-NMR data presentation has the signal of 20 carbon, comprises 1 ketone group carbon [δ 197.21 (C-7)], 1 methoxyl group carbon [δ 56.09 (C-9)], 1 methyl carbon [δ 32.52 (C-8)]; And sugar chain structure is primeverose: D-wood sugar (1 → 6) D-Glucose [D-glucose: δ 100.94 (C-1 '), 73.70 (C-2 '), 76.89 (C-3 '), 70.09 (C-4 '), 76.14 (C-5 '), 69.28 (C-6 '); D-xylose: δ 104.63 (C-1 "), 73.54 (C-2 "), 76.89 (C-3 "), 69.85 (C-4 "), 66.08 (C-5 ")].
By this compound and document
[1]report
1h-NMR and
13c-NMR data compare, and deterministic compound 1 is 2-primrose glycosyl-4-methoxy-acetophenone.
Compound 2:4-hydroxy-acetophenone
White powder,
211,260.
1aB Coupling System is there is: δ 7.86 (2H, d, J=8.1Hz, H-2,6), 6.87 (2H, d, J=8.1Hz, H-3,5) and 1 methyl δ 2.53 (3H, s, H-8) in this compound structure of H-NMR data presentation.
13c-NMR data presentation has the signal of 8 carbon, comprises 1 ketone group carbon [δ 195.15 (C-7)], 1 methyl carbon [δ 25.29 (C-8)].
Comprehensive above data, compare (table 1) and searching document with the spectral data of compound 3
[2], deterministic compound 2 is 4-hydroxy-acetophenone.
(solvent is DMSO-d to the nuclear magnetic data of table 1. compound 2,3 and 5
6)
Compound 3:4-O-β-D-Glucose base-methyl phenyl ketone
Pale yellow powder,
215,267.
1aB Coupling System is there is: δ 7.92 (2H, d, J=8.5Hz, H-2,6), 7.11 (2H, d, J=9.0Hz, H-3,5) and 1 methyl δ 2.53 (3H, s, H-8) in this compound structure of H-NMR data presentation; Simultaneously 1 sugared terminal hydrogen [δ 5.01 (1H, d, J=7.5Hz, H-1 ')] shows that this monose is beta comfiguration.
13c-NMR data presentation has the signal of 14 carbon, comprise 1 ketone group carbon [δ 196.95 (C-7)], 1 D-Glucose [δ 100.23 (C-1 '), 73.49 (C-2 '), 77.54 (C-3 '), 69.96 (C-4 '), 76.81 (C-5 '), 60.96 (C-6 ')] and 1 methyl carbon [δ 26.93 (C-8)].
By this compound and document
[3]report
1h-NMR and
13c-NMR data compare, and deterministic compound 3 is 4-O-β-D-Glucose base-methyl phenyl ketone, is commonly called as picein (picein).Compound 4:2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters
Pale yellow powder,
220,248.
1aBX Coupling System is there is: δ 7.68 (1H, d, J=8.5Hz, H-6) in this compound structure of H-NMR data presentation, 6.83 (1H, d, J=2.3Hz, H-3), 6.67 (1H, dd, J=8.5,2.3Hz, H-5) and 2 methoxyl group δ 3.81 (3H, s, H-9), 3.77 (3H, s, H-8) and 1 sugared terminal hydrogen [δ 4.91 (1H, d, J=7.3Hz, H-1 ')], show that this sugar is beta comfiguration.
13c-NMR data presentation has the signal of 15 carbon, comprises 1 carbonyl carbon [δ 166.35 (C-7)], 2 methoxyl group carbon [δ 55.82 (C-9), 52.43 (C-8)]; 1 molecule glucose [D-glucose: δ 101.74 (C-1 ') may be there is simultaneously, 73.71 (C-2 '), 76.75 (C-3 '), 70.23 (C-4 '), 76.26 (C-5 '), 61.08 (C-6 ')].
Comprehensive above data, infer that this compound structure is 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters.By retrieval CA, only there is document
[4]mention this structure, but do not report its NMR data.Therefore, by comparing (table 2) with the NMR data of primeverin, to the 1H of compound 4 and
13c signal belongs to.
(solvent is DMSO-d to the nuclear magnetic data of table 2. compound 4 and 6
6)
Note:
a)above signal is obtained by two-dimentional nuclear-magnetism supplementary means.
Compound 5:4-primrose glycosyl-methyl phenyl ketone
Pale yellow powder,
209,264.
1aB Coupling System is there is: δ 7.93 (2H, d, J=9.0Hz, H-2,6), 7.15 (2H, d, J=8.5Hz, H-3,5) and 1 methyl δ 2.53 (3H, s, H-8) in this compound structure of H-NMR data presentation; Simultaneously 2 sugared terminal hydrogens [δ 4.95 (1H, d, J=7.0Hz, H-1 '), 4.17 (1H, d, J=7.5Hz, H-1 ")] show that 2 monose are beta comfiguration.
13c-NMR data presentation has the signal of 19 carbon, comprises 1 ketone group carbon [δ 196.85 (C-7)], 1 methyl carbon [δ 26.04 (C-8)]; Sugar chain structure is primeverose: D-wood sugar (1 → 6) D-Glucose [D-glucose: δ 99.45 (C-1 '), 72.58 (C-2 '), 75.90 (C-3 '), 69.04 (C-4 '), 75.60 (C-5 '), 67.67 (C-6 '); D-xylose: δ 103.34 (C-1 "), 72.84 (C-2 "), 75.94 (C-3 "), 69.04 (C-4 "), 65.10 (C-5 ")].
Comprehensive above data, and compare (table 1) and searching document with the spectral data of compound 3
[5], determine that this compound structure is 4-primrose glycosyl-methyl phenyl ketone.
Compound 6:2-primrose glycosyl-4-methoxy-benzoic acid methyl esters
White powder,
222,253.ESI-MS spectrum provides quasi-molecular ion peak m/z499.2 [M+Na]
+, 515.1 [M+K]
+peak, in conjunction with
1h-NMR and
13c-NMR modal data, infer that its molecular weight is 476, molecular formula is C
20h
28o
12.
1aBX Coupling System is there is: δ 7.68 (1H, d, J=8.7Hz in this compound structure of H-NMR data presentation, H-6), 6.81 (1H, d, J=1.7Hz, H-3), 6.66 (1H, dd, J=8.7,1.7Hz, H-5) and 2 methoxyl group δ 3.82 (3H, s), 3.77 (3H, s); And sugar chain structure: 2 sugared terminal hydrogens [δ 4.92 (1H, d, J=6.5Hz, H-1 '), δ 4.15 (1H, d, J=7.5Hz, H-1 ")] show that 2 monose are beta comfiguration.
2 end group carbon: δs corresponding with sugar chain 2 anomeric protons 101.35 (C-1 ') can be obtained by HMQC spectrum, 104.63 (C-1 ").
131 carbonyl carbon: δ 166.18 (C-7) is there is in C-NMR data presentation structure, and sugar chain may comprise 1 molecule glucose [δ 73.71 (C-2 '), 76.73 (C-3 '), 70.11 (C-4 '), 76.16 (C-5 '), 69.16 (C-6 ')] and 1 molecule wood sugar [δ 73.84 (C-2 "); 77.04 (C-3 "), 69.97 (C-4 "); 66.11 (C-5 ")], and can H-1 be found in HMBC spectrum " (δ 4.15) and C-6 ' (δ 69.16) have reference point.Therefore determine that sugar chain structure is D-wood sugar (1 → 6) D-Glucose, i.e. primeverose (primverose).
This compound and document
[6]report
1h-NMR and
13c-NMR data consistent (table 3), therefore deterministic compound 6 is primeverin, is commonly called as primeverin (primeverin).
(solvent is DMSO-d to the nuclear magnetic data of table 3. compound 6
6)
Note: above signal is obtained by two-dimentional nuclear-magnetism supplementary means.
Compound 7:2-primrose glycosyl-5-methoxy-acetophenone
Brownish-yellow powder,
221,259.HRESI-MS provides molecular formula C
20h
28o
12na (measured value 483.1480, calculated value 483.1478), in conjunction with
1h-NMR and
13c-NMR data, determine that its molecular weight is 460, molecular formula is C
20h
28o
12, illustrate that this compound has 7 degrees of unsaturation.
1aBX Coupling System is there is: δ 7.32 (1H, d, J=9.0Hz in this compound structure of the H-NMR Notes of Key Data, H-3), 7.12 (1H, dd, J=9.0,3.0Hz, H-4), 7.08 (1H, d, J=3.0Hz, H-6) and 1 methoxyl group δ 3.74 (3H, s, H-9), 1 methyl δ 2.62 (3H, s, H-8) and 2 sugared terminal hydrogens: δ 4.83 (1H, d, J=7.0Hz, H-1 '), 4.19 (1H, d, J=7.5Hz, H-1 "), show that 2 monose are beta comfiguration.
13c-NMR data presentation has the signal of 20 carbon, comprises 1 ketone group carbon [δ 199.38 (C-7)], 1 methoxyl group carbon [δ 55.96 (C-9)], 1 methyl carbon [δ 32.47 (C-8)]; And sugar chain structure is: D-glucose: δ 101.97 (C-1 '), 73.76 (C-2 '), 76.96 (C-3 '), 70.10 (C-4 '), 76.82 (C-5 '), 68.74 (C-6 '); D-xylose: δ 104.31 (C-1 "), 73.70 (C-2 "), 76.53 (C-3 "), 69.94 (C-4 "), 66.05 (C-5 ").
Can H-1 be found in HMBC spectrum " (δ 4.19) and C-6 ' (δ 68.74) have reference point, meanwhile, by compound 26 sugar chain structure
13c-NMR data and compound 6 compare, and both are consistent, therefore infer that sugar chain structure is: D-wood sugar (1 → 6) D-Glucose, i.e. primeverose.
Meanwhile, NOESY spectrum display δ 3.74 (3H, s) and δ 7.12 (1H, dd, J=9.0,3.0Hz) have reference point, show to be in methoxyl group hydrogen and phenyl ring neighbour, the dual coupling in a position hydrogen at homonymy.
Comprehensive above data by 2D-NMR to it
1h,
13c signal carries out belonging to (see table 4), determines that the structure of this compound is 2-primrose glycosyl-5-methoxy-acetophenone.Through searching document, have no relevant report, compound 7 is a new compound.
The nuclear magnetic data of table 4. compound 7 and main phase OFF signal
Note: above signal is obtained by two-dimentional nuclear-magnetism supplementary means;
a, b, c)commutative.
Reference
[1]Kuwajima，Hiroshi；Shibano，Naoka；Baba，Toshifumi；et al.，An acetophenoneglycoside from Exacum affine，Phytochemistry，1996，41(1)：289-92.
[2]Srebotnik，Ewald；Jensen，Kenneth A.，Jr.；Kawai，Shingo；et al.，Evidence thatCeriporiopsis subvermispora degrades nonphenolic lignin structures by aone-electronoxi-dation mechanism.Applied and Environmental Microbiology，1997，63(11)：4435-40.
[3]Schroeder，Cornelia；Lutterbach，Ralf；Stoeckigt，Joachim.Preparativebiosynthesiss of natural glucosides and fluorogenic substrates for β-glucosidasesfollowed by in vivo
13C-NMR with high density plant cell cultures.Tetrahedron，1996，52(3)：925-34.
[4]Wagner，G.；Kuhmstedt，H.Phenol glycosides.XVIII.Fermentative hydrolysis ofβ-D-glucopyranosides of the resorcylic acid series.Archiv der Pharmazie，1960，29(3)：428-41.
[5]Li，Jun；Kadota，Shigetoshi；Kawata，Yukio；et al.，Constituents of the roots ofCynanchum bungei Decne.Isolation and structures of four new glucosides，bungeisideA，B，C，and D.Chemical&Pharmaceutical Bulletin，1992，40(12)：3133-7.
[6]Andrea Müller，Markus Ganzera and Hermann Stuppner.Analysis of phenolic glycosidesand saponins in Primula elatior and Primula veris(primula root)by liquidchromatography，evaporative light scattering detection and mass spectrometry，Journalof Chromatography A，2006，1112：218-223.
The preparation at primeverin and methyl phenyl ketone glycosides position in root of Maximowicz Primrose
1. the process of macroporous adsorbent resin
1.1 pre-treatment: get AB-8 type macroporous adsorbent resin, with acetone reflux wash-out, are eluted to noresidue after elutriant evaporate to dryness.
1.2 dress posts: with ethanol wet method dress post, continuation ethanol rushes post, frequently checks effluent liquid, (ethanol: water=1: 5) to mix not in white casse with water.Then ethanol is washed away with a large amount of distilled water, for subsequent use.
1.3 regeneration: resin uses once, when the general ethanol elution with 95% is extremely colourless, namely resin column regenerates, then removes ethanol with massive laundering, can carry out separation next time.After Reusability, polymeric adsorbent darkens, and when adsorption effect declines, available 0.1% ~ 0.5%NaOH and HCl carries out soda acid alternate treatment or soaks reasonable time, is advisable to resin close to native color, continues to wash with water to neutrality and can use again.
2. the extraction of medicinal material: material root of Maximowicz Primrose of getting it filled, is ground into meal, with 50% alcohol reflux 3 (8 times amount, 2 hours; 6 times amount, 1 hour; 6 times amount, 1 hour), filter, merging filtrate, low-temperature reduced-pressure reclaims ethanol to without alcohol taste, is adjusted to every milliliter of liquid is equivalent to 1.0g crude drug with water.
The qualitative selection of 2.1 wash-out solvents: get extracting solution and be added on processed good AB-8 type macroporous adsorptive resins, use successively distilled water, 30%, 50%, 90% ethanol gradient elution, Fractional Collections, decompression recycling ethanol is concentrated, polyam ide TLC chromatogram and HPLC chromatographic qualitative thereof check primeverin and methyl phenyl ketone methods of glycosides, containing methyl phenyl ketone glycosides in result display 30%.Therefore determine that extracting solution is after AB-8 type absorption with macroporous adsorbent resin, water elution removing water-soluble impurity, methyl phenyl ketone methods of glycosides in 30% ethanol elution enrichment root of Maximowicz Primrose.
The determination of 2.230% ethanol elution amount: extracting sample solution 20ml upper prop, after leaving standstill, first be eluted to colourless with distilled water, use 30% ethanol elution again, every 50ml is a, and every part concentrates a little, tlc qualitative detection primeverin, examine when result shows the 5th part and do not measure primeverin, therefore determine that wash-out solvent consumption is 200mL.
2.3 enrichment degrees are investigated: sample thief liquid 20ml, by above-mentioned condition upper prop wash-out, collect water elution liquid concentrate drying to constant weight, collect 30% ethanol eluate, decompression recycling ethanol, and drying under reduced pressure are to constant weight.Separately get extracting solution 20ml drying under reduced pressure to constant weight, measure the content of total solid substance respectively.And the content before pressing content determination mensuration upper prop and after upper prop, the results are shown in following table.
Result shows, with AB-8 type macroporous adsorbent resin for sorbent material, total solid before and after loading and wash-out and the content of primeverin are index, AB-8 type macroporous adsorbent resin effectively can remove impurity, the content of primeverin and methyl phenyl ketone methods of glycosides is increased substantially, and retention rate close, illustrate that AB-8 type macroporous adsorbent resin can be used for the enrichment of primeverin and methyl phenyl ketone methods of glycosides in root of Maximowicz Primrose.
Take primeverin as the applied sample amount of primeverin and methyl phenyl ketone methods of glycosides in metrics evaluation AB-8 type macroporous adsorbing resin for purification root of Maximowicz Primrose, result shows that macroporous adsorbent resin 20mL maximal absorptive capacity is 20ml root of Maximowicz Primrose liquid, and namely 20g root of Maximowicz Primrose crude drug is good.
3. the process of polymeric amide
3.1 pre-treatment: get 60-90 order polymeric amide, use alcohol heating reflux wash-out, are eluted to noresidue after elutriant evaporate to dryness.
3.2 dress posts: with 30% ethanol wet method dress post, continue to rush post with 30% ethanol, for subsequent use.
3.3 regeneration: resin uses once, when the general ethanol elution with 95% is extremely colourless, namely polyamide column regenerates, and then uses 30% ethanol elution, can carry out separation next time.After Reusability, absorption polymeric amide darkens, and when adsorption effect declines, available 0.1% ~ 0.5%NaOH and HCl carries out soda acid alternate treatment or soaks reasonable time, is advisable to polymeric amide close to native color, continues to wash with water to neutrality and can use again.
The qualitative selection of 3.4 wash-out solvents: get macroporous adsorbent resin 30% ethanol eluate part in [2], be concentrated into 1ml solution and be equivalent to 10g crude drug, be added on processed good polyamide column, with 10%, 30%, 50%, 90% ethanol gradient elution, Fractional Collections, decompression recycling ethanol is concentrated, and polyam ide TLC chromatogram and HPLC chromatographic qualitative thereof check primeverin and methyl phenyl ketone methods of glycosides, containing primeverin and methyl phenyl ketone glycoside in result display 30%.Therefore determine that 30% macroporous adsorbent resin elutriant is again after polycaprolactam, primeverin and methyl phenyl ketone methods of glycosides in 30% ethanol elution enrichment root of Maximowicz Primrose.
The determination of 3.5 30% ethanol elution amounts: extracting sample solution 20ml upper prop, use 30% ethanol elution, every 20ml is a (1 column volume), every part concentrates a little, tlc qualitative detection primeverin, examine when result shows the 6th part and do not measure primeverin, therefore determine that wash-out solvent consumption is 120mL.
3.6 enrichment degrees are investigated: sample thief liquid 20ml, by above-mentioned condition upper prop wash-out, collect 30% ethanol eluate, reclaim ethanol, and are dried to constant weight.Separately get extracting solution 20ml drying under reduced pressure to constant weight, measure the content of total solid substance respectively.And the content before pressing content determination mensuration upper prop and after upper prop, the results are shown in following table.
Result shows, take polymeric amide as sorbent material, total solid before and after loading and wash-out and the content of primeverin are index, polymeric amide effectively can remove flavonoid, the content of primeverin and methyl phenyl ketone methods of glycosides is increased substantially, and retention rate close, illustrate that polymeric amide can be used for the enrichment of primeverin and methyl phenyl ketone methods of glycosides in root of Maximowicz Primrose.
Take primeverin as the applied sample amount of primeverin and methyl phenyl ketone methods of glycosides in metrics evaluation polymeric amide enrichment root of Maximowicz Primrose, result shows that polymeric amide 20g maximal absorptive capacity be 200g root of Maximowicz Primrose is good.
Except the above, implement other operation involved in the process of the inventive method, the volatilization of such as extracting solution, volatilization of heating can be adopted, also can directly naturally volatilize or other feasible method by normal temperature, the concrete operations point getting and volatilize process belong to the basic skills of this area, and the anaesthetic medicine materical crude slice related to is processed, pulverizes, sieved and extraction etc., all can adopt this area routine operation or other feasible operation, the present invention is not particularly limited.By the determination method of any routine, the extract obtained can learn whether it meets the requirements, as tlc, ultraviolet spectrophotometry and other as " a Chinese Pharmacopoeia 2005 version annex the analytical procedure recorded, more than belong to this area routine operation, the present invention is also not particularly limited.
The methyl phenyl ketone glycosides of separation and purification and main component primeverin thereof from root of Maximowicz Primrose medicinal material, therefore as one of the material site of action and main component of root of Maximowicz Primrose.Set up good separating effect herein, quick and precisely, the HPLC method of favorable reproducibility, have detected primeverin and methyl phenyl ketone glycoside site component in root of Maximowicz Primrose to method.
Analyze containing primeverin and methyl phenyl ketone methods of glycosides
1 instrument and reagent
HP-1100 series of high efficiency liquid chromatograph; Methyl alcohol (chromatographically pure), acetonitrile (chromatographically pure), all purchased from Fisher Scientific; Commercially available pure water.
2 sample preparations
The preparation precision of reference substance solution takes 2-primrose glycosyl-4-methoxy-acetophenone reference substance, 4-O-β-D-Glucose base-methyl phenyl ketone reference substance, 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters reference substance, 4-primrose glycosyl-methyl phenyl ketone reference substance, primeverin reference substance, 2-primrose glycosyl-5-methoxy-acetophenone reference substance are appropriate, add methyl alcohol and make the solution of every 1ml containing 20 μ g, to obtain final product.
Above-mentioned preparation position sample 0.1g is got in the preparation of need testing solution, accurately weighed, puts in 100mL Erlenmeyer flask, add 70% methyl alcohol 50mL, weigh, ultrasonic (power 250W, frequency 50KHZ) processes 40 minutes, put to room temperature, add 70% methyl alcohol and supply weight, shake up, filter, get subsequent filtrate, to obtain final product.
Assay method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
3 chromatographic conditions and system suitability
Diamonsil C
18(5 μm, 150 × 4.6mm) chromatographic column; Methyl alcohol (A)-water (B) is moving phase, carries out gradient elution according to the regulation in following table; Determined wavelength is 254nm; Column temperature: 30 DEG C; Flow velocity: 1mL/min.
4 sample determinations
Get need testing solution described in 1, measure respectively with chromatographic condition described in 2.Positioned its position in the peak at position by the retention time of each monomeric compound, analytical results is in Table, and HPLC color atlas is shown in Fig. 1 ~ 5.
Reference substance is corresponding with sample chromatogram peak to be shown
On above-mentioned Research foundation, present invention also offers the pharmaceutical composition that can be used for primeverin and methyl phenyl ketone glycoside, comprise acceptable pharmaceutical adjuvant in anaesthetic primeverin and methyl phenyl ketone glycoside extracts and pharmaceutics, wherein primeverin and methyl phenyl ketone glycoside extracts in the composition weight ratio be at least not less than 10%, preferably be not less than 30%, more preferably more than 50%, be no more than 95%.
Aforementioned pharmaceutical compositions comprises the acceptable any formulation of pharmacy, as injection, tablet, capsule, granule and oral liquid etc., said medicine assistant agent also comprises the acceptable any auxiliary material of pharmacy, and the preparation technology of each formulation is this area routine operation, does not add repeat at this.
According to pharmaceutical composition of the present invention, this primeverin and methyl phenyl ketone glycoside position also can reach better result for the treatment of with other anaesthetic composition is composite, described composition can be the effective constituent in the bulk drugs such as the such as root of kudzu vine, ginseng, ginkgo pseudo-ginseng, Herba Erigerontis, the red sage root, and it can from any extracting method.
Accompanying drawing illustrates:
Fig. 1 primeverin reference substance HPLC color atlas
Fig. 2 root of Maximowicz Primrose medicinal material HPLC color atlas (p: primeverin)
Fig. 3 contains the HPLC color atlas at primeverin and methyl phenyl ketone glycosides position
Fig. 4 compound 3 (4-O-β-D-Glucose base-methyl phenyl ketone) and compound 5 (4-primrose glycosyl-methyl phenyl ketone) reference substance HPLC color atlas
Fig. 5 compound 7 (2-primrose glycosyl-5-methoxy-acetophenone), compound 1 (2-primrose glycosyl-4-methoxy-acetophenone), compound 6 (primeverin (primeverin)) and compound 4 (2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters) reference substance HPLC color atlas
Fig. 6 primeverin and methyl phenyl ketone glycosides compound structural formula
Fig. 7 2-primrose glycosyl-4-methoxy-acetophenone
1h-NMR spectrogram
Fig. 8 2-primrose glycosyl-4-methoxy-acetophenone
13c-NMR spectrogram
Fig. 9 4-hydroxy-acetophenone
1h-NMR spectrogram
Figure 10 4-hydroxy-acetophenone
13c-NMR spectrogram
Figure 11 4-O-β-D-Glucose base-methyl phenyl ketone
1h-NMR spectrogram
Figure 12 4-O-β-D-Glucose base-methyl phenyl ketone
13c-NMR spectrogram
Figure 13 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters
1h-NMR spectrogram
Figure 14 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters
13c-NMR spectrogram
Figure 15 4-primrose glycosyl-methyl phenyl ketone
1h-NMR spectrogram
Figure 16 4-primrose glycosyl-methyl phenyl ketone
13c-NMR spectrogram
The ESI-MS spectrogram of Figure 17 primeverin
Figure 18 primeverin
1h-NMR spectrogram
Figure 19 primeverin
13c-NMR spectrogram
The HMQC spectrogram of Figure 20 primeverin
The HMBC spectrogram of Figure 21 primeverin
The HRESI-MS spectrogram of Figure 22 2-primrose glycosyl-5-methoxy-acetophenone
Figure 23 2-primrose glycosyl-5-methoxy-acetophenone
1h-NMR spectrogram
Figure 24 2-primrose glycosyl-5-methoxy-acetophenone
13c-NMR spectrogram
Figure 25 2-primrose glycosyl-5-methoxy-acetophenone
1h-
1h COSY spectrogram
The HMQC spectrogram of Figure 26 2-primrose glycosyl-5-methoxy-acetophenone
The HMBC spectrogram of Figure 27 2-primrose glycosyl-5-methoxy-acetophenone
The NOESY spectrogram of Figure 28 2-primrose glycosyl-5-methoxy-acetophenone
Embodiment
About specific embodiment of the invention and result for the treatment of, will in conjunction with the preferred embodiments and accompanying drawing be described in detail, it should be noted that: the implication at term of the present invention " methyl phenyl ketone glycoside extracts ", " methyl phenyl ketone glycoside position " is identical.
Pharmaceutical composition embodiment 1: injection
Containing primeverin and methyl phenyl ketone glycoside extracts 400g
Phenylcarbinol 0.1g
Water for injection adds to 1000ml
Make injection 1000ml altogether.
Pharmaceutical composition embodiment 2: tablet
Containing primeverin and methyl phenyl ketone glycoside extracts 40g
Starch 20g
Icing Sugar 20g
Dextrin 20g
Conventional compression, suppresses 1000, every sheet 0.1g.
Pharmaceutical composition embodiment 3: capsule
Containing primeverin and methyl phenyl ketone glycoside extracts 40g
Dextrin 20g
Icing Sugar 20g
Starch 20g
Make 1000 altogether.
Pharmaceutical composition embodiment 4: granule
Ordinary method makes granule 100g, pack, every bag of 5g.
The foregoing describe the preferred embodiment for the present invention, so itself and be not used to limit the present invention.Those skilled in the art can not depart from improvement and the change of scope and spirit to embodiment disclosed herein.
Claims (7)
1. primeverin and methyl phenyl ketone glycoside extracts, it is characterized in that the primeverin that contains in every gram of extract and methyl phenyl ketone methods of glycosides are no less than 500 milligrams in primeverin, methyl phenyl ketone glycoside is selected from 2-primrose glycosyl-4-methoxy-acetophenone, 4-O-β-D-Glucose base-methyl phenyl ketone, 4-hydroxy-acetophenone, 2-O-β-D-Glucose base-4-methoxy-benzoic acid methyl esters, 4-primrose glycosyl-methyl phenyl ketone, 2-primrose glycosyl-5-methoxy-acetophenone.
2. primeverin as claimed in claim 1 and methyl phenyl ketone glycoside extracts, wherein, is no less than 50 milligrams containing primeverin in every gram of extract.
3. primeverin as claimed in claim 2 and methyl phenyl ketone glycoside extracts, wherein said primeverin and methyl phenyl ketone glycoside extracts be the every gram of extract extracted in root of Maximowicz Primrose contain primeverin be no less than 50 milligrams have finger printing as shown in Figure 3 containing primeverin and methyl phenyl ketone glycoside extracts, wherein the chromatographic condition of Fig. 3 is Diamonsil C
18: 5 μm of 150 × 4.6mm chromatographic columns; Methyl alcohol (A)-water (B) is moving phase, carries out gradient elution according to the regulation in following table, and determined wavelength is 254nm; Column temperature: 30 DEG C; Flow velocity: 1mL/min,
4. the preparation method of the primeverin according to any one of claim 1-3 and methyl phenyl ketone glycoside extracts, step is by root of Maximowicz Primrose pulverizing medicinal materials, extraction using alcohol, recycling design, concentrating and precipitating, filter, macroporous adsorbent resin on filtrate, uses organic solvent ethanol gradient elution, collect the composition that 10%-90% concentration elutes, upper polyamide column, with 20%-35% ethanol elution, obtains described primeverin and methyl phenyl ketone glycoside extracts.
5. pharmaceutical composition, comprises acceptable pharmaceutical adjuvant in primeverin in any one of claim 1-3 and methyl phenyl ketone glycoside extracts and pharmaceutics.
6. pharmaceutical composition as claimed in claim 5, wherein the part by weight of primeverin and methyl phenyl ketone glycoside extracts is not less than 10%.
7. the pharmaceutical composition as described in claim 5 or 6, wherein the part by weight of primeverin and methyl phenyl ketone glycoside extracts is 10% ~ 95%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927015A (en) * | 2004-09-10 | 2007-03-14 | 长谷川香料株式会社 | Method of preparing tea extract and tea flavor |
| WO2009083420A1 (en) * | 2007-12-28 | 2009-07-09 | Unilever Plc | Process for recovering aroma from tea |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927015A (en) * | 2004-09-10 | 2007-03-14 | 长谷川香料株式会社 | Method of preparing tea extract and tea flavor |
| WO2009083420A1 (en) * | 2007-12-28 | 2009-07-09 | Unilever Plc | Process for recovering aroma from tea |
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| Title |
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| Andrea Muller,等.Analysis of phenolic glycosides and saponins in Primula elatiorand Primula veris (primula root) by liquid chromatography, evaporative light scattering detection and mass spectrometry.《Journal of Chromatography A》.2005,第1112卷第218-223页. * |
| 曲功霖,等.胭脂花中樱草苷的HPLC测定.《中国医药工业杂志》.2007,第38卷(第12期),第866-867页. * |
| 曲功霖,等.胭脂花化学成分的研究II.《中国药学杂志》.2008,第43卷(第17期),第1300-1304页. * |
| 曲功霖,等.蒙药胭脂花的化学成分研究.《中草药》.2007,第38卷(第9期),第1308-1310页. * |
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