CN1537864A - Fern effective part and compound fern glycoside and its separaton and purification method - Google Patents

Fern effective part and compound fern glycoside and its separaton and purification method Download PDF

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CN1537864A
CN1537864A CNA03150986XA CN03150986A CN1537864A CN 1537864 A CN1537864 A CN 1537864A CN A03150986X A CNA03150986X A CN A03150986XA CN 03150986 A CN03150986 A CN 03150986A CN 1537864 A CN1537864 A CN 1537864A
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ring
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proton
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fiber crops
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CN1280304C (en
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蔡光明
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Beijing Meite Biotechnology Co Ltd
302th Hospital of PLA
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Beijing Meite Biotechnology Co Ltd
302th Hospital of PLA
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Abstract

A process for extracting the active component from potentilla anserine, the structure of potentilla anserine's glycoside, a process for separating and purifying it, and the application of said potentilla anserine's glycoside having anti-activity of hepatitis virus, inhibition of HbsAg and HbeAg and in protecting liver and gall and reducing transaminase are disclosed.

Description

Fern fiber crops efficient part and compound fern fiber crops glycosides and isolation and purification method
Technical field
The invention belongs to natural medicine technical field.Be specifically related to fern fiber crops efficient part and compound fern fiber crops glycosides and isolation and purification method.
Background technology
Chinese medicine fern fiber crops (Potentiila anserine L) belong to Rosaceae potentilla anserina, are used as medicine with root, and the different name Herba Herminii, the lotus cauliflower stabs agate (hiding name).This medicine beginning is stated from " herbal medicine is used in Tibet always ", is recorded by " Qinghai Province's drug standard (version in 1992) ", " Chinese medicine voluminous dictionary (descending) " and books such as " Chinese Higher plant illustrated handbook ", " Chinese medicine sea ", " Chinese natural tonic resource voluminous dictionary ".This product mainly is distributed in Hexi Corridor, west area one band.Qinghai, Gansu, Tibet are the main place of production.The fern fiber crops belong to highland and severe cold area growing plants, general grassland, the hillside of growing environment more than height above sea level 2000-3000m.This product medicine source is abundant, and can gather the whole year, generally concentrates on summer, autumn, and only area, Gannan, a Gansu Province year purchase volume can reach kiloton.In addition, Gannan, the Gansu state institute of agricultural sciences has finished the experimental study of fern fiber crops artificial cultivation techniques, has created essential condition for enlarging fern fiber crops resource.Fern fiber crops are owing to be rich in amino acid, VITAMIN, carbohydrate and various active composition, therefore, as nutritional supplements in the Northwest among the people being widely used.Outside inferior, the local report that the fern fiber crops is used for the treatment of malignant tumour that has among the people.The present situation of relevant fern fiber crops research has been carried out the documentary investigation of system.According to nearest document Investigation: bibliographical information Hu Benxiang etc. the fern fiber crops research of pharmacognosy identification: Wang Baoqi etc. have been carried out to fern fiber crops trace element germanium assay; Wang Jin etc. are to the research of nutritive ingredient VITAMIN, amino acid glucide composition in the fern fiber crops; The merchant keeps peaceful grade fern fiber crops oxygen lack resistant function has been carried out experimental study; Lin Na etc. have studied the researchs of fern fiber crops to immunologic hypofunction mouse influence: Mccutcheon-AR..et al. has reported that the water extract of potentilla Potentill argut can suppress to breathe the result of study of full cell space virus.About the research of fern fiber crops Chemical Composition, Yang Hua etc. have reported the research of polysaccharide component in the fern fiber crops and assay thereof; Morri wolfred, et al and P.Tunmann.Arch have carried out research and have reported result of study the equal platymiscium chemical ingredients of fern fiber crops.Look into new demonstration:, antihepatitis drug antiviral about the fern fiber crops imitated aspect and the separation of its corresponding Chemical Composition and the research of configuration aspects, still do not have report both at home and abroad.Since 9 years, the contriver has carried out the research of fern fiber crops main chemical and research antiviral, antihepatitis drug reason pharmacodynamics aspect thereof, wherein study emphasis to the extract of fern fiber crops different sites and reactive site thereof and main active ingredient experimental study at anti-hepatitis, anti-virus aspect.Experiment is whole hepatitis B virus resisting pharmacodynamics screening and assessment model, is external anti-hepatitis B virus test method with 2215 cell methods with dhbv dna (DHBVDNA) experimental animal model, the water extracts of fern fiber crops, 70% different extract parts such as ethanol extraction carried out antiviral, antihepatitis drug imitated and learn research.Through the test evaluation of system, important experimental result and progress have been obtained.The ethanol extraction of finding the fern fiber crops has anti-HBV effect in remarkable vitro and the body.Meanwhile, experimental studies results also further shows: the fern fiber crops anti-chmice acute liver injuries of alcohol extract (falling ALT) effect significantly, circulation ratio is good as a result.
Summary of the invention
Technical problem to be solved by this invention is to seek fern fiber crops efficient part (JMS), the method for design isolation and purification compound fern fiber crops glycosides.
The invention provides a kind of called after fern fiber crops glycoside compound
Title, chemical name, chemical structural formula, molecular formula and the molecular weight of compound (fern fiber crops glycosides)
(1) Chinese name: fern fiber crops glycosides
(2) Chinese phonetic alphabet: Jue Ma Gan
(3) English name: potentilla anserine side
(4) chemical structural formula:
(5) chinesization formal name used at school: 2 α, 3 β, 19 β-trihydroxy--12-alkene-ursane-28-carboxylic acid-28-O-β-D-galactopyranoside
(6) English chemical name: 2 α, 3 β, 19 β-trihydroxy-12-en-urs-28-oicacid-28-O-β-D-galactonopyranesyl ester
(7) molecular formula: C 36H 58O 10H 2O
(8) molecular weight: 650.848+18.015=668.86
One, for conclusive evidence the compound chemical structure purity of sample and the method for inspection purity
(1) source of trial-product
Trial-product is provided by PLA the 302nd hospital drug research center (fern fiber crops seminar).
Trial-product lot number: 20010526
(2) purity of trial-product
Trial-product purity is 98.85% (measuring method is HPLC, sees Fig. 1)
(3) inspection method of trial-product purity
(1) thin layer chromatography: see＜Chinese Pharmacopoeia 2000 editions〉an appendix P37.
(2) high performance liquid chromatography: see＜Chinese Pharmacopoeia 2000 editions〉an appendix P38.
Two, the method for conclusive evidence chemical structure
(1) physical behavior and physicochemical constant
Fusing point: DSC:231.7 ℃;
Specific optical rotation: [α] D 20=+3.0 ° (MeOH, C=0.5625%)
(2) qualitative identification
(1) Lieberman-Burchard is the red-purple positive reaction
(2) the Molish reaction is also positive
(3) ultimate analysis
1. two kinds of elements of analytical procedure: C, H adopt Flash EA 1112 type elemental analysers to measure.
2. mensuration element: this product contains C, H, three kinds of elements of O, and having measured wherein two kinds is C and H.
3. measurement result: see table 1 for details.
The ultimate analysis data of table 1 trial-product fern fiber crops glycosides
Elements C (%) H (%) remarks
(66.43 anhydride) * 8.98 (anhydride) *
Theoretical value
64.65 (a water thing) * * 9.04 (a water thing) * * * is by molecular formula C 36H 58O 10Calculate
1 64.49 9.08 * * are by molecular formula C 36H 58O 10H 2O calculates
Trial-product
2????64.23????????????????8.95
4. discuss and explanation
(1) this product contains C, H, three kinds of elements of O, except that O, has measured the content of C and two kinds of elements of H, and by table 1 as seen, the determination of elemental analysis result of trial-product fern fiber crops glycosides shows the numerical value and the theoretical value basically identical that contains a water thing of carbon; The numerical value of hydrogen is more approaching with the theoretical value that contains a water thing.Prove that this compound should contain a crystallization.
(2) this result is also confirmed by nuclear-magnetism and thermogravimetric analysis structural information, can think that the results of elemental analyses of this product conforms to the molecular formula that contains a water thing.
(4) infrared absorption spectrum (IR)
1. instrument model: U.S. Buddhist nun high-tensile strength (Nicolef) Nexus-470 of company type fourier transform infrared spectrometer.
2. instrumental correction
The infrared spectroscopy degree method of pharmacopeia two appendix 27 of version in 2000 and IVc is proofreaied and correct and calibration method, and the spectral results of recording polystyrene film (thickness is 0.05mm) sees Table 2.
Table 2 polystyrene film is proofreaied and correct the result
Actual measurement wave number (cm -1) standard wave number (cm -1) error (cm -1)
3026.0????????????????3027.1?????????????????????-1.1
2923.9????????????????2924.0?????????????????????-0.1
2849.6????????????????2840.7?????????????????????-1.1
1870.5????????????????1871.0?????????????????????-0.5
1802.4????????????????1801.0??????????????????????1.4
1601.3????????????????1601.3??????????????????????0
1583.2????????????????1583.1??????????????????????0.1
1181.5????????????????1181.4??????????????????????0.1
1154.7????????????????1154.3??????????????????????0.4
1069.3????????????????1069.1??????????????????????0.2
1028.5????????????????1028.0??????????????????????0.5
906.7?????????????????906.7???????????????????????0
697.2?????????????????698.9??????????????????????-1.7
As seen from the above table: at 4000-2000cm -1In the interval, error is at ± 8cm -1In, 2000-400cm -1In the interval, error is at ± 4cm -1In, requirement up to specification.
3. sample preparation methods
According to the preparation method of sample requirement in Chinese Pharmacopoeia Commission's " infrared preparation spectra collection of medicine " version explanation in 2000, adopt KBr pressed disc method recording light spectrogram.
4. measurement result: see table 3 and Fig. 2 for details.
The IR spectrum data and the parsing of table 3 fern fiber crops glycosides trial-product
5. discuss and explanation
(1) each data shows in table 3 and the accompanying drawing, each main functional group in the chemical structural formula of trial-product: three kinds of alkyl (CH 3,-CH 2-and
Figure A0315098600073
Three substituted alkenyls Ester carbonyl group
Figure A0315098600075
Two kinds of alcoholic extract hydroxyl groups With
Figure A0315098600077
And β-main infrared absorption peak and relevant peaks such as D-pyranose all exist, and illustrate that each functional group in this product chemical structural formula exists really and conforms to practical situation.
(2) since have association phenomenon to have (promptly have hydrogen bond form) in the molecule thus among the figure quite a few absorption peak all have to associate and widen phenomenon.
(3) ((1956) are introduced, v for I.L.Allsop, A.R.Cole etc.J.Chem.Soc.4968 according to document * C-OThe size of data of (-CHOH) can be used for distinguishing the configuration of the C-3 position 3-OH of triterpene compound.Owing to the influence of gem-dimethyl is arranged, the v of 3 β-OH on the C-4 position C-OBe slightly less than the v of 3 α-OH C-OAnd this β-OH is the e key, then at 1045-1050cm -1And 1013-1031cm -1There are two absorptions at the place.This product meets this situation (1050 and 1031cm -1).The hydrogen spectrum situation of this result and this product fits like a glove.About document shows: many now employing NMR distinguish the axial bond (a) of C-3 position and the substituting group of horizontal key (e).(P288) 3-OH in the six-ring of chair conformation and the configuration of 2-OH can utilize * for L.M.Jackmanetc " Application of NMR in Org.Chem. " 2nd Edition, PergammonPress NY (1969) 3J HHIt is axial bond or horizontal key that (H-2 and H-3) size decides the two.Common H-2 and H-3, as be 3Jaa=8-13Hz; As be 3Jae=2-6Hz, 3Jee=1-5Hz, the three has notable difference; At present, the most effective way is the correlationship in the sharp NOESY spectrum.According to above several respects information, its 2 OH of this pentacyclic triterpenoid should be 2 α-OH (horizontal keys).3 OH then are 3 β-OH (horizontal keys).By in the hydrogen spectrum as seen 3J H-2/H-3The correlation circumstance of=9.5z and NOESY spectrum also matches with hydrogen spectrum, infrared information.(seeing this data (six) for details)
(4) introduce 1380 (cm according to document * * * -1) be various-CH 3The principal character peak, its peak position of dissimilar methyl and peak shape, peak are strong etc. all certain difference.As for the carbon dimethyl (gem-dimethyl then is split into two than the peak and signal occurs at 1391-1381 and 1368-1366 place respectively, and latter's intensity be about the former 4/5), confirm to have in the molecule gem-dimethyl existence thus.
(5) introduce according to document * * *:
1. when not containing strong absorption functional group in the aglycon molecule, the spectrum of this glycosides is similar to sugar, promptly at 3400 and 1100 (cm -1) near strong v is arranged OHAnd δ C-OAbsorption peak.Infrared this situation that meets of this product.(v OHBe 3423cm -1, δ C-O1075cm -1And the two is a highest peak in the full spectrum).
2. be that hexa-atomic pyranose is then at 1100-1010 (cm as sugar -1) between 3 strong peaks (two strong peaks appear in five yuan of furanoses) this product occurs and meet this situation, (this product has 1074,1050 and 1,032 three strong peaks.)
3. so pyranose is that β-D configuration is then at 890cm -1Locate that absorption is arranged (and α-L configuration is at 840cm -1Near absorption is arranged).This product conforms to this situation (at 896cm -1There is absorption at the place).In addition: as being axial bond for its H-1 of beta configuration, its δ C-HAt 891 ± 7cm -1The place, ring vibration is 774 ± 9cm -1This product meets this situation (δ CHBe 896cm -1Ring vibration is 773cm -1).
4. three types (oleanane type, ursane type and tetracyclic triterpenes saponins) can utilize regional A (1355-1392cm in the infrared spectra in the triterpenoid sapogenin -1) and area B (1225-1330cm -1) the number and the peak position situation of absorption peak distinguished.The ursane type has 3 peaks in the A district, three peaks are also arranged in the B district.This product meets this situation.(its peak position is respectively 1390,1384,1390 and 1325,1262,1226cm -1)
Above situation shows:
(i) aglycon of this product is not for containing the ursane type pentacyclic triterpenoid of strong absorption group.
(ii) this product is hexa-atomic pyranose;
(iii) this product is β-D configuration pyranose.
(6) each symbol implication is respectively in the table 3:
v As---asymmetric (the anti-title) stretching vibration
v s---symmetrical stretching vibration
δ As---asymmetric bending vibration (in-plane bending vibration)
δ s---symmetric curvature vibration (in-plane bending vibration)
γ---out-of-plane deformation vibration (twisting vibration)
Vs---absorb very by force
S---the strong absorption
M---medium absorption
W---weak absorption
Br---wide absorption
(5) ultra-violet absorption spectrum (UV)
1. instrument model: HP8452A (U.S. Hewlett-Packard Corporation)
2. instrumental correction and calibrating
Press 2000 editions two appendix 26 page or leaf IV of Chinese Pharmacopoeia ARegulation check the accuracy and the stray light of optical density, the result meets the pharmacopeia requirement.
3. sample solution preparation
Neutral solution: get this product with CH 3OH is a solvent, is made into the solution (0.2mg/ml methyl alcohol system) of suitable concentration.
Acidic solution: getting this product is solvent with 0.1mol/L hydrochloric acid soln methyl alcohol, is made into the need testing solution (0.2mg/ml methyl alcohol system) of suitable concentration.
Basic solution: getting this product is solvent with 0.1mol/L sodium hydroxide methyl alcohol, is made into the need testing solution of suitable concentration.
4. measurement result
Press 2000 editions two appendix IV of Chinese Pharmacopoeia ASpectrophotometry is used in requirement, and in the wavelength region interscan of 200-400nm, (A=ε CL) calculates molar absorption coefficient ε by Beer's law, the results are shown in Table 4 and Fig. 3, Fig. 4 and Fig. 5.
The uv-spectrogram data and the parsing of table 4 fern fiber crops glycosides trial-product
Figure A0315098600101
5. discuss and explanation
This product is the pentacyclic triterpene sapogenin that contains isolated double bond, so the uv-absorbing of a medium tenacity is only arranged at the 205-220nm place, conforms to the practical situation of this product.
(6) nucleus magnetic resonance one peacekeeping two dimension hydrogen spectrum ( 1H-NMR, D 2The O exchange, 1H- 1HCOSY, DSNOE, NOESY and one dimension and two-dimentional TOCSY)
1. instrument model
Germany Brooker (Bruker) Avance of company 500 type NMR spectrometer with superconducting magnet, U.S.'s Varian (Varian) Inova of company 600 type NMR spectrometer with superconducting magnet.
2. condition determination
Solvent is DMSD-d 6, the explanation on the collection of illustrative plates of seeing Appendix of other condition.
3. reactive hydrogen replaces test
DMSO-d 6+D 2O
4. measurement result
See table 5 and Fig. 6 for details.
Figure A0315098600102
The one peacekeeping two dimension hydrogen spectrum data and the parsing of table 5 fern fiber crops glycosides trial-product
Figure A0315098600111
Table 5 (continuing)
Figure A0315098600121
Table 5 (continuing)
Figure A0315098600141
Table 5 (continuing)
Figure A0315098600151
Table 5 (continuing)
Figure A0315098600161
5. discuss and explanation
(1) by molecular formula (C as can be known 36H 58O 10H 2O): this product contains 60 protons altogether, and 7 CH are wherein arranged 3, 9-CH 2With 7-OH, all the other be all CH and=CH (totally 12), and water molecules.Hydrogen spectral integral result has 7-CH 3, 7 OH and 32 single proton peak, accumulative total totally 60 protons, after the heavy water exchange, 7 OH and a H 2The O signal all disappears or weakens, and showing has 9 active protons in the molecule.Its integration numerical value is 51 protons.Show that molecular formula conforms to practical situation.
(2) 5.19-5.15ppm place integration number is 5 protons in the trial-product collection of illustrative plates, wherein has the proton that a 12-alkene hydrogen and one sugar ring end group C-1 ' locates (H-1 ') to show that all the other 3 are the last three-OH proton (2 ', 3 ', 6 '-OH) of sugar ring.
(3) 5.16ppm place proton number is that 1 triplet (J=3.6Hz) is the alkene hydrogen proton signal on the C ring in the trial-product collection of illustrative plates. 1H- 1Bright its of H COSY stave has relevant with H-11ab and H-18 respectively; NOESY spectrum proof it have living space relevant with H-11, H-18, H-29 and 19-OH respectively.
(4) in the trial-product collection of illustrative plates 5.15ppm place proton number be 1 bimodally be 1 proton of sugar ring upper end hydrogen. 1H- 1Bright its H-2 ' of H COSY stave has coupling (J=8Hz) to show J H-1/H-2Be the coupling of aa key.NOESY spectrum proof its respectively with H-3 ', 5 ' and have living space relevant binding molecule model and stipulate that according to what distinguish that sugar encircles last α or β key it is H-1 ' α that this proton should be α (a) key of 4 ' and 2 '-OH, so glucoside should be O-β-D type.
(5) 4.40ppm place proton number is single broad peak D of 2 in the trial-product collection of illustrative plates 2O exchange postpeak disappears, and shows that this 2 proton should be the signal of 1 crystal water in the molecule. 1H- 1H COSY spectrum shows does not have relevant proton.
(6) 3.78ppm place proton number is a 19-OH signal on 1 the unimodal E ring in the trial-product collection of illustrative plates. 1H- 1H COSY spectrum show its with same ring on H-18 have relevant; H-12 alkene hydrogen proton on NOESY spectrum proof itself and the C ring, 16a proton on the D ring, each proton of H-21a, H-22a, H-29 and H-30 has living space relevant on the E ring.The binding molecule model can affirm that this 19-OH should be beta comfiguration, promptly should be 19 β-OH.In other words, then should be α-type, be 29 α-CH with 29 methyl on the C 3
(7) 3.58ppm place proton number is 1 in the trial-product collection of illustrative plates, and the bimodal of J=11.2Hz is 6 ' a proton signal of 6 ' primary alconol base on the sugar ring side chain; 1H- 1H COSY spectrum shows that it has relevant with H-6 ' a and H-5 '; The NOESY spectrum proves that itself and sugar encircle the H-2 ' on (F ring), 4 ', 5 ', 6 ' b and 4 '-OH, and 6 '-OH has living space relevant.
(8) 3.43ppm place proton number is 1 in the trial-product collection of illustrative plates, and the bimodal of J=10.8Hz is the proton signal of 6 ' b of primary alconol base on the sugar ring side chain, because the influence of 5 ' chirality C atom, obviously this methylene radical 6 '-CH 2On two protons be prochirality and non-equivalence.H-4 ' on the NOESY spectrum proof one F ring, 5 ', 6 ' a has living space relevant with 6 '-OH.
(9) 3.40ppm place proton number is 1 in the trial-product collection of illustrative plates, and the multiplet of J=10.5 and 4.2Hz is that A encircles 2 proton signals. 1H- 1H COSY spectrum show its respectively with same ring on H-1a, 1b has relevant with H-3; NOESY spectrum proof its respectively with last two the axial methyl H-24 of same ring and H-25 and axial 3-OH with have living space relevant with 2 '-OH on the carbon.The binding molecule model can judge that this proton should be axial bond, and promptly its configuration should be 2 β-H.In other words, it should be the α configuration with the 2-OH on the C and promptly should be 2 α-OH (calm).
(10) 3.31ppm place proton number is that single broad peak of 1 should be sugar ring (F ring) last 4 ' OH signal in the trial-product collection of illustrative plates, 1H- 1H COSY spectrum shows that it does not have relevant proton; The NOESY spectrum then proves upward H-2 ' of itself and same sugar ring, 4 ', and H-6 ' a, 6 ' b has living space relevant with 3 '-OH.Relevant explanation in (3) bar of binding molecule model and this section, this 4 '-OH should be axial β key, i.e. 4 ' β-OH.Then should be calm α key with it with the H-4 ' on the C, be 4 ' α-H.
(11) 3.17ppm place proton number is 1 in the trial-product collection of illustrative plates, and the multiplet of J=4.8 and 8.4Hz is the H-3 ' signal on the F sugar ring; 1H- 1H COSY spectrum show its with same ring on H-2 ' and two protons of H-4 ' have relevant; H-1 ' on NOESY spectrum proof itself and the same ring, H-4 ' has living space relevant with H-5 ' and 2 '-OH.Relevant explanation in binding molecule model and this section (3) bar, H-3 ' should be upright downward α key, and promptly 3 ' α-H then is 3 ' β-OH (calm) with 3 '-OH on the C.
(12) 3.11ppm place proton number is 1 in the trial-product collection of illustrative plates, and the multiplet of J=4.8 and 9.6Hz is a H-5 ' proton signal on the F ring; 1H- 1H COSY spectrum show its with same ring on H-4 ' relevant with H-6 ' ab proton; NOESY spectrum then proves H-1 ' on itself and the same ring, 3 ', and H-6 ' a, H6 ' b has living space relevant with 6 '-OH.Relevant explanation in binding molecule model and this section (3) and (9) bar: H-5 ' should be axial α key and is 5 ' α-H.
(13) 3.08ppm place proton number is 1 in the trial-product collection of illustrative plates, the double doublet of J=4.8 and 8.4Hz.Be H-4 ' proton signal on the sugar ring; 1H- 1H COSY spectrum show its with same ring on H-3 ' relevant with H-5 ' b; H-1 ' on NOESY spectrum proof itself and the same ring, 3 ' has living space relevant with 5 ' H.Relevant explanation in binding molecule model and this section (3) bar: H-4 ' should be calm α key, is 4 ' α-H.
(14) 3.06ppm place proton number is 1 in the trial-product collection of illustrative plates, and the double doublet of J=8.4 and 4.8Hz is a H-2 ' proton signal on the F ring; 1H- 1H COSY spectrum show its with same ring on H-1 ' and H-3 ' have relevant; NOESY spectrum then proves 3 '-OH on itself and the same ring, and 4 '-OH has living space relevant with H-6 ' ab.Relevant explanation in binding molecule model and this section (3) bar: H-2 ' should be axial β key, is 2 ' β-H.Then should be calm α key with it with 2 ' of C-OH, be 2 ' α-OH.
(15) 2.72ppm place proton number is 1 in the trial-product collection of illustrative plates, and J=9.6Hz's is bimodal, is 3 protons on the A ring; 1H- 1H COSY spectrum shows that it has relevant with H-2 on the same ring; The NOESY spectrum proves it and goes up H-1a with ring that 5,23 have living space relevant with 2-OH.The binding molecule model is confirmed therewith the proton relevant H-1a that has living space, and 5 is upright downward α key, and H-23 and 2-OH also be the α key, so the H-3 proton also should be upright downward α key, so and its should be calm β key with the 3-OH on the C, promptly should be 3 β-OH.
(16) 2.52ppm place proton number is 1 in the trial-product collection of illustrative plates, and the multiplet of J=9.6 and 3.6Hz is a 16a proton signal on the D ring, because 16-CH 2-be that the methylene radical of set collar is so two proton is the Δ δ that non-equivalence and the effect of anisotropy that is subjected to adjacent ester carbonyl group influence the two HValue is big (0.94ppm). 1H- 1H COSY spectrum show this proton respectively with same ring on H-15a, 15b and 16b have coupling relevant; The NOESY spectrum then proves H-15a, the 16b proton with ring, the 22a of E ring, and 26 methyl protons of b and C-8 all have living space relevant with it.
(17) in the trial-product collection of illustrative plates 2.36ppm place proton number be 1 unimodal for connecting 18 proton signals that D ring and E encircle. 1H- 1H COSY spectrum shows H-16b on its D ring and the 22b on the E ring, and 12 alkene hydrogen protons on the C ring and 19-OH have coupling to be correlated with; NOESY spectrum then proves the H-12 of C ring, H-15b, H-27 on the D ring, and protons such as the H-20 on the E ring, H-22a and H-29 have living space relevant with it; Binding molecule model validation wherein H-15a, H-20, H-22a and H-27 and H-29 etc. is all upright downward α key, so proton (H-18) then also should promptly should be 18 α-H for upright downward α key.
(18) 1.88ppm place proton number is 2 in the trial-product collection of illustrative plates, and the broad peak of J=9.6Hz is two protons of H-11 methylene radical on the C ring.Be subjected to the influence of 9 adjacent chirality C, this methylene radical should have certain prochirality, the δ value of two protons should have certain pass different, so its signal is being differentiated when better or is being carried out necessary expansion and should be able to observe it and split the branch situation, be that a broad peak also has the branch of splitting slightly herein, also can find out two differences between proton.Also measure its J=9Hz on the 1600MHz collection of illustrative plates. 1H- 1H COSY spectrum shows itself and the H-9 and the H-11a that examine together, has coupling relevant between b; The H-1a of NOESY spectrum proof A ring, b, the H-25 on the B ring, 26 all has living space relevant with it with the H-9 on the ring with H-12.Wherein H-1a and H-9 binding molecule model know that it is upright downward α key, and H-25,26 be upright β key, obviously two protons (H-11a and H-11b) on this methylene radical the two be not of equal value fully with have identical configuration.
(19) 1.78ppm place proton number is 1 in the trial-product collection of illustrative plates, and the wide double doublet of J=10.6 and 4.2Hz is a upright downward H-1a proton on the A ring, owing to be subjected to the influence of H-2 chirality.A ring has different (the Δ δ value=1.03ppm) of bigger δ value difference for die two prochirality protons on 1 methylene radical of fixed stiffening ring in addition. 1H- 1H COSY spectrum shows it and upward H-1b and H-2 have coupling relevant with ring; NOESY spectrum then proof with H-1b, H-3 on the ring, 5, H-23 all has living space relevant with it with H-9 on the C ring with H-11a.Binding molecule model H-3, H-5, H-9 and H-23 are all upright downward α key, promptly should be H-1 α so proton obviously also should be upright downward α key.
(20) 1.75ppm place proton number is that the multiplet of 1 J=13.2 and 4.8Hz is the H-15a proton signal on the D ring in the trial-product collection of illustrative plates. 1H- 1H COSY spectrum show its with H-15b, H-16a on the ring, b has coupling relevant with H-26 on the C ring; NOESY spectrum proof H-7a, H-11, H-15b, 16b have coupling relevant with H-26 on the C ring, and NOESY composes proves that H-7a, H-11, H-5b, 16b have living space relevant with H-26,27.
(21) 1.65ppm place proton number is that the wide multiplet of 1 J=12.0Hz is a H-22a proton signal on the E ring in the trial-product collection of illustrative plates; 1H- 1H COSY spectrum shows that it has coupling relevant with H-22b with it with the H-21a on the ring; The NOESY spectrum then proves the H-16a on the D ring, and b, H-18 and H-27 go up H-20 with ring, and 21b, 22b all have living space relevant with it.Because E ring is the fixed stiffening ring, influenced by the effect of anisotropy of the two keys of adjacent ester carbonyl group C=O, two proton H-22a and H-22b on this methylene radical also have bigger Δ δ HValue (0.16ppm), obviously H-22a is that upright hydrogen H-22b is open and flat hydrogen.
(22) 1.62ppm place proton number is that 1 J value is 9.6 and the multiplet of 10.0Hz in the trial-product collection of illustrative plates, is H-21a proton signal on the E ring.Be subjected to the influence of 20 adjacent chirality C, this methylene radical also has prochirality, is on set collar in addition, so two proton H-21a and H-21b have bigger Δ δ HValue (0.48ppm). 1H- 1H COSY spectrum shows it and has coupling relevant with H-20, H-21b and H-22a on the ring; H-16a on the NOESY spectrum proof D ring, b and H-21b, H-22b, H-30 and 19-OH on the E ring all have living space relevant, and binding molecule model, H-30 and 19-OH are all the β key and are 21 β-H so proton also should be axial β key.Obviously H-21b should be 21 α-H.
(23) 1.60ppm place proton number is that the triplet of 1 J=9.6Hz should be H-9 signal on the C ring in the trial-product collection of illustrative plates. 1H- 1H COSY spectrum shows it and encircles adjacent H-11a together that two protons of b have coupling relevant; NOESY composes proof, H-1a, the H-5 of A ring; The H-7a of B ring, with the H-11b on the ring and H27 all with its spatial correlation.Binding molecule model validation H-1a, H-5, H-7a, H-27 are all upright downward α key, and obviously H-9 also should be upright downward α key and promptly should be H-9 α.
(24) 1.58ppm place proton number is the signal that the multiplet of 1 J=3.6 and 12.0Hz should be H-16b proton on the D ring in the trial-product collection of illustrative plates.In this section (15), point out: because on the set collar and to be subjected to two the proton H-16a and the H-16b that influence on this methylene radical of each and the opposite effect of adjacent ester carbonyl group C=O be non-equivalence, its Δ δ HValue is big (0.94ppm). 1H- 1H COSY spectrum shows it and goes up H-15a with ring that b, H-16a have coupling relevant with H-18; The NOESY spectrum then proves H-15a, and b, H-16a have living space relevant with H-21a with it.
(25) 1.49ppm place proton number is that 1 J=6.6 and the multiplet of 12.6Hz are H-22b proton signal on the E ring in the trial-product collection of illustrative plates.Pointed out in this section (21) because E ring is that set collar is influenced by the effect of anisotropy of adjacent ester carbonyl group C=O in addition, two protons on 22 methylene radical be non-equivalence and Δ δ HValue has certain difference (0.16ppm). 1H- 1HCOSY spectrum show its with same ring on H-18, H-21a and H-22a have coupling relevant.The NOESY spectrum then proves H-16a, and 21ab has living space relevant with 22a with it.
(26) 1.47ppm place proton number is that 1 J=6.6 and the multiplet of 8.4Hz are the signal of H-7a on the B ring in the trial-product collection of illustrative plates.Two proton H-7a of methylene radical and H-7b also are non-equivalence because B ring also is a set collar.Its Δ δ HValue=0.32ppm. 1H- 1H-5 on demonstration of H COSY spectrum and the same ring, 6ab has coupling relevant with H-7; NOESY spectrum proof is all had living space relevant with it with the H-27 on H-5, H-6b, H-9 and the C ring on the ring, binding molecule model H-5, H-9 and H-27 are all upright downward α key, so H-7a also should be downward axial α key, promptly the obvious H-7b of H-7 α should be H-7 β.
(27) 1.43ppm place proton number is that 1 J=6.5 and the multiplet of 5.5Hz should be H-6a proton signal on the B ring in the trial-product collection of illustrative plates.Because 6 methylene radical are that C-5 adjacent in addition on fixed B ring is chirality C, so methylene radical also is a prochirality, two proton H-6a and H-6b are non-equivalence, its Δ δ H=0.13ppm. 1H- 1H COSY spectrum show its with same ring on H-5 and H-6b and H-7a, b has coupling to be correlated with; H-24 on the NOESY spectrum proof A ring, 25 and same ring on H-6b, 7b has living space relevant with H-26.Binding molecule model H-24,25 and 26 are all upright β key, so H-6a also should be upright β key, i.e. H-6 β.Obviously H-6b then should be calm H-6 α.
(28) 1.30ppm place proton number is that 1 multiplet is the signal of H-6b proton on the B ring in the trial-product collection of illustrative plates.Prosthomere has pointed out that 6 H-6a and H-6b on the methylene radical are the non-equivalence proton, its Δ δ H=0.13ppm. 1H- 1H COSY spectrum shows itself and the H-5 and H-6a and the H-7a that encircle together, and b has coupling relevant; NOESY spectrum then proves except that above several protons, also with the A ring on H-23 have living space relevant.Should be calm H-6 α as pointed this proton of prosthomere.
(29) the proton number H-27 methyl signals that to be 3 the unimodal C of being link to each other with C-14 with D ring junction in 1.27ppm place in the trial-product collection of illustrative plates. 1H- 1H COSY spectrum show its with the D ring on 15 methylene radical on two proton H-15a, b has coupling to be correlated with; H-7a and H-9 proton on the NOESY spectrum proof B ring, H-15b, H-16a on the D ring, b and H-18 proton, the H-22a proton on the E ring all has living space relevant with it with an OH proton on sugared the ring.Binding molecule model validation H-7a, H-9, H-18 and H-22a are all upright downward α key, so methyl also should be upright downward α key, are 27 α-CH 3
(30) 1.21ppm place proton number is that 1 J=9.0 and the multiplet of 9.6Hz are the signal of H-20 proton on the E ring in the trial-product collection of illustrative plates; 1H- 1H COSY spectrum shows it and goes up H-21a with ring that b has coupling relevant with H-30; Upward H-18, H-21b, H-22a have living space relevant with H-30 NOESY spectrum proof with ring.Binding molecule model and relevant data can confirm that H-18, H-22a and H-29, H-30 are all upright downward or calm α key, are H-20 α so proton obviously also should be the α key, and obviously the H-30 methyl on the C same with it should be the β key, i.e. 30 β-CH 3
(31) 1.15ppm place proton number is that the multiplet of 1 J=6.5Hz should be H-7b proton signal on the B ring in the trial-product collection of illustrative plates.As described in (27) bar in this section, because this methylene radical is on fixed B ring, so H-7a and H-7b are the proton (Δ δ H=0.32ppm) of two non-equivalences. 1H- 1H COSY spectrum shows it and goes up H-6a with ring, and b and H-7a have with nuclear phase and close; NOESY spectrum confirms and goes up H-6a with ring, and H-15b and H-27 have living space relevantly on b, H-7 and H-26 and the D ring, and preceding this proton of having pointed out is the β key, i.e. 7 β-H.
(32) 1.14ppm place proton number is that 1 J=6.0 and the multiplet of 10.2Hz are H-21b proton signal on the E ring in the trial-product collection of illustrative plates.As described in this section (23) bar, because the influence of 20 ' chirality C and set collar, two protons (H-21a and H-21b) on this methylene radical have bigger Δ δ HValue (0.48ppm). 1H- 1H COSY spectrum shows it and has coupling relevant with H-20, H-21a and H-22ab on the ring; NOESY spectrum proof goes up except that above-mentioned several protons with ring, also has living space relevant with H-30.Before pointed out that this proton is H-21 α for the α key.
(33) in the trial-product collection of illustrative plates 1.08ppm place proton number be 3 unimodal be 29-CH in the E ring 3Signal. 1H- 1H COSY spectrum shows that it does not have relevant proton; NOESY composes proof: C and encircles 12 alkene hydrogen protons; D encircles 18 protons; H-20, H-30 all have living space relevant with it with 19-OH on the E ring; Each relevant data of binding molecule model and front can confirm that H-18, H-20 are all downward axial α key, and the 19-OH on the same C has proved that it is upright β key, is 29 α-CH so methyl should be the α key 3
(34) 0.98ppm place proton number is single broad peak of 1 in the trial-product collection of illustrative plates, is 2-OH proton signal on the A ring. 1H- 1H COSY spectrum shows that it does not have relevant proton; The NOESY spectrum confirms that H-1b, H-2, H-3 have living space relevant with 3-OH on its same ring.Binding molecule model and front relevant data confirmed that H-3 is upright downward α key, and H-2 go up for C same with it and have confirmed as upright β key, so 2-OH should be calm α key, promptly should be 2 α-OH.
(35) in the trial-product collection of illustrative plates 0.91ppm place proton number be 3 unimodal, should be A and encircle 23 methyl proton signals. 1H- 1H COSY spectrum shows that it does not have relevant proton; NOESY spectrum proves that then H-5 and H-6b and its have living space relevant binding molecule model and the front relevant data on H-3, H-24 on the same ring, 3-OH and the B ring must confirm that H-3 and H-5 are that uprightly downward α key, H-6b also is α key (calm), so the H-23 methyl also should be the α key, is 23 α-CH 3
(36) in the trial-product collection of illustrative plates 0.89ppm place proton number be 3 unimodally be H-24 methyl signals on the A ring. 1H- 1H COSY spectrum shows that it does not have relevant proton; NOESY composes proof: the H-2 on the A ring, H-23, and 25, the H-6a on the 3-OH B ring, b all has living space relevant with it with H-26.Binding molecule model and aforementioned relevant data have confirmed that H-2, H-6a, H-25, H-26 are upright β key, and 3-OH is calm β key, and H-23 is and its calm α key with C, so methyl also must be upright β key, i.e. 24 β-CH 3
(37) 0.84ppm place proton number is that 3 J=9.0Hz bimodal should be on the E ring and the 30-CH of 20 α-H on same C in the trial-product collection of illustrative plates 3Signal. 1H- 1H COSY spectrum shows that it has coupling relevant with H-20; NOESY composes proof: with H-20, the 21a on the ring, and b, 29 all have living space relevant with it with 19-OH.Binding molecule model and aforesaid each relevant data have confirmed that H-21a, 19-OH are upright β key, H-29 and H-21b then are the α key quite approaching with its space, H-20 then is and its upright downward α key with C certainly, so think that this methyl is calm β key certainly, promptly should be 30 β-CH 3
(38) 0.78ppm place proton number is that single broad peak of 1 is the 3-OH signal on the A ring in the trial-product collection of illustrative plates. 1H- 1H COSY spectrum shows that it does not have relevant coupling proton, but NOESY spectrum proof, with H-2, the H-23 on the ring, 24,25 with the B ring on H-26 all have living space relevant with it.The binding molecule model determines that wherein H-2, H-24, H-25 and H-26 are all upright β key, has determined that it is axial α key with its H-3 on same C in addition, so 3-OH should be calm β key, is 3 β-OH (e).
(39) 0.75ppm place proton number is that 1 J=12.0 and the multiplet of 5.4Hz should be the signals of H-1b proton on the A ring in the trial-product collection of illustrative plates.Pointing out in this section (18) bar: owing to be subjected to the influence of 2 adjacent chirality C, 1 methylene radical is a prochirality, and the A ring so the non-equivalence of H-1a and H-1b has sizable difference, is added adjacent 25-CH for set collar in addition 3And 24-CH 3Steric influence, make the two Δ δ HValue is up to 1.03ppm. 1H- 1The HCOSY spectrum shows the H-1a on this proton and the same ring, and H-2 has coupling relevant with H-25; NOESY composes proof: the H-9 on the same ring on H-1a, H-25,2-OH and the C ring all has living space relevant with it with H-11b.Each relevant data of binding molecule model and front has assert that H-9, H-11b and 2-OH are axial or calm α key.Although H-2, H-25 are upright β key, its locus is then quite approaching with this proton, and H-1a so proton should be calm β key, is 1 β-H then for to have determined and its upright downward α key proton on same C.
(40) 0.73ppm place proton number is 1 J=5.4 and the multiplet of 6.6Hz in the trial-product collection of illustrative plates, should be the H-5 proton signal of A and B two ring junctions. 1H- 1H COSY spectrum show its respectively with A ring on H-23 and the H-6a on the B ring, b has coupling to be correlated with; NOESY spectrum is proof then: the H-1a on the A ring, H-3 and H-23, the H-6b on the B ring, H-7a, the H-9 relation of all having living space with it.Binding molecule model and aforementioned each related data, more than each proton all confirmed as or upright (H-1a, H-3a, H-7a and H-9) or calm (H-23 and H-6b) α key downwards, so proton can only be upright downward α key, promptly just be 5 α-H.
(41) in the trial-product collection of illustrative plates 0.70ppm place proton number be 3 unimodally be the 25-CH that links to each other with A, B junction C-10 3Signal. 1H- 1H COSY spectrum shows that it has coupling relevant with H-1b on the A ring.NOESY composes proof: the H-1b on the A ring, H-2, H-24 and 3-OH, the H-6a on the B ring, H-2b all have living space relevant with it with H-11a on the C ring.Binding molecule model and aforesaid each relevant data have confirmed that H-2, H-6a, H-11a, H-24 and H-26 are upright β key, and H-1b and 3-OH are calm β key, so methyl also must be upright β key, are 25 β-CH 3
(42) 0.66ppm place proton number is 3 unimodal 26-CH for linking to each other with 8 C on the B ring in the trial-product collection of illustrative plates 3 1H- 1H COSY spectrum shows no hydrogen related; H-6a on the same ring of NOESY spectrum proof, 7b, the H-24 on the A ring, 25, the H-11 on the C ring, 12, the H-15a on the D ring, b all have living space relevant with it.Each relevant data of binding molecule model and front has been confirmed in above-mentioned each spatial correlation proton, H-24, and 25, H-6a, 11a, 15a wait to be all upright β key, and H-7b, H-12 and H-15b are non-axial β key or the α keys close with its space.So can affirm this methyl should be upright β key, promptly should be 26 β-CH 3
(43) results verification of the two-dimentional TOCSY of this product spectrum A, B, C, D is in the dependency between each proton in the same big spin system in six kinds of rings of E and F.That is: the H-1a during A encircles, H-1b, H-2, H-3, H-23, H-24, H-5 etc.; H-5 in the B ring, H-6a, b, H-7a, b, H-9, H-25, H-26 etc.; H-9 in the C ring, H-11a, b, H-12, H-27 etc., the H-15a in the D ring, b, H-16a, b and H-18 etc.; H-18 in the E ring, H-19 (OH), H-29, H-30, H-20, H-21a, b, H-22a, b etc., the result of this result and one dimension hydrogen spectrum and 1H-1HCOSY spectrum matches.
(44) result of the one dimension TOCSY of this product has confirmed that the ownership of sugared ring (β ring) is correct, they (H-1 ', 2 ', 3 ', 4 ', 5 ', 6 ' a is b) in same big spin system.
(45) the one dimension NOE result of this product also conforms to 2D NOESY result.
Conclusion: in sum, the molecular structure of this product should be:
Figure A0315098600251
Therefore compound is a new compound that is obtained by extraction in the Chinese medicine fern fiber crops, separation, purifying first, so it is fern fiber crops glycosides for name, wherein cultural formal name used at school can be decided to be: 2 α, 3 β, 19 β-trihydroxy--12-alkene-ursane-28-carboxylic acid-28-O-β-D-galactopyranose glucoside; But or called after: 2 α, 3 β, 19 β-trihydroxy--12-black bearberry-28-acid-28-O-β-D-galactopyranose glucoside.
This result has also obtained: the relevant information (seeing the related content of each several part in this data for details) of other relevant spectrum (as carbon spectrum, mass spectrum etc.) in (1) this data; (2) analysis-by-synthesis in this data (seeing aftermentioned for details); (3) the seemingly strong supports such as relevant data of structural compounds of known class among the document *.
Therefore this structure is identified and should be conformed to actual.
* attached: the related data in the relevant documents and materials.
1. document I:Acta Pharmacentica Sinica 1996,31 (11): 844-848
(i) hydrogen of compd A spectrum data.(2 α, 3 β, 19 β-trihydroxy--ursane-12-alkene-28-carboxylic acid-28-O-β-D-glucopyranoside, C 36H 58O 10):
1H-NMR (500MHz, CD 3COCD 3, δ ppm): 5.36 (1H, d, J=8.1Hz, the end group H on the sugar, H-1 '), 5.28 (1H, brs, H-12), 3.75-3.22 (6H, m, the H on the sugar), 3.62 (1H, m, H-2), 2.90 (1H, d, J=9.5Hz, H-3), 2.52 (1H, s, H-18), 1.31,1.17,0.98,0.97,0.78,0.76 (each 3H, s, 6 * CH 3), 0.91 (3H, d, J=6.5Hz, 30-CH 3).
(ii) the hydrogen of compd B is composed data (2 α, 3 α, 19 β-trihydroxy--ursane-12-alkene-28-carboxylic acid-28-O-β-D-glucopyranoside, C 36H 58O 10):
1H-NMR (500MHz, DMSO-d 6, δ ppm): 5.17 (1H, brs, H-12), 5.14 (1H, d, J=8.1Hz, the terminal hydrogen of sugar, H-1 '), 3.42 (1H, m, H-2), 3.9-3.05 (m, the H on the sugar, H-2 ', 3 ', 4,5 ' and 6 '), 3.13 (1H, brs, H-3), 2.35 (1H, s, H-18), 1.28,1.07,0.87,0.76,0.64 (each 3H, s, 6 * CH 3), 0.83 (3H, d, J=6.5Hz, 30-CH 3).
(iii) the hydrogen of Compound C is composed data (2 α, 3 β, 19 β-trihydroxy--ursane-12-alkene-28-carboxylic acid, C 30H 48O 5):
1H-NMR (500MHz, DMSO-d 6, δ ppm): 5.16 (1H, s, H-12), 3.40 (1H, m, H-2), 2.73 (1H, d, J=9.3Hz, H-3), 2.35 (1H, s, H-18), 1.28,1.11,0.91, O.70,0.69 (each 3H, s, 6 * CH 3), 0.83 (3H, d, J=4.5Hz, 30-CH 3).
2. document II: CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998 the 23rd volume the 1st phase P37-38:
(iv) the hydrogen of Compound D is composed data (2 α, 3 α, 19 α-trihydroxy--12-ursane-28-carboxylic, C 30H 48O 5):
1H-NMR (400MHz, C 5D 5N, 6ppm): 5.57 (1H, brs, H-12), 4.29 (1H, brs, J=11Hz, 2 β-H), 3.75 (1H, brs, 3 β-H), 3.02 (1H, brs, H-18), 1.45,1.28,1.10 (each 3H, s, 3 * Me), 1.10 (3H, d, J=7.5Hz, Me), 0.96,0.88,0.80 (each 3H, s, 3 * Me).
8. document III: Botany Gazette, 1988,30 (4): 409-413:
(v) the hydrogen of compd E is composed data (2 α, 3 α, 19 α-trihydroxy-black bearberry-12-alkene-28-carboxylic acid-28-O-β-D-glucopyanoside, C 36H 58O 102H 2O):
1H-NMR (100MHz,
Figure A0315098600261
δ ppm): 5.55 (1H, d, J=8Hz, C 1 '-H), 5.40 (1H, m, C 12-H), and 4.60-3.24 (m, on the sugar ring-OH), 4.28 (1H, m, C 2-H), 3.77 (1H, d, J=3Hz, C 3-H), 3.06 (1H, s, C 19-H), 2.74 (1H, s, C 18-H), 1.67 (3H, s, C 19-CH 3), 1.46 (3H, s, C 29-CH 3), 1.44 (3H, s, C 27-CH 3), 1.29 (3H, s, C 25-CH 3), 1.15 (3H, d, J=7Hz, C 30-CH 3), 1.13 (3H, s, C 24-CH 3), 1.01 (3H, s, C 23-CH 3), 0.93 (3H, s, C 26-CH 3).
(vi) the hydrogen of compound F 17-hydroxy-corticosterone spectrum data (2 α, 3 β, 19 α-trihydroxy--black bearberry-12-alkene-28-carboxylic acid-28-O-β-D-glucopyanoside,
Figure A0315098600262
1H-NMR (100MHz, C 5D 5N, 6ppm): 5.52 (1H, d, J=8Hz, C 1 '-H), 5.50 (1H, m, C 12-H), and 3.90-3.24 (m, on the sugar ring-OH), 4.76 (1H, d, J=10Hz, C 2-H), 4.27,4.02 (2H, m, C 6-H), 3.80 (1H, m, C 5-H), 2.89 (1H, s, C 18-H), 1.62 (3H, s, C 29-CH 3), 1.42 (3H, s, C 27-CH 3), 1.22 (3H, s, C 25-CH 3), 1.10 (3H, d, J=7Hz, C 30-CH 3), 1.06 (6H, s, C 23, C 24-2 * CH 3), 1.01 (3H, s, C 26-CH 3).
4. document IV: Acta Pharmaceutica Sinica, 18 (4): 314-316,1983:
(the vii) hydrogen of compound G spectrum data (2 α, 3 α, 19 α-trihydroxy--ursolic acid-(28-1)-β-D-glucose ester, C 36H 58O 10):
1H-NMR (JEDL FX-60Q, CDCl 3, δ ppm): 5.32 (1H, m, C 12-H), 5.12 (1H, t, J=10 and 4.5Hz, C 2-H), 2.90 (1H, d, J=9.6Hz, C 3-H), 2.51 (1H, s, C 18-H), 1.33 (3H, s), 1.20 (3H, s), 1.00 (6H, s), 0.87 (3H, s), 0.80 (3H, s), 0.77 (3H, s).
(7) nucleus magnetic resonance one peacekeeping two dimension carbon spectrum ( 13C-NMR:PBB, DEPT, HMQC and HMBC)
1. instrument model
Germany Brooker (Bruker) the Avance 125MHz of company NMR spectrometer with superconducting magnet; U.S.'s Varian (Varian) Inova 150MHz of company NMR spectrometer with superconducting magnet.
2. condition determination
Solvent is DMSO-D 6, the explanation on the collection of illustrative plates of seeing Appendix of other condition.
3. measurement result
See table 6 for details.
Table 6 fern fiber crops glycosides trial-product nuclear-magnetism one peacekeeping two dimension carbon spectrum data and parsing
Figure A0315098600272
Table 6 (continuing)
Figure A0315098600291
Table 6 (continuing)
4. discuss and explanation
(1) by trial-product molecular formula C 36H 58O 10H 2O this product as can be known contains 36 C atoms altogether, and carbon is composed in the spectrum (PBB) of uncoupling entirely 36 spectral lines, illustrates that 36 C all are non-equivalence, conform to practical situation.
(2) as can be known, this product has 7 uncle C, 9 secondary C, 11 uncle C (in 1 alkene uncle C is arranged) and 9 season C (in an ester carbonyl group is arranged) by the trial-product structural formula.The result of DEPT spectrum conforms to practical situation.
(3) the 175.53ppm peak is the signal of 28 ester carbonyl group C atoms in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C; The HMQC spectrum does not find that any C therewith has directly related proton; Anomeric proton H-1 ' on the HMBC spectrum proof sugar ring, the H-18 on the E ring, H-22a, b, H-16a, protons such as b have long-range heteronuclear relevant with it, and the result conforms to actual.
(4) the 138.18ppm peak is a C-13 alkene C signal in the C ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C; Do not find proton any and its directly related company in the HMQC spectrum; H-11,12 on the HMBC spectrum proof C ring, 26 with 27 with the D ring on H-18 have long-range heteronuclear relevant with it.
(5) the 126.94ppm peak is the alkene C signal of C-12 in the C ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle's alkene C; HMQC spectrum find itself and hydrogen compose in 5.16ppm (H-12) signal correction; Two protons of H-18 have long-range heteronuclear relevant with it on H-11 on the HMBC spectrum proof C ring and the D ring.
(6) the 94.05ppm peak is that sugar ring is gone up end group C (C-1 ') signal in the trial-product collection of illustrative plates; The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum find itself and hydrogen compose in 5.15ppm (H-1 ') signal correction; H-2 ' proton on the HMBC spectrum proof sugar ring has long-range heteronuclear relevant with it.
(7) the 82.31ppm peak is a C-3 signal on the A ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum find its with the hydrogen spectrum in 2.72ppm (H-3) signal correction, HMBC spectrum proof A ring (with ring) middle H-2,3-OH, H-5, H-23 all have heteronuclear long-range relevant with it with H-24.
(8) the 77.58ppm peak is that the sugar ring is gone up C-5 ' signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum find itself and hydrogen compose in 3.11ppm (H-5 ') signal correction; It goes up H-4 ' with the sugar ring HMBC spectrum proof, 6 ' a, and 6 ' b has heteronuclear long-range relevant with H-1 '.
(9) the 76.70ppm peak is that the sugar ring is gone up C-3 ' signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum find itself and hydrogen compose in 3.17ppm (H-3 ') signal correction; It goes up H-5 ' with the sugar ring HMBC spectrum proof, and each proton of H-4 ' and H-2 ' has heteronuclear long-range relevant.
(10) the 72.24ppm peak is that the sugar ring is gone up C-2 ' signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum confirm itself and hydrogen compose in 3.06ppm (H-2 ') signal correction; The HMBC spectrum shows that it goes up H-1 ' with the sugar ring and two protons of H-3 ' have heteronuclear long-range relevant.
(11) the 71.65ppm peak is a C-19 signal on the E ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C; The HMQC spectrum is not found the proton directly related with it; The HMBC spectrum confirms it and has heteronuclear long-range relevant with encircling to go up between H-18, H-29 and H-30 and each proton of 19-OH.
(12) the 69.51ppm peak is that the sugar ring is gone up C-4 ' signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C; HMQC spectrum find itself and hydrogen compose in 3.08ppm (H-4 ') signal correction; The HMBC spectrum proves that it goes up H-3 ' with the sugar ring and H-5 ' proton has heteronuclear long-range relevant.
(13) the 67.13ppm peak is a C-2 signal on the A ring in the trial-product collection of illustrative plates.It is uncle C for the identification of DEPT spectrum.The HMQC spectrum shows that it is relevant with the multiplet (H-2) of 3.40ppm in the hydrogen spectrum.The HMBC spectrum confirms that each proton such as H-1b, H-3, H-5, H-23 and 3-OH have heteronuclear long-range relevant with it on the same ring.
(14) the 60.64ppm peak is a C-6 ' signal on the sugar ring side chain in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is secondary C.3.58 is relevant with two groups of signals of 3.43ppm (H-6 ' a and H-6 ' b) during HMQC spectrum was found to confirm this signal and hydrogen is composed.H-5 ' on the HMBC spectrum proof sugar ring has long-range heteronuclear relevant with H-4 ' with it.
(15) the 54.82ppm peak is the C-5 signal of A ring and B ring connecting place in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C.The HMQC spectrum shows that it is relevant with 0.73ppm signal (H-5) in the hydrogen spectrum.H-23,24 on the HMBC spectrum proof A ring, the H-6b on the B ring has heteronuclear long-range relevant with all protons of H-7a and its.
(16) the 53.15ppm peak is the C-18 signal of D ring and E ring junction in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C.HMQC spectrum show itself and hydrogen compose in 2.36ppm (H-18) signal correction.HMBC spectrum confirms H-12, H-20, the H-21a on the E ring on the C ring, and protons such as b, H-22a and H-29 all have heteronuclear long-range relevant with it.
(17) the 47.33ppm peak is another connecting place C-17 signal of D ring and E ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C.Bright its of HMQC stave do not have relevant proton.H-21a has heteronuclear long-range relevant with it respectively with all protons of 22ab on the H-16a on the HMBC spectrum proof D ring, b, H-18 and E ring.
(18) the 47.09ppm peak is a C-1 signal on the A ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is secondary C.1.78ppm (H-1a) and two groups of signal corrections of 0.75ppm (H-1b) during bright its of HMQC stave composed with hydrogen respectively.Protons such as H-9, H-25 have heteronuclear long-range relevant with it on H-2, H-3, H-5 and the B ring on the HMBC spectrum proof A ring.
(19) the 46.71ppm peak is a B ring and C ring both junction C-9 signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C.1.60ppm (H-9) signal correction in bright itself and the hydrogen spectrum of HMQC stave.H-1, H-7a, the H-25 on the H-5 B ring on the HMBC spectrum proof A ring, each proton of H-11 on the 26 C rings all has long-range heteronuclear relevant with it.
(20) the 41.18ppm peak is a C-20 signal on the E ring in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is uncle C.The HMQC spectrum finds to show that itself and hydrogen spectrum go up 1.21ppm (H-20) signal correction; H-18,21,29,30 all has long-range heteronuclear relevant with it with 19-OH on the same ring of HMBC spectrum proof.
(21) the 41.10ppm peak is C ring and D ring junction C-14 signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C.Bright its of HMQC stave do not have directly related proton.H-9, H-12 and H-26 on the HMBC spectrum proof C ring, the H-15a on the D ring, b, 16a, b all has long-range heteronuclear relevant with it with all protons of H-27.
(22) the 40.00ppm peak is C-4 signal on the A ring for (this peak and solvent peak have overlapping slightly) in the trial-product collection of illustrative plates.The DEPT spectrum shows that it is season C.Bright its no directly related proton of HMQC stave.HMBC spectrum proof is with H-3, H-23 on the ring, 24,25 with the B ring on protons such as H-9, H-5 all have long-range heteronuclear relevant with it.
(23) the 38.88ppm peak is A ring and B ring connecting place C-10 signal in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C; Bright its of HMQC stave do not have directly related proton; H-1a on the HMBC spectrum proof A ring, b, H-23,24, the H-5 on the B ring, H-9 all has heteronuclear long-range relevant with it with all protons of H-25.
(24) the 37.53ppm peak is the C-8 signal of connection B ring and C ring both in the trial-product collection of illustrative plates.The DEPT spectrum is confirmed that it is season C; The HMQC spectrum shows its no directly related proton; H-1,5 on the HMBC spectrum proof A ring, the H-6 on the B ring, 7,9,26, the H-15 on the C ring all has heteronuclear long-range relevant with it with 27 all protons.
(25) the 36.55ppm peak is a C-22 signal on the E ring in the trial-product collection of illustrative plates.The DEPT spectrum shows that it is secondary C; Bright this signal of HMQC stave respectively with hydrogen spectrum in 1.65ppm (H-22a) and two groups of proton signals of 1.49ppm (H-22b) have directly related; H-15ab, H-16ab on the HMBC spectrum proof D ring all has long-range heteronuclear coupling relevant with it with the H-20,21 on the E ring with 30 all protons.
(26) the 32.50ppm peak is a C-7 signal on the B ring in the trial-product collection of illustrative plates.Bright its of DEPT stave is secondary C.1.47ppm (H-7a) and two groups of proton signals of 1.15ppm (H-7b) had directly related during the HMQC spectrum showed it and hydrogen is composed.H-5, H-6, H-9 and H-26 proton on the same ring of HMBC spectrum proof all have long-range heteronuclear relevant with it.
(27) the 28.77ppm peak is a C-23 signal on the A ring in the trial-product collection of illustrative plates.The DEPT spectrum shows that it is uncle C.The HMQC stave bright its with hydrogen spectrum in the 0.91ppm proton signal have directly related; H-3, H-5, each proton of H-24 and 3-OH all have heteronuclear long-range relevant with it on the same ring of HMBC spectrum proof.
(28) the 28.00ppm peak is a C-15 signal on the C ring in the trial-product collection of illustrative plates.The DEPT spectrum shows that it is secondary C.Bright its of HMQC stave has directly related with 1.75ppm (H-15a) and two groups of proton peak of 0.87ppm (H-15b) on the hydrogen spectrum.HMBC spectrum proof is with the H-16a on the ring, and b, H-18 all have long-range heteronuclear relevant with it with all protons of H-27.
(29) the 26.37ppm peak is a C-29 signal on the E ring in the trial-product collection of illustrative plates.Bright its of DEPT stave is uncle C.During HMQC spectrum shows it and hydrogen is composed 108ppm (H-29) unimodal have directly related; Each proton of H-18,20,21ab and 19-OH on the same ring of HMBC spectrum proof all has long-range heteronuclear relevant with it.
(30) the 25.77ppm peak is a C-21 signal on the E ring in the trial-product collection of illustrative plates, and bright its of DEPT stave is secondary C.1.62ppm (H-21a) and two groups of proton signals of 1.14ppm (H-21b) had directly related during the HMQC spectrum showed it and hydrogen is composed.H-18,20,22,29 on the same ring of HMBC spectrum proof all has long-range heteronuclear relevant with it with 30 each proton.
(31) the 25.08ppm peak is a C-16 signal on the D ring in the trial-product collection of illustrative plates.Bright its of DEPT stave is secondary C.2.52ppm (H-16a) and two groups of proton signals of 1.58ppm (H-16b) had directly related during the HMQC spectrum showed it and hydrogen is composed.Each proton of H-22ab all has long-range heteronuclear relevant with it on H-15ab, H-18 on the HMBC spectrum proof D ring and the E ring.
(32) the 23.82ppm peak is the methyl (C-27) on the C-14 that connects C and D ring in the trial-product collection of illustrative plates; The DEPT spectrum shows that it is uncle C.The HMQC stave bright its with hydrogen spectrum in 1.27ppm (H-27) signal have directly related.H-7ab on the HMBC spectrum proof B ring, the H-15a on the D ring, protons such as b and H-18 have long-range relevant with it.
(33) the 23.22ppm peak is a C-11 signal on the C ring in the trial-product collection of illustrative plates; The DEPT spectrum shows that it is secondary C.The HMQC stave bright its with hydrogen spectrum in 1.88ppm (H-11ab) have directly related.H-7a on the HMBC spectrum proof B ring, b has heteronuclear long-range relevant with 26 with it with the H-9,12 on the ring.
(34) the 18.09ppm peak is a C-6 signal on the B ring in the trial-product collection of illustrative plates.The DEPT spectrum shows that it is secondary C.The HMQC stave bright its with hydrogen spectrum in 1.43ppm (H-6a) and two proton signals of 1.30ppm (H-6b) have directly related.H-1 on the HMBC spectrum proof A ring, 3,5,23 and the B ring on H-7ab, H-26 all has heteronuclear long-range relevant with it.
(35) the 17.09ppm peak is that A ring and B encircle methyl (C-25) signal on the C-10 of junction in the trial-product collection of illustrative plates.Bright its of DEPT stave is uncle C.HMQC spectrum show its with the hydrogen spectrum on 0.70ppm (H-25) unimodal have directly related.H-1a on the HMBC spectrum proof A ring, b and H-5, each proton of the H-9 on the B ring all has long-range indirect correlation with it.
(36) the 16.43ppm peak is a C-26 signal in the C ring in the trial-product collection of illustrative plates.Bright its of DEPT stave is uncle C.HMQC composes and shows that itself and hydrogen spectrum go up 0.89ppm (H-26) signal and have directly related.H-7a in the HMBC spectrum proof B ring, b, H-9 on the C ring, each proton of H-15ab and H-27 all has the long-range indirect correlation of heteronuclear on the D ring with it.
(37) the 16.39ppm peak is a C-24 signal on the A ring in the trial-product collection of illustrative plates.Bright its of DEPT stave is uncle C.0.66ppm (H-24) signal on the HMQC spectrum shows it and hydrogen is composed has directly related.Each proton of H-3, H-5, H-23 and 3-OH on the same ring of HMBC spectrum proof all has long-range indirect correlation with it.
(38) the 16.22ppm peak is a C-30 signal on the E ring in the trial-product collection of illustrative plates.It is uncle C for a DEPT spectrum proof.The HMQC stave bright its with hydrogen spectrum in 0.84ppm (H-30) signal have directly related.H-20 and H-21a on the same ring of HMBC spectrum proof, each proton of b has long-range indirect correlation with it.
Conclusion: in sum, this product 13The result of each peacekeeping two-dimensional spectrum (PBB, DEPT, HMQC and HMBC etc.) of C-NMR conforms to fully with practical structures.This conclusion is not only consistent with relevant information such as peacekeeping two dimension hydrogen spectrum, and with relevant document * in known class like the relevant data of structural compounds compare also confirmed this conclusion rationally and reliability.
* attached: the related data in the relevant documents and materials:
1. document I:Acta Pharmacentica Sinica 1996,31 (11): 844-848
(i) compd A (2 α, 3 β, 19 β-trihydroxy--ursane-12-alkene-28-carboxylic acid-28-O-β-D-glucopyanoside, C 36H 58O 10) carbon spectrum data:
13C-NMR (125MHz, CD 3COCD 3, δ ppm): 176.8 (C-28), 139.2 (C-13), 128.8 (C-12), (95.1 the end group C on the sugar, C-1 '), 83.9 (C-3), 79.0 (C-5 '), 78.7 (C-3 '), 73.7 (C-2 '), 73.1 (C-19), 71.2 (C-4 '), 68.8 (C-2 '), 62.5 (C-6 '), 56.2 (C-5), 54.4 (C-18), 48.8 (C-17), 48.1 (C-1), 47.7 (C-9), 42.2 (C-14), 41.3 (C-20), 40.9 (C-8), 39.8 (C-10), 38.9 (C-4), 37.8 (C-22), 33.8 (C-7), 29.3 (C-15), 29.2 (C-23), 27.1 (C-21), 26.7 (C-29), 26.2 (C-16), 24.5 (C-27), 24.4 (C-11), 19.2 (C-6), 17.4 (C-25), 17.0 (C-26), 16.5 (C-30).
(ii) compd B (2 α, 3 α, 19 α-trihydroxy--ursane-12-alkene-28-carboxylic acid-28-O-β-D-glucopyanoside, C 36H 58O 10) carbon spectrum data:
13C-NMR (125MHz, DMSO-d 6, δ ppm): 175.4 (C-28), 138.1 (C-13), 127.0 (C-12), (94.0 the end group C on the sugar, C-1 '), 77.7 (C-3), 77.4 (C-3 '), 76.6 (C-5 '), 72.1 (C-2 '), 71.7 (C-19), 69.4 (C-4 '), 64.5 (C-2 '), 60.6 (C-6 '), 53.1 (C-18), 47.6 (C-18), 47.6 (C-5), 47.3 (C-17), 46.5 (C-9), 41.6 (C-1), 41.3 (C-20), 39.9 (C-14), 39.8 (C-8), 37.9 (C-4), 37.8 (C-10), 36.5 (C-22), 32.5 (C-7), 28.7 (C-15), 27.9 (C-23), 26.3 (C-21), 26.3 (C-29), 25.7 (C-16), 23.9 (C-27), 23.1 (C-11), 21.8 (C-6), 17.6 (C-26), 16.4 (C-30), 16.1 (C-25), 16.1 (C-24).
(iii) Compound C (2 α, 3 β, 19 α-trihydroxy--ursane-12-alkene-28-carboxylic acid, C 30H 48O 5) carbon spectrum data:
13C-NMR(125MHz，DMSO-d 6，δppm)：178.9(C-28)，138.6(C-13)，126.7(C-12)，82.2(C-3)，71.6(C-19)，67.1(C-2)，54.8(C-5)，53.1(C-18)，46.9(C-17)，46.8(C-1)，46.7(C-9)，41.4(C-20)，41.1(C-14)，39.0(C-8)，38.9(C-4)，37.6(C-10)，37.2(C-22)，32.6(C-7)，28.8(C-23)，28.0(C-15)，26.4(C-29，25.9(C-21)，25.1(C-16)，23.9(C-27)，23.2(C-11)，18.1(C-6)，17.1(C-26)，16.6(C-30)，16.3(C-25)，16.3(C-24)。
2. document II: CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998 the 23rd volume the 1st phase P37-38:
(iv) Compound D (2 α, 3 α, 19 α-trihydroxy--12-ursane-28-acid, C 30H 48O 5) carbon spectrum data:
13C-NMR(100MHz，C 5D 5N，δppm)：180.0(C-28)，142.0(C-13)，128.0(C-12)，79.3(C-3)，72.7(C-19)，66.1(C-2)，54.6(C-18)，48.8(C-5)，48.3(C-17)，47.6(C-9)，42.9(C-1)，42.4(C-20)，42.4(C-14)，40.6(C-8)，38.8(C-10)，38.7(C-4)，38.5(C-22)，33.5(C-7)，29.5(C-23)，29.5(C-15)，27.1(C-29)，27.0(C-16)，26.4(C-21)，24.7(C-27)，24.1(C-11)，23.3(C-24)，18.8(C-6)，17.3(C-26)，16.8(C-25)，16.6(C-30)。
3. document III: Acta Pharmaceutica Sinica, 18 (4): 314-316,1983:
(v) compd E (2 α, 3 α, 19 α-trihydroxy--ursolic acid-(28-1)-β-D-glucose ester, C 36H 58O 10) carbon spectrum data:
13C-NMR (JEOL FX-60Q, CD 3OD, δ ppm): 178.4 (sugar ester C=O), 139.5 (s), 129.5 (d), 95.7 (d), 84.5 (s), 78.4 (d), 78.3 (d), 73.8 (d), 73.6 (d), 71.2 (d), 69.5 (d), 62.5 (t).
4. document IV: Botany Gazette, 1988,30 (4): 409-413:
(vi) compound F 17-hydroxy-corticosterone (2 α, 3 α, the male fruit-12-alkene of 19 α-trihydroxy--28-carboxylic acid-28-O-β-D-glycoside, C 36H 58O 102H 2O) carbon spectrum data:
13C-NMR(JEOL?FX-100Q，C 5D 5N，δppm)：42.0(t，C-1)，65.5(d，C-2)，78.3(d，C-3)，38.0(s，C-4)，47.9(d，C-5)，17.3(t，C-6)，32.7(t，C-7)，40.1(s，C-8)，46.9(d，C-9)，38.0(s，C-10)，23.9(t，C-11)，127.5(d，C-12)，138.3(s，C-13)，41.5(s，C-14)，28.8(t，C-15)，26.0(t，C-16)，47.9(s，C-17)，53.6(d，C-18)，71.9(s，C-19)，42.0(d，C-20)，25.6(t，C-21)，36.9(t，C-22)，28.8(q，C-23)，21.6(q，C-24)，16.1(q，C-25)，16.8(q，C-26)，23.2(q，C-27)，176.2(s，C-28)，26.4(q，C-29)，16.1(q，C-30)，95.0(d，C-1’)，73.1(d，C-2’)，78.3(d，C-3’)，70.5(d，C-4’)，78.0(d，C-5’)，61.6(t，C-6’)。
(8) mass spectrum
1. instrument model
The Britain Zabspec of VG company mass spectrograph.
2. condition determination
HS/MS (polymer mass spectrum), FAB/MS (fast atom bombardment mass spectroscopy(FABMS)), EI/MS (electron impact mass spectra).
3. measurement result
See table 7 and Fig. 7 for details
Table 7 fern fiber crops three kinds of mass spectrum data of glycosides and parsing
Mass spectrum mode mass-to-charge ratio (m/z) is kurtosis (%) fragmention chemical constitution remarks relatively
FAB +651 [C 36H 59O 10 7] +The M+I peak
FAB -649 [C 36H 57O 10 7] +The M-1 peak
EI 488 15 [C 30H 48O 5 7] +The M ' of M-glycosyl (162) glucoside unit
470??????????????5????????????????????[C 30H 46O 4 7] +?????????????M’-H 2O(18)
442??????????????30???????????????????[C 29H 44O 2 7] +?????????????M’-HCOOH(46)
424??????????????10???????????????????[C 29H 42O 7] +???????????????M’-HCOOH-H 2O
264??????????????6????????????????????[C 16H 24O 3 7] +?????????????M’-224
246??????????????20???????????????????[C 16H 22O 2 7] +
223??????????????18???????????????????[C 14H 23O 2 7] +
219??????????????20???????????????????[C 15H 23O 7] +
205??????????????32???????????????????[C 14H 21O 7] +
201??????????????30???????????????????[C 15H 21 7] +
189??????????????20???????????????????[C 14H 21 7] +
146??????????????100??????????????????[C 11H 14 7] +
119??????????????34???????????????????[C 9H 11 7] +
4. discuss and explanation
(1) as seen, the negative spectrum of FAB ortho-spectrum (651) and FAB (649) shows that the molecular ion peak of this product should be 650, and promptly the molecular weight of this product anhydride should be 650, its elementary composition C that should be by table 7 36H 58O 10
(2) m/z 488 peaks are the molecular weight that molecular ion peak removes glycosyl (162) back resulting glucoside unit, and the complementary fragmention of total mass number 162 shows that this glycosyl should be hexose.
(3) C in this product 12-C 13Two keys are important cracking factors, are easy to carry out the RDA cracking at the electron impact lower C shape ring and molion is divided into contains A, B ring and contain D, two portions of E ring.Promptly 223 (A, B ring) and 264 (D, E encircles), two big fragmentions.The further cracking of this two big fragmention respectively 189 (20) and 246 (20), 219 (25), 205 (35), 201 (30), 146 (100) fragmentions such as 119 (34) are arranged.The relative kurtosis of each fragmention of the latter is all bigger, and wherein 146 is base peak (100).This base peak be m/z 264 fragments at the substituting group that loses the C-17 position and a H atom, so encircle E ring and carry out RDA and split and be situated between and lose a C 4H 8Behind the O fragment and form.This is a Δ 12-Usu alkene triterpenoid has distinctive fragmention after β-OH is arranged when 19 or 20.
(4) above each fragmention lytic pathway and mechanism may be as follows:
(9) powder X-ray diffraction spectrum
1. instrument
Japan's Rigaku D/MAX RC type powder crystal diffraction spectra of science.
2. sample
Trial- product 1,2,3
3. condition determination
CuKo 1Plant (λ α)=1.54056A), monochromatic radiation, 50kV, 80mA excites.
4. measurement result
See table 8 and Fig. 8 for details.
Table 8 fern fiber crops glycosides trial-product powder X-ray diffraction spectrum data and parsing
Sample 2 θ (°) interplanar distance d (A) relative intensity (I/Io,
％)
Trial-product 1 5.800 15.225 21
9.300??????????9.502???????????35
12.240?????????7.225???????????64
13.880?????????6.375???????????100
14.360?????????6.163???????????95
15.600?????????5.676???????????48
16.820?????????5.267???????????37
19.440?????????4.562???????????38
22.780?????????3.901???????????46
24.320?????????3.657???????????39
Trial-product 2 5.740 15.384 21
9.180??????????9.626???????????36
12.440?????????7.110???????????65
13.760?????????6.430???????????100
14.200?????????6.232???????????94
15.540?????????5.698???????????49
16.660?????????5.317???????????35
19.300?????????4.595???????????38
22.760?????????3.904???????????44
24.200?????????3.675???????????35
Trial-product 3 5.800 15.225 22
9.240??????????9.563???????????38
12.200?????????7.249???????????63
13.820?????????6.403???????????100
14.300?????????6.189???????????96
15.580?????????5.683???????????55
16.740?????????5.292???????????39
19.420?????????4.567???????????42
22.820?????????3.894???????????46
24.200?????????3.675???????????39
Conclusion: this product is a crystalline powder, and the sample crystal formation of different batches is identical.
(10) differential scanning calorimeter and thermogravimetric analysis (DSC and TG)
1. instrument: NETZSCH DSC 204
2. sample: trial-product
3. condition determination: 40 ℃ of starting temperatures, 300 ℃ of final temperatures, 10.0 ℃/min of heat-up rate.
4. measurement result:
5. conclusion: 1. the fusing point of trial-product is 231.7 ℃ (DSC); 2. the water content of trial-product is 3.4%, illustrates in the molecule also should to contain a crystal water (being equivalent to 2.7%) (TG) except that surperficial hygroscopic water.
Integration analysis
One, the deduction of molecular formula and molecular weight and definite
1. the deduction of molecular weight and definite
(1) three kind of mass spectrum (FAB +, FAB -, HS) result shows that the molecular formula of this product anhydride should be C 36H 58O 10
(2) by the result of ultimate analysis (EA): C%64.49, H% 9.08; The hydrogen spectrum ( 1H-NMR and D 2O) Jiao Huan result: 60 protons (wherein totally 9 active H); Thermogravimetric analysis (TG); 3.4% grade shows has a crystal water to exist in the molecule.
Conclusion: the accurate molecular formula of this product should be: C 36H 58O 10H 2O (or C 36H 60O 10).
2. the deduction of molecular weight is with definite:
(1) the anhydride molecular formula of this product is C 36H 58O 10, its molecular weight should be 650.85.
(2) a water thing molecular formula of this product is C 36H 58O 10H 2O, its molecular weight should be 668.87.
Two, the deduction of structural formula
1. the calculating of degree of unsaturation and deduction
(1) calculate:
According to degree of unsaturation calculation formula: Ω=(2+2n IV+ n III-n I)/2
The insatiable hunger degree Ω of this product should be 8.
(2) infer:
1. react by qualitative identification, and IR, 1H-NMR, 13C-NMR and MS judge that for information about this product is urson (ursolic acid) type pentacyclic triterpenoid.Be 5 cycloaliphatic rings (A, B, C, D, E) to be arranged in the molecule and be present between C-12 and C-13 one two key, and an ester group on the C-28 position.Thus, they contribute 7 degrees of unsaturation altogether.
2. by hydrogen spectrum, carbon spectrum, mass spectrum with infrared etc. confirm to have in the molecule sugar ring for information about, this ring is also contributed a degree of unsaturation.
Conclusion: the calculating of this product degree of unsaturation conforms to molecular formula, structural formula and the practical situation of this product fully with the inference structure.
2. the existence of basic structural unit and definite
By relevant information of the qualitative identification (positive reaction of Molish and Libermann-Burchard) of this product and its each spectrum as can be known: it is glucoside unit and the Ester Saponin of a monose molecule be combined into by urson (ursolic acid) the type pentacyclic triterpene that replaces that this product is one.So can think that the molecular structure only actually of this product is made up of two macrostructure unit (being urson triterpene and a part glycosyl).
(1) structure of aglycon part is inferred and is determined:
1. the type inference of parent nucleus is with definite:
(i) positive reaction of Molish and Libermann-Buurchard shows the pentacyclic triterpenoid of the aglycon of this product.
(ii) since in this product pentacyclic triterpene aglycon part hydrogen spectrum and carbon compose and show that all a methyl (δ is arranged on the C-19 position H: 1.08ppm, S, 3H and δ C: 26.67ppm, uncle C, HMBC spectrum shows itself and H-18,20,21ab and 19-OH have the long-range heteronuclear relevant), C-28 is ester carbonyl group (δ C: 175.53, season C, with H-1 ', 18,22ab has long-range relevant), be Usu acids pentacyclic triterpene so show this aglycon.
2. the deduction of the position of three substituted hydroxies and configuration and definite on the parent nucleus:
Through hydrogen spectrum and D 2O exchange back confirms in this product molecule 9 reactive hydrogens are arranged, remove four-OH on a water molecules and the sugar ring after, confirm should to have on this product parent nucleus three replacements-OH.
(i) determining of 2 α-OH:
Relevant information (single broad peak of 0.98ppm, D by hydrogen spectrum and carbon spectrum 2It is active proton for an O exchange proof, NOESY spectrum shows itself and H-1b, H-2, H-3,3-OH has living space relevant, H-1b and H-3 are the α key, H-2 and 3-OH be the β key so OH should be the α configuration, the numerical value of δ C-2 (67.13ppm) and type (uncle C) have also further been confirmed alpha-hydroxy existence on the C-2 position.
(ii) 3 β-OH's is definite:
δ C-3Numerical value (82.31ppm) and type (uncle C) show the very big gene of an electronegativity arranged on this C and (OH) exist; The 0.78ppm of hydrogen spectrum, D 2The O exchange is confirmed that it is active proton; NOESY spectrum shows itself and H-1a, 2,24,25,26 have living space relevant; These protons are all axial β key, and the binding molecule model shows that this OH also is the β key.Confirmed the existence of 3 β-OH calm on the C-3 position.This conclusion also confirms (seeing the parsing of this data IR spectra part for details) for infrared relevant information.Be proved to be result correct reliable that has also confirmed 3 β-OH for upright downward α key with its H-3 on same C.
(iii) 19 β-OH's is definite:
δ C-19Numerical value (71.65ppm) and type (season C) show also have on this C an electronegativity big-OH exists; Its hydrogen spectrum δ H3.78ppm, D 2The O exchange is confirmed that it is an active proton, bright its H-18 of COSY stave has relevant, and NOESY spectrum confirms itself and H-12,16a, 21a, 22a, 29,30 each proton relation of all having living space, H-16a wherein, 21a, 22a and H-30 are the β key, and the binding molecule model can confirm that 19-OH also should be upright beta configuration on this C-19 position.Be proved to be to calm α key with its C-29 methyl on same C and show further that also this result's is reliable.
3. the deduction of the position of 7 angular methyl(group)s and configuration and definite on the parent nucleus:
The angular methyl(group) that 7 replacements should be arranged on carbon spectrum (DEPT) and hydrogen spectrum confirmation this product parent nucleus.
(i) 23 α-CH 3With 24 β-CH 3Determine:
The DEPT spectrum confirms that C-4 (40.00ppm) is season C on the A ring; HMBC spectrum shows itself and H-3,5, and 6ab, H-23,24 have long-range relevant with 3-OH; In the carbon spectrum uncle C (C-23) the HMQC stave of 28.77ppm bright its with H-23 (0.91ppm, s, 3H) continuous; HMBC spectrum shows itself and H-3,5,24, and 3-OH has long-range relevant; Bright H-23 of NOESY stave (methyl) and H-3,5,6b, 24 with 3-OH have living space relevant, H-3 wherein, 5,6b is the α key, the binding molecule model can be confirmed this 23-CH 3It also is the α key; With its 24-CH on same C 3, its IR (1384,1370cm -1), hydrogen spectrum (0.89ppm, s, 3H), NOESY spectrum (with H-2,3-OH, H-6ab, H-23,25,26 all have living space relevant), carbon spectrum (16.43ppm, uncle C and H-24 have directly related, and with H-3,5,23,3-OH has long-range being correlated with) etc. all show this 24 CH 3It is a β type methyl.More than various information show that 4 season of this pentacyclic triterpene A ring is connected with a α-CH respectively on the C atom 3(C-23) and a β-CH 3(C-24).IR also proves the together existence of C gem-dimethyl in the molecule.
(ii) 25 β-CH 3With 26 β-CH 3Determine:
The DEPT spectrum confirms that the two is all uncle C (δ C-25: 17.09ppm, δ C-26: 16.39ppm), HMQC spectrum proof the two respectively with δ H 0.70ppm (s, 3H) and 0.66ppm (s, 3H) continuous; HMBC spectrum shows itself and H-1a, b, and H-5 has long-range relevant with H-9; The bright 25-CH of NOESY stave 3Respectively with H-1b, 2,6a, 11a, 24,26,3-OH has living space relevant; 26-CH 3Respectively with H-6a, 7b, 11,112,15a, b, 24,25 have living space relevant.The binding molecule model shows that the two is all axial β key.Prove 25 β-CH in conjunction with relevant information 3Should be connected on the season C of C-10; 26 β-CH 3Then should be connected on the season C atom of C-8 position.
(iii) 27 α-CH 3With 29 α-CH 3Determine:
The DEPT spectrum confirms that the two is all uncle C (δ C-27: 23.82ppm, δ C-29: 26.67ppm); HMQC spectrum confirm the two respectively with δ H-27(1.27ppm, s, 3H) and δ H-29(1.08ppm, s is 3H) relevant; The methyl peak of the bright 23.82ppm of HMBC stave respectively with H-7a, b, 15a, b has long-range relevant with 18 protons; Hydrogen spectrum 1.27ppm (s, 3H) peak in two-dimentional NOESY spectrum respectively with H-7ab, H-9, H-15b, H-16a, b, H-18, protons such as H-22a have living space relevant, H-9 wherein, H-15b, H-18 and H-22a are upright downward α key, and binding molecule model validation C-27 methyl also should be upright downward α key.In like manner, the bright 26.67ppm methyl of HMBC stave respectively with H-18, H-20, H-21a, b has long-range relevant with 19-OH; 1.08ppm in the hydrogen spectrum (s, 3H) methyl peak show in the NOESY spectrum respectively and H-12, H-18, and H-20, H-30 and 19-OH have living space relevant, and wherein, H-18 and H-20 also be the α key, and 19-OH has turned out to be the die 29-CH of on same C (C-19) of β key 3Also should be the α key, this result also can be proved from molecular model.Prove 27 α-CH in conjunction with relevant information 3Should be connected on the season C atom of C-14 position; And 29 α-CH 3Then should be connected on the season C atom of C-19 position.
(iv) 30 β-CH 3Determine:
(3H) peak has confirmed H-20 proton (1.21ppm to hydrogen spectrum 0.84ppm for d, J=9.0Hz, m, J=9.0,9.6Hz, 1H) coupling is arranged, carbon spectrum 16.22ppm peak, the DEPT spectrum confirms as uncle C, and HMQC spectrum proof has directly related with 0.84ppm methyl peak, HMBC stave bright itself and H-20, H-21a, b have long-range relevant, 30-CH in the NOESY spectrum 3Respectively with H-20,21a, b, H-29 and 19-OH have living space relevant, and the binding molecule model can confirm that this angular methyl(group) should be the β key and should be connected on the uncle C atom of C-20 position.
4. the determining of carboxylic acid group's position on the parent nucleus:
DEPT spectrum confirms that the 175.53ppm peak is a season C atom, and δ value shows it is carboxyl C atom, and its HMBC spectrum is confirmed itself and H-1 ' (the sugar ring is gone up end group C atom), and H-18, H-22a, b have long-range being correlated with.The δ value and the situation (with H-16a, b, H-18, H-22ab has long-range relevant with H-21a) of HMBC spectrum of C atom can confirm that this carboxyl should be on 17 season C atom in conjunction with 17 season.
5. the deduction of the position of four α protons (5,9,18 and 20) and configuration and definite:
0.73ppm during (i) hydrogen is composed (m, br, J=5.4,6.6Hz, 1H) signal HMQC spectrum confirms that its uncle C atom with C-5 (54.82ppm) position links to each other.NOESY stave bright itself and H-1a, 3,6b, 7a, 9 have living space relevantly with 23 each α proton, and it also should be the binding molecule model validation and is connected in upright downward α proton on the C-5 position.
(1H) peak confirms that in the HMQC spectrum its uncle C-9 C atom links to each other to 1.60ppm during (ii) hydrogen is composed for t, 9.6Hz; Show in the NOESY spectrum its respectively with the H-1a of α key, 5,7a, 11b has living space relevant with 27 each protons.This proton of binding molecule model validation also should be and is connected in the upright downward α proton in C-9 position.
2.36ppm during (iii) hydrogen is composed (s, 1H) peak confirms that in the HMQC spectrum it links to each other with uncle C-18 C atom; The NOESY stave bright its respectively with the H-5 of α key, 11a, 15b, 20,22a, 27 have living space relevant with 29 each Nan Zi peak.This proton of binding molecule model validation also should be upright downward α key.
(iv) the 1.21ppm signal confirms that in the HMQC spectrum it links to each other with the uncle C atom of 41.18ppm in the hydrogen spectrum; The NOESY stave bright its respectively with the H-18 of α key, 29 have living space relevantly, prove that this proton also should be connected in the upright downward α proton in C-20 position.
In sum, the aglycon part that can confirm this product should have following structure:
This structure has also obtained 488 (15), 442 (35) and 424 (10) and has waited providing powerful support for of each important fragmention in the mass spectrum of this product.
(2) deduction of the existence of glycosyl and glycosyl type and definite:
1. the existence of glycosyl is determined:
(i) each relevant information such as polymer mass spectrum, ultimate analysis, hydrogen spectrum and thermogravimetric analysis have confirmed that the molecular formula of this product is: C 36H 58O 10H 2O.Remove the front composition C with confirmed glucoside unit (pentacyclic triterpene) has been discussed 30H 48O 5And H 2Behind the O, a C is arranged still in the molecule 6H 10O 5Group is according to the δ of this group HAnd δ CNumerical value can judge that this group is a monose molecule.The δ of 6 C wherein CValue is respectively 94.05 (uncle C, C-1 '), 77.85 (uncle C, C-5 '), 76.70 (uncle C, C-3 '), 72.24 (uncle C, C-2 '), 69.51 (uncle C, C-4 ') and 60.64 (secondary C, C-6 '); The δ H value of corresponding proton is respectively: 5.15 (d, 8.4Hz, 1H, H-1 '), 3.58 (d, 11.2Hz, 1H, H-6 ' a), 3.43 (d, 10.8Hz, 1H, H-6 ' b), (3.17 m, 4.8,8.4Hz, 1H, H-3 '), (3.11 m, 4.8,8.4,1H, H-5 '), (3.08 dd, 8.4,4.8Hz, 1H, H-4 '), (3.06 dd, 8.4,4.8Hz, 1H, H-2 ').
(ii) 5 OH on the sugar ring have one to link to each other with glucoside unit, and the position of all the other 4 OH confirms through hydrogen spectrum and heavy water exchange, and three hydroxyls are arranged, and (2 '-OH, 3 '-OH and 6 '-OH) is concentrated to be distributed in the 5.20-5.15ppm place that hydrogen is composed.(4 '-OH) at the 3.31ppm place for another.Confirmed the existence of a glycosyl in the molecule thus.
2. the glycosyl configuration is definite:
According to the chemical shift (5.15ppm) of this sugared anomeric proton (H-1 ') and coupling constant ( 3J 1,2=8.4Hz) show that the H-1 ' of this glycosyl and H-2 ' are axial a key, promptly 3J 1,2Be a, a intersects, so glycosyl should be beta comfiguration.This result also by H-1 ' and the H-2 ' situation in one dimension DSNOE and two-dimentional NOESY spectrum confirmed (H-1 ' and 3 ', 4 ' and 2 '-OH relation of having living space; H-2 ' and H-6 ' a, b, 3 '-OH and 4 '-OH relation of having living space).
3. the glycosyl type is definite:
Result according to one dimension NOE difference spectrum (DSNOE) and two-dimentional NOESY spectrum can judge:
(i) H-1 ' is upright downward α key, and H-2 ' is upright β key, and the J=8.4Hz of the two proves the Jaa coupling; In DSNOE and NOESY spectrum H-1 ' respectively with H-3 ', 4 ' and 2 '-OH has living space relevant, prove the three be all the α key (H-3 ' and H-4 ' be a key, 2 '-OH is the e key).
(ii) H-3 ' and 4 '-OH is respectively axial α key and β key, and H-4 ' and 3 '-OH then is respectively calm α key and β type.In DSNOE and NOESY spectrum H-3 ' respectively with H-1 ', 4 ' and 2 '-OH relation of having living space; H-4 ' respectively with H-3 ', 5 ', 3 '-OH and 4 '-OH relation of having living space.4 '-OH then respectively with H-2 ', 3 '-OH, 6 ' a, 6 ' b and the 6 '-OH relation of having living space.
(iii) H-5 ' is the α key, and H-6 ' ab then is a β type a key.In DSNOE and NOESY spectrum, H-5 ' respectively with H-4 ', 4 '-OH, 6 ' a, b and 6 '-OH relation of having living space; H-6 ' ab then respectively with H-2 ', 5 ', 3 '-OH and 4 '-OH relation of having living space.
The structure of solid the above this product glycosyl type is as follows:
Figure A0315098600451
Obviously this structure should be β-galactopyranose.Each δ of this glycosyl CValue is respectively: 94.05 (C-1 '), 77.58 (C-5 '), 76.70 (C-3 '), 72.24 (C-2 '), 69.51 (C-4 ') and 60.64 (C-6 ').These numerical value and single β-each δ of D-semi-lactosi CValue is compared: 97.7 (C-1 '), and 76.31 (C-5 '), 74.2 (C-3 '), 73.3 (C-2 '), its numerical values recited of 70.1 (C-4 ') and 62.3 (C-6 ') conforms to substantially with the ordering situation.Further supported the reasonable reliability of this glycosyl structure.
(3) deduction of aglycon and glycosyl tie point and definite:
Show that according to the C-28 (ester carbonyl group) that shows in the HMBC spectrum and H-1 ' long-range relevant the pentacyclic triterpenoid saponin unit of this product obviously is the ester carbonyl group by 28
Figure A0315098600452
The saponin that is connected with end group carbon in the glycosyl (C-1 ') and forms.So the complete correct structure of saponin(e should be:
Figure A0315098600461
According to this structure, full name of this product should be:
2 α, 3 β, 19 β-trihydroxy--12-alkene-ursane-28-carboxylic acid-28-O-β-D-galactopyranoside
Owing to this compound is the new monomeric compound with saponin(e structure that extraction from medicinal plant fern fiber crops, separation and purification first obtain, so life is fern fiber crops glycosides.Its molecular formula is C 36H 58O 10H 2O.
Illustrate: in (1) this molecular formula the existence of a part water can by:
1. the result of ultimate analysis (C%64.49, H%9.08 conform to 9.04 (H%) with the calculated value 64.65 (C%) that contains a part water).
2. (integrated value is 60 protons to the result of hydrogen spectrum and heavy water exchange before the heavy water exchange, heavy water exchange back integrated value is 51 protons, and having 7 in 9 reactive hydrogens is obviously provided by a crystal water for remaining two the active protons of-OH proton (the replacement OH on 4 sugared OH and 3 parent nucleus)).
(crystal water is at C for the result of 3. thermogravimetric analysis (TG) 36H 58O 10H 2The shared quantity of O is 2.7%, measured value be 3.4% surpass 0.7% account for 1/4 water molecules may be surperficial hygroscopic water fail to dry fully so.
The analytical results of obvious three kinds of methods all confirms should contain in this product a crystal water.
(2) the reasonable reliability of this molecular formula and structural formula is removed and has been obtained various peacekeeping two dimensional NMR spectrum (PMR, D 2O, DSNOE, 1D-TOCSY, COSY, NOESY, 2D-TOCSY; PBB, DEPT, HMQC and HMBC etc.) various information confirm and support outside, EA, IR, FAB/MS, HSMS, EI/MS, PXR, all kinds of relevant informations such as DSC and TG have also given strong support.Especially mass spectral 651 (M-H 2O+1), 649 (M-H 2O-1), 488 (M-glycosyls 1162), 470 (M-glycosyl-H 2O), 442 (M-H 2O-HCOOH), 424 (M-H 2O-HCOOH-H 2The chemical constitution of O) etc. various important fragmentions and the sapogenin that splitting mechanism has all further been confirmed this saponin(e is formed and the composition situation of glycosyl.
Conclusion: the structure of this product is identified beyond the question
According to above-claimed cpd structure qualification result, by international online retrieval, search international STN system relevant data storehouse (chemical substance registered database, U.S. chemical abstract) and domestic online information retrieval through ISTIC Research ﹠ Traning Center.Confirm the new compound of this compound for finding and identify first.
Another object of the present invention has provided the isolation and purification method of above-mentioned fern fiber crops efficient parts (JMS) and fern fiber crops glycoside compound, and this method comprises the following steps, sees schema, sees Fig. 9.
Description of drawings
The HPLC collection of illustrative plates of Fig. 1 fern fiber crops glycosides trial-product
The IR collection of illustrative plates of Fig. 2 fern fiber crops glycosides trial-product
The UV of Fig. 3 fern fiber crops glycosides trial-product under neutrallty condition absorbs collection of illustrative plates (neutrality)
The UV of Fig. 4 fern fiber crops glycosides trial-product under acidic conditions absorbs collection of illustrative plates (acidity)
The UV of Fig. 5 fern fiber crops glycosides trial-product under alkaline condition absorbs collection of illustrative plates (alkalescence)
Fig. 6 fern fiber crops glycosides trial-product ' H-NMR schemes (0-7.5ppm)
The mass spectrum (HSMS) of Fig. 7 fern fiber crops glycosides trial-product
Mass spectrum powder X-ray-ray diagram the III and the data thereof of Fig. 8 fern fiber crops glycosides trial-product
The isolation and purification method flow diagram of Fig. 9 fern fiber crops glycoside compound
Embodiment
Embodiment 1
Get fern fiber crops crude drug and pulverized 20 mesh sieves, soaked into 4-6 hour with 70% ethanol, the 70%-95%7 alcohol reflux extracts 3 times, each 1.5 hours, united extraction liquid, concentrating under reduced pressure gets medicinal extract, medicinal extract is with macroporous resin column D-101 type resin on 1: 5 aqueous solution: crude drug ratio 1.5: 1, water elution is monitored terminal point with TCL, and the 30-95% ethanol elution is with TCL monitoring terminal point, collect pure wash-out part, put vacuum-drying and get fern fiber crops efficient part (JMS), get JMS and make 10% solution with water dissolution, last silicagel column carries out VLC decompression gradient column chromatography, eluent: CH 2CL 2CH 3OH:7: 3, substep is collected elutriant, concentrating under reduced pressure, last silicagel column normal pressure chromatography is with CH 2CL 2CH 3OH:20: 1 is elutriant, and substep is collected elutriant, and concentrating under reduced pressure gets fern fiber crops glycosides crude product purity＜80%, and crude product is made 10% solution with methyl alcohol, last silica gel column chromatography eluent: sherwood oil: acetone 5: 2 gets fern fiber crops glycosides crude product purity＞85%; The HPLC preparative chromatograph; HP1100 USA; Moving phase: CH 3OH: H 2O 65: 35, substep is collected elutriant, concentrating under reduced pressure, and recrystallization is handled (sherwood oil: methyl alcohol) get the pure product purity of fern fiber crops glycosides＞98%.
The evaluating drug effect of fern fiber crops efficient part of the present invention is as follows:
(1) evaluation of fern fiber crops reactive site (hereinafter to be referred as JMS) hepatitis virus resisting activity.
1. material
(1) for the white imperfect crystal formation of reagent thing fern fiber crops reactive site (JMS) (the drug research chamber preparation of pharmacy portion of No.302 Hospital, P.L.A., lot number: 0010526).Draw the miaow furan fixed, specification 100mg/ sheet, (Britain Wellcome fumdation product, lot number: 990812);
(2) foetal calf serum (U.S. Gibco company product); G-418 (U.S. Sigma company product); HBeAg, HBsAg stationary phase radioimmunoassay box (Beifang Inst. of Immune Reagents, Chinese Isotopes Co.'s product); A-32P-dCTP (the auspicious biotechnology of Beijing good fortune engineering corporation, lot number: 981206): nick translation medicine box (Promega company, lot number: 990426); Duck hepatitis B virus is duck hepatitis B virus DNA (DHBV-DNA) strong positive Shanghai sheldrake serum (Viral Laboratory, Beijing Medical University of Chinese Academy of Medical Sciences Institute of Medicinal Biological Technique provides ,-70 ℃ of preservations)
(3) the 2.2.15 cell full genome of HBV (Aayw subunit) that has been transfection, can secrete HBsAg, HBeAg, the female oncocyte of HBV-DNA particulate people liver system, U.S. Mount sinai medical center makes up, the cultivation of going down to posterity of medical biotechnology institute of Chinese Academy of Medical Sciences Viral Laboratory)
(4) dimension duck in animal 1 age in days Beijing is available from Nangyuan District, Beijing duckery
2. method:
(1) toxicity test of medicine pair cell:
(2) different concns pastille nutrient solution is added 96 porocyte culture plates, every concentration 4 holes, every 4d changes same concentration liquid, establishes no medicine cell control group.With the observation of cell pathology is index, the 8th day microscopically observation of cell pathology, and completely destroy is that 4,75% to destroy be that 3,50% to destroy be 2,25% to be 1, anosisly becomes 0.Calculate every concentration liquid average cell lesion degree and inhibiting rate.Press Reed Meuench method and calculate TC50, TC0.
(3) (11 2.2.15 cells are cultivated according to a conventional method with the DMEM substratum that contains 10% foetal calf serum and G418 (380ug/ml) in 2.2.15 cell cultures and medicinal application.When beginning to test, the 2.2.15 cell is dispersed into the individual cells suspension with 0.25% trypsinase, 3 ' 105 * L-1, use the pastille nutrient solution behind the 24h instead, every 4d renews bright pastille nutrient solution, collects 4d, 8d supernatant liquor, and-20C preserves, and is to be checked.
(4) duck hepatitis B virus infection and pharmacological agent [2] 1 age in days Beijing ducklings are through shin intravenous injection DHBV-DNA duck serum seed culture of viruses, every 0.3ml.Get blood after 7 days, separation of serum ,-70 ℃ of preservations, to be checked.After duckling DHBV infects 7d serum test positive, random packet, 6 every group, carry out the pharmacological agent test, the course of treatment 10d.That JMS divides is high, in low 3 dosage groups, i.e. 0.15g/kg day-1.d-1,0.3g/kg. day-1.d-1,0.6g/kg. day-1.d-1, ig, bid; The positive control group of lamivudine, 0.1g/kg. day-1.d-1, ig, bid; If the physiological saline group is the DHBV control group, ig, bid.Get blood respectively at 3 days (P3) after 5 days (T5), 10 days (T10) and the drug withdrawal after the medication from duck shin vein, separation of serum, 70 ℃ of preservations, to be measured.
(5) 2.2.15 emiocytosis HBsAg, HBeAg detect the solid phase radioimmunoassay box detection that the northern immunoreagent of employing institute produces, and the method by specification is measured every hole soup min-1 (cpm) value with 9 mouthfuls of counters.
(6) nick translation reagent specification sheets method is pressed in the detection of duck serum DHBV-DNA, with 32P mark DHBV-DNA probe, do direct dot hybridization of serum and radioautograph by methods such as Chen Yuanqing, measure the OD value of radioautograph diaphragm hybridization spot with enzyme-linked immunosorbent assay instrument (spectral filter goes into to be 490nm), with this as duck serum specimen DHBV-DNA level value.With the average OD value of serum DHBV-DNA before and after the duck medication on the same group and not on the same group the average inhibiting rate of serum DHBV-DNA of same time of duck compare the judgement curative effect of medication.DHBV-DNA inhibiting rate calculation formula:
3. evaluation of result:
(1) JMS suppresses external virus marker thing excretory
(2) JMS is to the toxic action of 2.2.15 cell: respectively containing JMS 2,1.5,1,0.5, the substratum of 0.25mg/ml and 2.2.15 cell co-cultivation are observed the toxic action of different concns medicine pair cell.The result shows that the maximal non-toxic concentration of JMS is 1mg/ml, and it is 1.38mg/ml that the product pair cell produces 50% toxic concentration, sees Table 9.
(mending table)
(3) JMS to the low dose of JMS of the restraining effect of 2.2.15 emiocytosis HBsAg (250 μ g * ml-1) to the inhibiting rate of 2.2.15 emiocytosis HBsAg at 4d, be respectively 16.5% and 51% mutually during two of 8d, in dosage JMS (inhibiting rate of phase is respectively 26.5% and 44.8% when 500 μ g * ml-1) at 4d, two of 8d.Compare with the physiological saline control group, low dose of and middle dosage JMS group inhibiting rate to HBsAg when 8d has significance meaning (P＜0.05).Heavy dose of JMS (1000 μ g * ml-1) in the time of two, all can significantly suppress duplicating and expressing of HBsAg mutually.(P＜0.05, P＜0.01) sees Table 10.
(4) JMS removes low dose of JMS to the restraining effect of the HBeAg of 2.2.15 emiocytosis (HBeAg to 2.2.15 emiocytosis when the 4d of 250 μ g * ml-1) does not have obvious restraining effect (P＞0.05), and all the other each dosage groups all have the significance restraining effect to the secretion of HBeAg.(P＜0.05，P＜0.01，P＜0.001)。Have tangible dose-dependently and time-dependent manner simultaneously, see Table 11.
(5) JMS restraining effect duckling that DHBV-DNA is duplicated infects DHBV-DNA total positives behind the hepatitis B virus.JMS height, middle dosage group are in administration 5 days (T5), 10 days (fourths 10), and no matter (laterally) compared in the administration front and back is still compared (vertically) with control group, and the DHBV-DNA level all significantly reduces (P＜0.05, P＜0.01) in the duck serum.And be regular hour dependency and dose-dependently.Low dose group does not then have obvious restraining effect (P＞0.05), but drug withdrawal after 3 days inhibiting rate rise to some extent.Though inhibiting rate rising degree and virus control group comparison there was no significant difference have embodied JMS and have suppressed the time-dependent manner that DHBV-DNA duplicates.The positive drug lamivudine is done can significantly suppress duck DHBV-DNA during the medication, but drops to 18.63% after the drug withdrawal, " knock-on " phenomenon occurs, sees Table 12.
Table 9 JMS is to the toxic action of 2.2.15 cell
Table 10 JMS is to restraining effect x ± s of 2.2.15 emiocytosis HBsAg
Table 11 JMS is to inhibiting rate effect x ± s of 2.2.15 emiocytosis HBeAg
Table 12 JMS is to restraining effect x ± s of duck serum DHBVDNA
4. the evaluation of compound monomer one fern fiber crops glycosides hepatitis virus resisting effect
(1) test method: 2.2.15 cell method
HBsAg, HBeAg positive controls are established in test, negative control group, cell control group and contain the medicine group of different ferns fiber crops glycosides concentration.2.2.15 cell is inoculated in 24 porocyte culture plates with 1 * 106L-1, every hole 1ml, 37 ℃ of 5%CO2 cultivated 24 hours, the following 2 times of dilutions of test soup non-toxic concn, 4 extent of dilution are respectively 100,50,25,12.5 μ g/ml, every concentration 4 holes, 37 ℃ of 5%CO2 cultivate, changed the original content soup in per 4 days and cultivate, results nutrient solution in the time of the 8th day ,-20 ℃ of stored frozen.Carry out the inhibition test of fern fiber crops glycosides respectively to 2.2.15 cell HBsAg and HBeAg expression; Fern fiber crops glycosides is to HBV-DNA dot hybridization test in the 2.2.15 cell conditioned medium and to HBV-DNA Southern blot test in the 2.2.15 cell.
(2) evaluation of result: see Table 13,14,15,16,17,18,19
Table 13 fern fiber crops glycosides pair cell toxicity and HbsAg in 2215 cell cultures, the restraining effect of HbeAg
The experiment batch Cytotoxicity HBV antigen is suppressed
??TC50(μg/ml ????TC0(μg/ml) ?????????????HbsAg ??????????????HBeAg
??IC50(μg/ml) ????SI ????IC50(μg/ml) ????SI
1 2 3 ??317.48 ??282.84 ????200 ????200 ???81.69 ???74.91 ???94.71 ????3.67 ????4.01 ????3.17 ????87.7 ????88.2 ????98.18 ????3.42 ????3.4 ????3.06
Three batches average 300.16±24.49 ????200±0 ????83.77±10.06 ??3.62±0.42 ????91.36±5.91 ??3.29±0.20
Two batches of lamivudine lamivudines are average 1130.03 1267.91 1198.97± 97.50 ????800 ????800 ????800±0 ????＞800 ????＞800 ????＞800 ??- ??- ??- ????＞800 ????＞800 ????＞800 ??- ??- ??-
Table 14 fern fiber crops glycosides in 2215 cell cultures to the restraining effect (%) of HbsAg and HbeAg
The experiment batch Silverweed cinquefoil essence concentration (μ g/ml) Suppress %
????HbsAg ????HBeAg
1 ????100 ????50 ????25 ????12.5 ????33.15 ????21.21 ????15.63 ????14.18 ????26.56 ????21.76 ????18.29 ????18.00
2 ????100 ????50 ????25 ????12.5 ????31.58 ????21.24 ????18.85 ????16.63 ????23.21 ????19.59 ????20.81 ????15.58
3 ????100 ????50 ????25 ????12.5 ????26.29 ????15.89 ????19.14 ????10.00 ????20.57 ????19.57 ????19.06 ????14.37
Three batches of empirical averages of silverweed cinquefoil essence ????100 ????50 ????25 ????12.5 ????30.37±3.61 ????19.43±3.06 ????17.87±1.97 ????13.6±3.34 ????23.47±3.01 ????20.3±1.27 ????19.4±1.28 ????16.0±1.83
Lamivudine ????400 ????200 ????100 ????50 ????12.67 ????16.35 ????17.48 ????13.20 ????8.10 ????2.35 ????12.05 ????6.24
Lamivudine ????400 ????200 ????100 ????50 ????13.32 ????19.31 ????14.19 ????14.99 ????0 ????8.99 ????5.15 ????0.65
Two batches of lamivudines are average ????400 ????200 ????100 ????50 ????19.55±2.43 ????23.50±0.25 ????20.53±3.09 ????16.60±7.29 ????4.05±5.73 ????5.67±4.7 ????8.6±4.88 ????3.45±3.95
Table 15 fern fiber crops glycosides in 2215 cell culture supernatants to effect HBV-DNA spot density value/inhibiting rate % of HBV-DNA:
Drug level (μ g/ml) Cell conditioned medium liquid HBV-DNA extension rate/inhibiting rate %
Stoste (CPM) Inhibiting rate % Dilution 1/2 (CPM) Inhibiting rate %
The contrast of 100 50 25 12.5 cells ??412.948 ??419.516 ??589.36 ??687.988 ??892.751 ????53.74 ????53.01 ????33.98 ????22.94 253.409 268.488 403.193 394.847 472.899 ????46.41 ????43.23 ????14.74 ????16.51
IC50/SI: former: IC50 45.13 μ g/ml/SI6.65 : IC50 63.06 μ g/ml/SI4.76
Table 16 lamivudine in 2215 cell cultures to supernatant liquor in effect HBV-DNA spot relative density value/inhibiting rate % of HBV-DNA:
Drug level (μ g/ml) Cell conditioned medium liquid HBV-DNA extension rate/inhibiting rate %
Stoste (CPM) Inhibiting rate % Dilution 1/2 (CPM) Inhibiting rate %
The contrast of 400 200 100 50 cells ????1767.02 ????1573.28 ????2063.2 ????2406.2 ????4344.61 ????59.3285 ????63.7878 ????52.5039 ????44.6164 305.135 161.533 243.224 911.185 1488.76 ????79.5041 ????89.1498 ????83.6626 ????38.7957
IC50/SI: former: IC50 119.61 μ g/ml/SI 10.02 : IC50 71.66 μ g/ml/SI 16.733
The inhibition data of table 17 fern fiber crops glycosides HBV-DNA Southern Blot in 2215 cell cultures
First ??100μg/ml Suppress % ??50μg/ml Suppress % ??25μg/ml Suppress % ??12.5μg/ml Suppress % The cell contrast
??Rows ??(IOD) ??(IOD) ??(IOD) ??(IOD) ????(IOD)
??r1 ??2834.7 ??36.66 ??2974.4 ??33.53 ??2293.1 ??48.76 ??1571.4 ??64.89 ????4475.1
??r2 ??668.81 ??92.01 ??569.93 ??94.63 ??719.53 ??91.41 ??603.2 ??92.8 ????8379.5
??r3 ??2850.3 ??91.96 ??4645.7 ??86.9 ??4543.3 ??87.19 ??2431.8 ??93.14 ????35457
??Sum ??6353.9 ??86.85 ??8070 ??83.3 ??7555.9 ??84.36 ??4003.2 ??91.71 ????48311
??In?Lane ??14445 ??84.18 ??18366 ??79.88 ???21513 ??76.44 ??22063 ??75.83 ????91293
The restraining effect that total HBV-DNA in|ane calculates in the different concns silverweed cinquefoil essence inoculating cell: IC50:57.13 μ g/ml SI:5.25
Effect is counted in the inhibition of table 18 fern fiber crops glycosides HBV-DNA Southern Blot in 2215 cells
First 400μg/ml Suppress % 200μg/ml Suppress % ??100μ ??g/ml Suppress % 50μg/ml Suppress % The cell contrast
??Rows ??(IOD) ??(IOD) ??(IOD) ??(IOD) ??(IOD)
??r1 ??368 ??70.37 ??526.41 ??57.62 ??530.96 ??57.26 ??619.28 ??50.15 ??1954.3
??r2 ??303.91 ??72.31 ??314.07 ??71.39 ??271.12 ??75.30 ??311.56 ??71.62 ??2367
??r3 ??214.35 ??82.10 ??330.9 ??72.36 ??416.24 ??65.24 ??465.81 ??61.10 ??2498.4
??Sum ??976.26 ??77.05 ??1171.4 ??72.46 ??1218.3 ??71.36 ??1396.6 ??67.17 ??2786.6
??In?Lane ??1954.3 ??76.10 ??2367 ??71.05 ??2498.4 ??69.44 ??2786.6 ??65.91 ??8175.3
The restraining effect that total HBV-DNA in lane calculates in the different concns silverweed cinquefoil essence inoculating cell: IC50:72.81 μ g/ml SI:16.47
Table 19 fern fiber crops glycosides and lamivudine in 2215 cell cultures to HBV restraining effect conclusive table
Medicine Cytotoxic T C50 μ g/ml ?????????HbsAg ?????????HBeAg ????HBV-DNA ????Dot?Blot ??HBV-DNA ??Southern?Blot
IC50 μg/ml SI IC50 μg/ml SI IC50 μg/ml SI IC50 μg/ml SI
Silverweed cinquefoil essence 300.16 ± 24.49 83.77 ± 10.06 3.62 ± 0.42 91.36 ±5.91 3.29 ± 0.20 54.44 ± 13.17 5.68 ±1.37 62.17 ±7.13 4.76 ± 0.55
Lamivudine 1198.9 7 ± 97.50 ＞800 - ＞800 - 122.13 ±3.56 9.82 ±0.28 77.49 ±6.62 15.53 ± 1.33
5.JMS the evaluation of anti-acute liver damage effect.
Materials and methods
(1) material
Medicine: JMS, white unformed powder is made into the suspension of proper concn with distilled water.Face the time spent jolting.Lot number: 020526); Bifendate Tablet (biphenyl dimethyldicarboxylate, BDD, 1.5mg. grain-1, Beijing consonance pharmaceutical factory, lot number 990918); Yinzhihuang oral liquid (mattress Cape jasmine Huang, 0.4g.10ml-1, Beijing the 4th pharmaceutical factory, lot number 990824); Tetracol phenixin (ccl4, analytical pure, Beijing Chemical Plant, lot number 980712); α ANIT (ANIT, analytical pure, chemical defence institute of the Chinese People's Liberation Army); Gpt test kit (Beijing northization fine chemicals limited liability company, lot number 000321); Total bilirubin determination reagent kit (Beijing Zhongsheng Biological Engineering High Technology Company, lot number 000301).Animal: Kunming mouse, body weight 18-22g, Military Medical Science Institute's animal center provides, conformity certification number: the moving word BDW-95007 of association of army.
Instrument: 4010 semi-automatic biochemical analyzers (Beijing biochemical analyzer factory) pathological section machine, powerful microscope (Japanese Olympus company).
Method:
(1) mouse ccl4 liver injury model
60 of healthy mices, if normal control group, ccl4 liver injury model group, Biphenylylmethylcarbinol group (BDD, 0.058g.kg-1, g-1) and 3 groups of the high, medium and low dosage of JMS (0.33,0.165,0.0825g.kg-1.g-1) totally 6 groups, 10 of every group of mouse, each organizes equal ig administration, every each 0.4mi, bid.Normal control group and model group are given with volume physiological saline.Administration the 3rd day, except that the normal control group, each organizes the equal ip 0.1%ccl4 of mouse sweet oil 20ml.kg-11 time, all animals is (36-40h afterwards poisons) behind the 7th day last administration 1h of experiment, win eyeball of mouse, get eye socket venous plexus blood, collect blood sample, detect Serum ALT levels with the biochemical measurement instrument, and cut liver specimens and do the pathology inspection.
(2) grouping of mouse ANIT liver injury model animal is with the ccl4 model, and positive drug control group is a Yinzhihuang oral liquid, 0.4L.kg.d-1, and administration the 3rd day, except that the normal control group, each organizes equal ig ANIT sesame oil liquid 12mg.kg-1.Behind the poisoning 24h, get mouse orbit venous pulse clump blood, measure SB level in the serum with the biochemical measurement instrument.
(3) statistical treatment experimental result is represented with x ± s, and is carried out the t check.
The result
(1) JMS is to the aminotransferase in mice level affects
Compare ccl4 model group mice serum transaminase obviously raise (P＜0.01) with the normal control group.Biphenylylmethylcarbinol group and JMS high dose group (0.33g.kg-1.d-1), middle dosage group (0.165g.kg-1.d-1) can significantly reduce ALT level (P＜0.01) in the ccl4 liver injury mouse blood, and JMS low dose group (0.0825g.kg-1.d-1) is to not influence (P＞0.05) of ALT.Middle and high dosage group JMS causes the damage mouse liver to ccl4 and has obvious provide protection.See Table 9.
(2) JMS influences the mouse bilirubin level
The yellow group of positive drug mattress Cape jasmine, JMS high dose group (0.33g.kg-1.d-1) can significantly reduce the mice serum bilirubin rising (P＜0.01) due to the ANIT.Dosage group (0.165g.kg-1.d-1) low dose group (0.0828g.kg-1.d-1) is to mice serum SB do not make significant difference (P＞0.05) among the JMS.Liver injury has provide protection (P＞0.05) and sees Table 10 high dose group FGN to the ANIT induced mice.
6.JMS the evaluation of choleretic effect.
60 of Wistar rats are divided into 6 groups at random by body weight, 10 every group, establish three dosage groups of normal control group, liver injury model group, Ursofalk group 0.135g/kgX day and JMS high, medium and low (0.6g, 0.3g, 0.15g/kg * day).After the administration 5 times, except that the normal control group, other each group is irritated stomach with 4%ANIT finish 1.75ml/kg (70mg/kg), causes rat bile alluvial model, and administration is continued in the back of poisoning.Behind the modeling type 48 hours, adopt combined anesthesia (ketamine: anesthetized rat diazepam=1: 1), separate ductus choledochus, insert the bile collection tube, collect 4 hours bile.Calculate each time period, every animal, every 100g body weight bile flow.The result shows, JMS can make 4 hours choleresis of ANIT liver injury rat increase to 3.19 ± 0.36 (high dosages), 2.52 ± 0.52ml (middle dosage), 2.05 ± 0.47ml (high dosage) by 1.38 ± 0.58ml of model group, and prompting JMS has choleretic effect.
Embodiment 2
Fern fiber crops crude drug was pulverized 40 mesh sieves, soak into more than 4 hours with 45% ethanol, with 70%-% alcohol reflux three times, each 2 hours, united extraction liquid, concentrating under reduced pressure gets medicinal extract, medicinal extract with 20%-ethanol in 1: 10 ratio thorough mixing, leave standstill then more than 12 hours, remove precipitation, macroporous adsorption resin chromatography post on the supernatant liquor (resin model D-101) sample size goes up pillar by portions of resin crude drug amount (1.5: 1), doubly measure pure water wash-out reject water lotion with 5-7, with (3-5 doubly measures) 30% ethanol elution, the reject elutriant is with (6-8 doubly measures) 60%-80% ethanol elution, collect elutriant, concentrating under reduced pressure gets powdered extract powder (fern fiber crops reactive site), remainder, the separation of remaining compound fern fiber crops glycosides, purification process is with reference to embodiment one.
Embodiment 3
Get fern fiber crops crude drug and pulverized 40 mesh sieves, soak into more than 4 hours with 50% ethanol, 60%-80% alcohol reflux three times, each 2 hours, united extraction liquid, concentrating under reduced pressure gets medicinal extract, medicinal extract, leaves standstill more than 12 hours in 1: 20 ratio thorough mixing then with 20%-45% ethanol, removes precipitation, macroporous adsorptive resins on the supernatant liquor (resin model D-101) sample size goes up pillar by portions of resin crude drug amount (1.5: 1), doubly measure the pure water wash-out with 5-7, the reject water lotion is with (5-7 doubly measures) 40-50% ethanol elution, the reject elutriant, with (6-8 doubly measures) 60%-90% ethanol elution, collect elutriant, concentrating under reduced pressure eliminates solvent, get powdered extract powder (fern fiber crops reactive site), the separation of remaining compound fern fiber crops glycosides, purification process is with reference to embodiment one.
Embodiment 4
Get fern fiber crops crude drug and pulverized 40 mesh sieves, soak into more than 4 hours with 50% ethanol, 50%-60% alcohol reflux secondary, each 2 hours, united extraction liquid, concentrating under reduced pressure gets medicinal extract, medicinal extract, leaves standstill more than 12 hours in 1: 25 ratio thorough mixing then with 30%-50% ethanol, removes precipitation, macroporous adsorptive resins on the supernatant liquor (resin model D-101) sample size goes up pillar by portions of resin crude drug amount (1.5: 1), doubly measure the pure water wash-out with 5-7, the reject water lotion is with (5-7 doubly measures) 40%-50% ethanol elution, the reject elutriant is with (6-8 doubly measures) 55%-85% ethanol elution, collect this elutriant, concentrating under reduced pressure eliminates solvent, get powdered extract powder (reactive site), the separation of remaining compound fern fiber crops glycosides, purification process is with reference to embodiment one.

Claims (3)

1. fern fiber crops glycoside compound is characterized in that this compound has following feature:
(1) chemical structural formula:
Figure A031509860002C1
(2) chinesization formal name used at school: 2 α, 3 β, 19 β-trihydroxy--12-alkene-ursane-28-carboxylic acid-28-O-β-D-galactopyranoside
(3) English chemical name: 2 α, 3 β, 19 β-trihydroxy-12-en-urs-28-oicacid-28-O-β-D-galactonopyranesyl ester
(4) molecular formula: C 3H 58O 10H 2O
(5) molecular weight: 650.848+18.015=668.86
2. the separation purification method of a fern fiber crops glycoside compound as claimed in claim 1 is characterized in that this method comprises the following steps:
Get fern fiber crops crude drug and pulverized 20 mesh sieves, soaked into 4-6 hour with 70% ethanol, 70% one 95% alcohol reflux 3 times, each 1.5 hours, united extraction liquid, concentrating under reduced pressure gets medicinal extract, medicinal extract was with macroporous resin column D-101 type portions of resin crude drug ratio on 1: 5 aqueous solution 1.5: 1, water elution is monitored terminal point with TCL, and the 30-95% ethanol elution is with TCL monitoring terminal point, collect pure wash-out part, put vacuum-drying and get fern fiber crops efficient part, get JMS and make 10% solution with water dissolution, last silicagel column carries out VLC decompression gradient column chromatography, eluent: CH 2CL 2CH 3OH:7: 3, substep is collected elutriant, concentrating under reduced pressure, last silicagel column normal pressure chromatography is with CH 2CL 2CH 3OH:20: 1 is elutriant, and substep is collected elutriant, and concentrating under reduced pressure gets fern fiber crops glycosides crude product purity＜80%, and crude product is made 10% solution with methyl alcohol, last silica gel column chromatography eluent: sherwood oil: acetone 5: 2 gets fern fiber crops glycosides crude product purity＞85%; The HPLC preparative chromatograph; HP1100USA; Moving phase: CH 3OH: H 2O 65: 35, substep is collected elutriant, concentrating under reduced pressure, with sherwood oil: recrystallizing methanol is handled, the pure product purity of fern fiber crops glycosides＞98%.
3. the application of a fern fiber crops glycoside compound as claimed in claim 1 in the preparation anti-hepatitis virus medicament.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100391983C (en) * 2006-06-27 2008-06-04 西北师范大学 Method for preparing radiation resistant fern amylose
CN101269086B (en) * 2008-05-09 2011-04-06 兰州大学 Use of plant extract
CN104958393A (en) * 2015-06-08 2015-10-07 中国科学院西北高原生物研究所湖州高原生物资源产业化创新中心 Extract of potentilla anserina rhizome and medical application thereof
CN104961791A (en) * 2015-06-05 2015-10-07 中国科学院西北高原生物研究所 Novel triterpene compound in potentilla anserina, and preparation method and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100391983C (en) * 2006-06-27 2008-06-04 西北师范大学 Method for preparing radiation resistant fern amylose
CN101269086B (en) * 2008-05-09 2011-04-06 兰州大学 Use of plant extract
CN104961791A (en) * 2015-06-05 2015-10-07 中国科学院西北高原生物研究所 Novel triterpene compound in potentilla anserina, and preparation method and application thereof
CN104961791B (en) * 2015-06-05 2017-03-08 中国科学院西北高原生物研究所 A kind of new triterpenoid, preparation method and purposes in Radix potentillae anserinae
CN104958393A (en) * 2015-06-08 2015-10-07 中国科学院西北高原生物研究所湖州高原生物资源产业化创新中心 Extract of potentilla anserina rhizome and medical application thereof

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