CN103175928B - Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root - Google Patents
Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root Download PDFInfo
- Publication number
- CN103175928B CN103175928B CN201110285363.0A CN201110285363A CN103175928B CN 103175928 B CN103175928 B CN 103175928B CN 201110285363 A CN201110285363 A CN 201110285363A CN 103175928 B CN103175928 B CN 103175928B
- Authority
- CN
- China
- Prior art keywords
- gromwell root
- circular dichroism
- radix macrotomiae
- liquid phase
- sherwood oil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention relates to the authenticate technology of radix macrotomiae and gromwell root, the peaceful enantiomter of the card utilizing radix macrotomiae and gromwell root to contain respectively, inverse relationship in liquid phase-circular dichroism spectra (LC-CD), establish the method that liquid phase-circular dichroism spectra (LC-CD) identifies radix macrotomiae and gromwell root medicinal material and extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer) exactly, thus, provide the method that radix macrotomiae and gromwell root medicinal material and extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer) were identified and distinguished to one exactly.
Description
Technical field
The present invention relates to the authenticate technology of radix macrotomiae and gromwell root, relate to the method that liquid phase-circular dichroism spectra (LC-CD) identifies radix macrotomiae and gromwell root medicinal material and extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer) exactly especially.
Background technology
Asian puccoon has long medicinal history in China, its sweet-salty, cold in nature, thoughts of returning home envelope Liver Channel, have cool blood, invigorate blood circulation, heat-clearing, the effect such as removing toxic substances, tcm clinical practice is mainly used in the diseases such as moist macula, purpura, blood urine, stranguria with turbid discharge and bloody flux, constipation with heat retention, burn, eczema, erysipelas, large carbuncle.
Asian puccoon (RadixArnrbiae, RadixLithospermi) is the dry root of Boraginaceae (Boraginaceae) plant lithospermum euchromum Royle Arnebiaeuchroma (Royle) Johnst., Asian puccoon LithospermumerythrorhizonSieb.etZucc. or arnebia guttata Bunge ArnebiaguttataBunge
[1].Wherein lithospermum euchromum Royle has another name called radix macrotomiae, and Asian puccoon or arnebia guttata Bunge are also known as gromwell root, and radix macrotomiae and gromwell root are the main sources of redroot.
Bibliographical information shows
[2,3], containing number of chemical composition in Asian puccoon, comprise the materials such as Gronwell naphthaquinone, phenolic acid class, alkaloids, phenol and benzoquinones class, triterpenic acid and sterols, flavonoids and polysaccharide.Wherein most study is alkannin derivatives, Chinese scholars is separated and obtains multiple naphthoquinone compound from the root of different genera Asian puccoon plant, as: beta-hydroxyisovalerylshiderivative, alkannin, 2, 3-dimethyl pentene acyl alkannin, Acetylshikonin, β, beta-dimethyl-acryloyl alkannin, isobutyryl shikonin, Alpha-Methyl-positive butyryl alkannin, IVS, dehydrogenation AK (dehydrogenation iso-alkannin), deoxyshikonin, β, beta-dimethyl-acry-lalkannin, β-acetoxyl group isovaleryl AK, beta-hydroxy isovaleryl AK etc.Naturally occurring Asian puccoon naphthoquinones has two kinds of optical isomers, and one is R type (called after alkannin class), and one is S type (called after alkannin), and they are enantiomorph each other
[4,5].
Alkannin class alkannin
Radix macrotomiae and gromwell root are all difficult to differentiate from aspects such as the ratios of mode of appearance and contained naphthoquinones constituents, and general being difficult to distinguishes radix macrotomiae and gromwell root exactly.Radix macrotomiae is different with effect with gromwell root effect, if accurately do not distinguished, by confusion reigned in market circulation and process of clinical application, the efficacy and saferry of medicine can be had a strong impact on, therefore, find a kind of accurately differentiation and identify radix macrotomiae and gromwell root medicinal material, especially radix macrotomiae and gromwell root extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer), will have important theory significance and actual application value.
Summary of the invention
The present invention is separation and purification 7 pairs of naphthoquinones enantiomters from radix macrotomiae and gromwell root respectively: AK (1), CAN1 (2), β-acetoxyl group isovaleryl AK (3), deoxidation AK (4), isobutyryl AK (5), β, beta-dimethyl-acry-lalkannin (6), isovaleryl AK (7), Alpha-Methyl-positive butyryl AK (8), alkannin (10), beta-hydroxyisovalerylshiderivative (11), Acetylshikonin (12), deoxyshikonin (4), isobutyryl shikonin (14), β, beta-dimethyl-acryloyl alkannin (15), IVS (16), Alpha-Methyl-positive butyryl alkannin (17).Determine their circular dichroism spectra (CD) respectively, find that they all have following feature: the CD collection of illustrative plates (partial spectrum is shown in Fig. 1-7) of the alkannin naphthoquinone analogs in radix macrotomiae is at 210-240nm, 250-300nm, 325-400nm and 450-600nm provides negative Cotton effect respectively, at 200-210nm, 240-250nm, 300-325nm, 400-450nm provide positive Cotton effect respectively; The CD collection of illustrative plates of the alkannin class naphthoquinone analogs in gromwell root is at 210-240nm, 250-300nm, 325-400nm and 450-600nm provides positive Cotton effect respectively, at 200-210nm, 240-250nm, 300-325nm, 400-450nm provide negative Cotton effect respectively, and the CD absorption curve of both compositions is just the opposite.
We find, if utilize the liquid chromatography technologies such as HPLC, compartment analysis goes out the naphthaquinone derivatives in Asian puccoon, recycle the circular dichroism that circular dichroism spectra (CD) detects them, just can distinguish and identify radix macrotomiae and gromwell root medicinal material well, especially radix macrotomiae and gromwell root extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer).
For this reason, we study and have found that good separation analyzes liquid phase-circular dichroism spectra (LC-CD) condition of the naphthaquinone derivatives in Asian puccoon, find the wavelength coverage (210-240nm detecting circular dichroism spectra (CD) several the best of radix macrotomiae and gromwell root, 250-300nm, 325-400nm and 450-600nm, 200-210nm, 240-250nm, 300-325nm, 400-450nm) and optimal wavelength (220nm, 245nm, 260nm, 310nm, 360nm, 430nm and 550nm).Thus, provide the method that radix macrotomiae and gromwell root medicinal material and extract (comprising the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer) were identified and distinguished to one exactly.
The present invention relates to the authentication method utilizing liquid phase-circular dichroism spectra (LC-CD) technology to distinguish radix macrotomiae and gromwell root, to identifying radix macrotomiae and gromwell root exactly.To avoid the confusion be all difficult to from aspects such as the ratios of mode of appearance and contained naphthoquinones constituents due to radix macrotomiae and gromwell root the market circulation differentiating to cause and process of clinical application, ensure the efficacy and saferry of clinical middle Asian puccoon.
The present invention establishes the analytical approach utilizing liquid phase-circular dichroism spectra (LC-CD) technology to distinguish radix macrotomiae and the qualification of gromwell root: with HPLC for the liquid phase analysis of representative is separated the naphthoquinones constituents contained by Asian puccoon, with circular dichroism spectra (CD) for detecting, in conjunction with UV detect, analyze radix macrotomiae and gromwell root medicinal material and extract and (comprise the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer), analyze the chromatogram obtained, consistent with standard medicinal material, or it is consistent with the radix macrotomiae that the present invention sets up and gromwell root liquid phase-circular dichroism spectra (LC-CD) feature separately, all can differentiate exactly.
The configuration of reference substance solution: get naphthoquinones constituents reference substance precision and take in right amount, be mixed with appropriate reference substance solution with methyl alcohol.
The collocation method 1 of sample solution: precision takes radix macrotomiae or gromwell root sample (medicinal material, extract, chemical composition monomer, herbal mixture etc. containing Asian puccoon) in right amount each, with methyl alcohol (or chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the aqueous solution of the organic solvents such as cyclohexane and variable concentrations thereof) extract, reclaim extract, methyl alcohol (or chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the aqueous solution of the organic solvents such as cyclohexane and variable concentrations thereof) dissolve, through the little chromatographic column pre-service of SPE of filling ODS (dodecyl bonded silica gel), the removal of impurities of water (or low-concentration organic solvent) wash-out, the use that continues acetonitrile (or methyl alcohol, ethyl acetate, acetone, chloroform, sherwood oil, the aqueous solution of the organic solvents such as cyclohexane and variable concentrations thereof) washing, merge eluent, methyl alcohol dissolves, constant volume, filter, obtain.
The collocation method 2 of sample solution: it is in right amount each that precision takes radix macrotomiae or gromwell root sample (medicinal material, extract, chemical composition monomer, herbal mixture etc.) containing Asian puccoon, extract with methyl alcohol (or aqueous solution of the organic solvent such as chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane and variable concentrations thereof), reclaim extract, methyl alcohol dissolves, constant volume, filter, to obtain final product.
The collocation method 3 of sample solution: it is in right amount each that precision takes radix macrotomiae or gromwell root sample (medicinal material, extract, chemical composition monomer, herbal mixture etc.) containing Asian puccoon, extract with methyl alcohol (or aqueous solution of the organic solvent such as chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane and variable concentrations thereof), filter, to obtain final product.
Method of testing: carry out HPLC analysis, with circular dichroism spectra (CD) for detecting, detects in conjunction with ultraviolet (UV), record chromatogram.Because naphthoquinones constituents has absorption peak at 200-300nm or 400-600nm in ultraviolet spectrum, can measure within the scope of these two; And in circular dichroism spectra, the cotton effect difference within the scope of 200-600nm (see Fig. 8,9) of each enantiomter, can in 200-600nm scope, selected wavelength measures (210-240nm, 250-300nm, 325-400nm and 450-600nm arbitrarily, 200-210nm, 240-250nm, 300-325nm, 400-450nm).
The present invention considers the factor such as convenience and sensitivity of Simultaneously test, best mensuration wavelength chooses is 220nm, 245nm, 260nm, 310nm, 360nm, 430nm and 550nm, wherein, 320nm, 360nm two wavelength place uv absorption are more weak, and 430nm wavelength place circular dichroism spectra is more weak, is not too beneficial to Simultaneously test ultraviolet spectrum and circular dichroism spectra.Therefore, optimum is 220nm, 245nm, 260nm and 550nm.
After mensuration, liquid phase-ultraviolet (LC-UV) chromatogram that radix macrotomiae and each wavelength place of gromwell root sample measure is posivtive spike.
Radix macrotomiae is different with the symbol of chromatographic peak in liquid phase-circular dichroism spectra (LC-CD) chromatogram that gromwell root sample each wavelength place measures, and specific features is as follows:
Liquid phase-circular dichroism spectra (LC-CD) chromatogram measured at 220nm, 260nm, 360nm and 550nm place
In radix macrotomiae, each chromatographic peak is negative peak, and in gromwell root, each chromatographic peak is posivtive spike.
Liquid phase-circular dichroism spectra (LC-CD) chromatogram measured at 245nm, 310nm and 430nm place
In radix macrotomiae, each chromatographic peak is posivtive spike, and in gromwell root, each chromatographic peak is negative peak.
1) with the LC-UV chromatographic peak area of compound each in sample, compared with chromatographic peak area corresponding to standard control sample, according to calibration curve method (or external standard 1 method, external standard 2 methods etc.), carry out quantitative test, calculate, obtain the total content of two enantiomters of each naphthoquinones constituents in sample.
2) positive and negative with the LC-UV chromatographic peak area of compound each in sample and chromatographic peak, chromatographic peak area corresponding to standard control sample is compared with the positive and negative of chromatographic peak, according to calibration curve method (or external standard 1 method, external standard 2 methods etc.), carry out quantitative test, calculate, obtain the ratio of two enantiomters of each naphthoquinones constituents in sample.
Also can according to the chromatographic peak in above-mentioned method of testing positive and negative rule, qualification radix macrotomiae and gromwell root.(Figure 10,11 is respectively radix macrotomiae and gromwell root liquid phase-ultraviolet chromatogram (LC-UV) figure and liquid phase-circular dichroism spectra (LC-CD) figure of 274nm place mensuration).From in figure: can very clearly distinguish radix macrotomiae and gromwell root according to liquid phase-circular dichroism spectra (LC-CD) figure.
The present invention also to comprise in radix macrotomiae and gromwell root the separation and purification method of 7 pairs of naphthoquinones enantiomters:
1) AK (1) in radix macrotomiae, CAN1 (2), β-acetoxyl group isovaleryl AK (3), deoxyshikonin (4), isobutyryl AK (5), β, the preparation method of beta-dimethyl-acry-lalkannin (6), isovaleryl AK (7), Alpha-Methyl-positive butyryl AK (8) is as follows:
Find that the naphthoquinones constituents polarity in Asian puccoon is less according to our research, be soluble in the organic solvent such as ethanol, sherwood oil, especially find that this constituents is to light, thermally labile, so screening is determined to extract Gronwell naphthaquinone composition with 95% ethanol cold soaking.
Radix macrotomiae medicinal extract, through thin-layer chromatographic analysis and silica gel column chromatography test, finds with sherwood oil: ethyl acetate carries out column chromatography gradient elution, can be separated Gronwell naphthaquinone composition preferably.The separation method of each compound is as follows:
The forward part of sherwood oil wash-out merges stream part, carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient (100: 1), can obtain isovaleryl AK (7) and Alpha-Methyl-positive butyryl AK (8);
The rear section of sherwood oil wash-out merges stream part, carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient (100: 0.5), can obtain deoxidation AK (4) and β, beta-dimethyl-acry-lalkannin (6);
Sherwood oil: the forward part of ethyl acetate (100: 0.5) wash-out merges stream part, carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, 100: 0.5, CAN1 (2) and β can be obtained, beta-dimethyl-acry-lalkannin (6);
Sherwood oil: the rear section of ethyl acetate (100: 0.5) wash-out merges stream part, carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, collect 100: 2 stream parts, utilize preparation liquid phase (ODS, methanol-water (65: 35) wash-out), separablely obtain isobutyryl AK (5);
Sherwood oil: part is flowed in the merging of ethyl acetate (100: 1) wash-out, carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient, collect stream part of 100: 2, silica gel column chromatography repeatedly, can obtain AK (1);
Sherwood oil: part is flowed in the merging of ethyl acetate (100: 2) wash-out is eluent with methanol-water, utilizes ODS to carry out separation and purification, methanol-water (60: 40) wash-out, can obtain β-acetoxyl group isovaleryl AK (3).
2) because be only enantiomter, so its physicochemical property is consistent with chromatographic behavior in the naphthoquinones constituents in gromwell root and radix macrotomiae.Therefore, alkannin (10) in gromwell root, beta-hydroxyisovalerylshiderivative (11), Acetylshikonin (12), deoxyshikonin (4), isobutyryl shikonin (14), β, the preparation method of beta-dimethyl-acryloyl alkannin (15), IVS (16), Alpha-Methyl-positive butyryl alkannin (17) is consistent with the preparation method of its enantiomter in radix macrotomiae, and concrete grammar is as follows:
Gronwell naphthaquinone composition is extracted with 95% ethanol cold soaking.
Gromwell root medicinal extract, through thin-layer chromatographic analysis and silica gel column chromatography test, finds with sherwood oil: ethyl acetate carries out column chromatography gradient elution, can be separated Gronwell naphthaquinone composition preferably.The separation method of each compound is as follows:
Sherwood oil: ethyl acetate (100: 0.5) wash-out, part is shunted in the front portion merging sherwood oil wash-out, carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, sherwood oil: ethyl acetate (100: 0.5) wash-out obtains deoxyshikonin (4)
Sherwood oil: ethyl acetate (100: 0.5) wash-out, part is shunted at the middle part merging sherwood oil wash-out, carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, sherwood oil: ethyl acetate (100: 0.5) obtains IVS (16) and Alpha-Methyl-positive butyryl alkannin (17);
Sherwood oil: ethyl acetate (100: 0.5) wash-out, part is shunted at the rear portion merging sherwood oil wash-out, carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, sherwood oil: ethyl acetate (100: 0.5) stream part, utilize preparation liquid phase separation, at acetonitrile: water (75: 25) obtains isobutyryl shikonin (14) and β, beta-dimethyl-acryloyl alkannin (15);
Sherwood oil: ethyl acetate (100: 1) wash-out, merge stream part, recrystallization obtains Acetylshikonin (12);
Sherwood oil: ethyl acetate (100: 7) wash-out, merges stream part, dextran gel column chromatography, methanol-eluted fractions, collects red stream part, obtains alkannin (10) by preparation liquid phase separation;
Sherwood oil: ethyl acetate (100: 15) wash-out, merges stream part, ODS purification by column chromatography, methanol-water gradient elution, and Fractional Collections redness stream part, obtains beta-hydroxyisovalerylshiderivative (11) by preparation liquid phase separation.
Accompanying drawing explanation
Fig. 1: the CD collection of illustrative plates of alkannin, AK.
Fig. 2: β, beta-dimethyl-acryloyl alkannin, β, the CD collection of illustrative plates of beta-dimethyl-acry-lalkannin.
Fig. 3: the CD collection of illustrative plates of Acetylshikonin, CAN1.
Fig. 4: the CD collection of illustrative plates of isobutyryl shikonin, isobutyryl AK.
Fig. 5: compound 7+8, the CD collection of illustrative plates of compound 16+17.
The CD collection of illustrative plates of Fig. 6: β-acetoxyl group isovaleryl AK.
Fig. 7: the CD collection of illustrative plates of beta-hydroxyisovalerylshiderivative.
Fig. 8: the circular dichroism spectrogram of naphthoquinones constituents.
Fig. 9: the ultraviolet spectrum of naphthoquinones constituents.
Figure 10: liquid phase-ultraviolet chromatogram (LC-UV) figure of gromwell root, radix macrotomiae.
Figure 11: liquid phase-circular dichroism spectra (LC-CD) figure of gromwell root, radix macrotomiae.
Figure 12: reference substance chromatogram.
Note: be followed successively by (a) isovaleryl AK+α-positive butyryl AK, (b) β from top to bottom, beta-dimethyl-acry-lalkannin, (c) isobutyryl AK, (d) deoxyshikonin, (e) β-acetoxyl group isovaleryl AK, (f) CAN1, (g) beta-hydroxyisovalerylshiderivative, (h) AK, (i) mix reference substance.
Figure 13: radix macrotomiae extraction and isolation process flow diagram.
Figure 14: gromwell root extraction and isolation process flow diagram.
Embodiment
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1: the qualification of radix macrotomiae and gromwell root
Chromatographic condition: chromatographic column is ScienhomeKromasilC18 (4.6mm × 200mm, 5 μm) and pre-column, mobile phase is acetonitrile (A)-0.3% phosphoric acid water (B), gradient elution process (A gradient) 0-10 minute 70%, 10-30 minute 70%-75%, 30-31 minute 75%-95%, 31-36 minute 95%, 36-37 minute 95%-70%, 37-50 minute 70%.Determined wavelength 274nm, column temperature 30 DEG C, flow velocity 1.0mL/min, number of theoretical plate is not less than 3000 in naphthoquinones.The regression equation of its ultraviolet spectrum, the range of linearity, lowest detectable limit and quantitative limit the results are shown in Table 1.
Table 1 nine kinds of naphthoquinone compound regression equations, the range of linearity, lowest detectable limit and quantitative limit result
Table1Regressionequations.correlationcoefficients.linearrange.LODandLOQfornineanalytes
The preparation of reference substance solution: accurate configuration reference substance is mixed with reference substance solution and the mixing reference substance solution of every milliliter of (1) AK 12.32 μ g, (2) CAN1 4.20 μ g, (3) β-acetoxyl group isovaleryl AK 82.5 μ g, (4) deoxidation AK 26.4 μ g, (5) isobutyryl AK 2.07 μ g, (6) β, beta-dimethyl-acry-lalkannin 40.95 μ g, (8) Alpha-Methyl-positive butyryl AK 83.26 μ g, (9) hydroxyhopanone 156.00 μ g.Accurate configuration reference substance is mixed with every milliliter of (10) alkannin 12.32 μ g, (11) beta-hydroxyisovalerylshiderivative 4.20 μ g, (12) Acetylshikonin 82.5 μ g, (14) isobutyryl shikonin 2.07 μ g, (15) β, the reference substance solution of beta-dimethyl-acryloyl alkannin 40.95 μ g, (16) IVS 83.26 μ g, (17) Alpha-Methyl-positive butyryl alkannin 156.00 μ g and mixing reference substance solution.Reference substance chromatogram is shown in Figure 12 (note: each enantiomter appearance time is consistent).
The preparation of need testing solution: get Asian puccoon meal and be about 0.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50mL, and close plug, shakes up, weighed weight, ultrasonic process 30min, lets cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, filter with miillpore filter (0.22 μm).
Method of testing: carry out HPLC analysis, with circular dichroism spectra (CD) for detecting, detects in conjunction with ultraviolet (UV), record chromatogram.
Also can according to the chromatographic peak in above-mentioned method of testing positive and negative rule, qualification radix macrotomiae and gromwell root.(Figure 10,11 is respectively radix macrotomiae and gromwell root liquid phase-ultraviolet chromatogram (LC-UV) figure and liquid phase-circular dichroism spectra (LC-CD) figure of 274nm place mensuration).From in figure: can very clearly distinguish radix macrotomiae and gromwell root according to liquid phase-circular dichroism spectra (LC-CD) figure.
Embodiment 2: the preparation of naphthoquinone derivatives in radix macrotomiae
Radix macrotomiae meal 1.8kg, 95% ethanol cold soaking extracts, and 10 times amount extract three times, each 24h, merges extract, utilizes Rotary Evaporators recycling design to obtain radix macrotomiae medicinal extract 105g.
Get radix macrotomiae medicinal extract 95g, silica gel 400g, with sherwood oil: ethyl acetate carries out column chromatography gradient elution, every 700mL is first-class part, is identified by silica gel thin-layer chromatography, merges same stream part.
Sherwood oil wash-out merges stream part 12-13 (2.7g), carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient (100: 1), obtains compound 7 (1g) and 8 (1g); Stream part 17 (0.97g), carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient (100: 0.5), obtains compound 4 (15mg), 6 (300mg); Stream part 20 (2.98g), carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, obtain compound 2 (2g), 6 (100mgs) at 100: 0.5; Stream part 21 (2.9g), carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient, collects 100: 2 stream part 0.5g, utilizes preparation liquid phase separation to obtain compound 5 (150mg); Stream part 26 (1.1g), carries out silica gel column chromatography, sherwood oil: ethyl acetate gradient, and collect stream part 0.21g of 100: 2, silica gel column chromatography repeatedly, obtains compound 1 (20mg); Merge 100: 2 streams part 29 (1.8g), be eluent with methanol-water, utilize ODS to carry out separation and purification, obtain compound 3 (20mg), 9 (10mg).Utilize the compound structure authenticate technologies such as NMR, MS, be accredited as respectively: AK (1), CAN1 (2), β-acetoxyl group isovaleryl AK (3), deoxidation AK (4), isobutyryl AK (5), β, beta-dimethyl-acry-lalkannin (6), isovaleryl AK (7), Alpha-Methyl-positive butyryl AK (8), hydroxyhopanone (9).
Radix macrotomiae extraction and isolation flow process is shown in Figure 13, and the structure of radix macrotomiae chemical composition and authentication method are in table 2.
The structure of table 2 radix macrotomiae chemical composition and authentication method
Table2ThecompoundsisolatedfromArnebiaenchroma(Royle)Johnst.dryroot
Embodiment 3: the preparation of naphthoquinone derivatives in gromwell root
Gromwell root meal 1.0kg, 95% ethanol cold soaking extracts, 10 times amount extract three times, each 24h, merge extract, Rotary Evaporators recycling design is utilized to obtain gromwell root medicinal extract 18g, medicinal extract 1L aqueous dispersion becomes suspension, with equal-volume petroleum ether extraction three times, and each 12h, reclaim sherwood oil partial extraction liquid, recycling design concentrates to obtain sherwood oil part medicinal extract 5.7g.
Get gromwell root sherwood oil part medicinal extract 5g, column chromatography silica gel 100g, with sherwood oil: ethyl acetate carries out column chromatography gradient elution, every 300mL is first-class part, is identified by silica gel thin-layer chromatography, merges same stream part.
Sherwood oil: ethyl acetate (100: 0.5) wash-out, merge stream part 2-4 (0.3g), carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, obtain compound 13 (5mg) at 100: 0.5; Merge stream part 5-10 (0.5g), carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, obtain compound 16+17 (25mg) at 100: 0.5; Merge stream part 17-25 (1.2g), carry out silica gel column chromatography, sherwood oil: ethyl acetate gradient, collect 100: 0.5 stream parts, utilize preparation liquid phase, at acetonitrile: water (75: 25) obtains compound 14 (5mg), 15 (25mg); Sherwood oil: ethyl acetate (100: 1) wash-out, merge stream part 55-70 (1.8g), recrystallization obtains compound 12 (1.7g); Sherwood oil: ethyl acetate (100: 7) wash-out, merges stream part 86-95 (0.4g), dextran gel column chromatography, methanol-eluted fractions, collects red stream part, obtains compound 10 (15mg) by preparation liquid phase separation; Sherwood oil: ethyl acetate (100: 15) wash-out, merge stream part 99-105 (0.6g), ODS purification by column chromatography, methanol-water gradient elution, Fractional Collections redness stream part, obtains compound 11 (15mg) by preparation liquid phase separation.Utilize the compound structure authenticate technologies such as NMR, MS, be accredited as respectively: alkannin (10), beta-hydroxyisovalerylshiderivative (11), Acetylshikonin (12), deoxyshikonin (4), isobutyryl shikonin (14), β, beta-dimethyl-acryloyl alkannin (15), IVS (16), Alpha-Methyl-positive butyryl alkannin (17).
Gromwell root extraction and isolation flow process is shown in Figure 14, and the structure of gromwell root chemical composition and authentication method are in table 3.
The structure of table 3 gromwell root chemical composition and authentication method
Table.3ThecompoundsisolatedfromLithos-permumerythrorhizonSieb.dryroot
Claims (3)
1. liquid phase-circular dichroism spectra the authentication method of a radix macrotomiae and gromwell root, it is characterized in that i. chromatographic condition: chromatographic column is ScienhomeKromasilC18,4.6mm × 200mm, 5 μm and pre-column, mobile phase is acetonitrile (A)-0.3% phosphoric acid water (B), gradient elution process: 0-10 minute 70%, 10-30 minute 70%-75%, 30-31 minute 75%-95%, 31-36 minute 95%, 36-37 minute 95%-70%, 37-50 minute 70%, determined wavelength 274nm, 220nm, 245nm, 260nm, 310nm, 360nm, 430nm or 550nm, column temperature 30 DEG C, flow velocity 1.0mL/min, number of theoretical plate is not less than 3000 in naphthoquinones, the regression equation of its ultraviolet spectrum, the range of linearity, lowest detectable limit and quantitative limit meet metrical error requirement, ii. the collocation method 1 of sample solution: precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or in right amount each containing the herbal mixture of Asian puccoon, with methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the extraction with aqueous solution of cyclohexane or more organic solvent variable concentrations, reclaim extract, methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the aqueous dissolution of cyclohexane or more organic solvent variable concentrations, through the little chromatographic column pre-service of SPE of filling dodecyl bonded silica gel, water or the removal of impurities of low-concentration organic solvent wash-out, continue with acetonitrile, methyl alcohol, ethyl acetate, acetone, chloroform, sherwood oil, the solution washing of cyclohexane or more organic solvent variable concentrations, merge eluent, methyl alcohol dissolves, constant volume, filter, obtain, iii. the collocation method 2 of sample solution: it is in right amount each that precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or the herbal mixture containing Asian puccoon, by the extraction with aqueous solution of methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane or more organic solvent variable concentrations, reclaim extract, methyl alcohol dissolves, constant volume, filter, to obtain final product, iv. the collocation method 3 of sample solution: it is in right amount each that precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or the herbal mixture containing Asian puccoon, by the extraction with aqueous solution of methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane or more organic solvent variable concentrations, filter, to obtain final product, v. method of testing: carry out efficient liquid phase chromatographic analysis is detect with circular dichroism spectra, record chromatogram, radix macrotomiae is different with the symbol of chromatographic peak in liquid phase-circular dichroism spectra chromatogram that gromwell root sample each wavelength place measures, specific features is as follows: at 220nm, 274nm, liquid phase-circular dichroism spectra chromatogram that 360nm and 550nm place measures, in radix macrotomiae, each chromatographic peak is negative peak, and in gromwell root, each chromatographic peak is posivtive spike, liquid phase-circular dichroism spectra the chromatogram measured at 245nm, 310nm and 430nm place, in radix macrotomiae, each chromatographic peak is posivtive spike, and in gromwell root, each chromatographic peak is negative peak, utilize positive and negative CHARACTERISTICS IDENTIFICATION radix macrotomiae and the gromwell root at the peak in circular dichroism chromatogram.
2. liquid phase-circular dichroism spectra the authentication method of a radix macrotomiae and gromwell root, it is characterized in that: i. chromatographic condition: chromatographic column is ScienhomeKromasilC18,4.6mm × 200mm, 5 μm and pre-column, mobile phase is acetonitrile (A)-0.3% phosphoric acid water (B), gradient elution process: 0-10 minute 70%, 10-30 minute 70%-75%, 30-31 minute 75%-95%, 31-36 minute 95%, 36-37 minute 95%-70%, 37-50 minute 70%, determined wavelength 274nm, 220nm, 245nm, 260nm, 310nm, 360nm, 430nm or 550nm, column temperature 30 DEG C, flow velocity 1.0mL/min, number of theoretical plate is not less than 3000 in naphthoquinones, the regression equation of its ultraviolet spectrum, the range of linearity, lowest detectable limit and quantitative limit meet metrical error requirement, ii. the collocation method 1 of sample solution: precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or in right amount each containing the herbal mixture of Asian puccoon, with methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the extraction with aqueous solution of cyclohexane or more organic solvent variable concentrations, reclaim extract, methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, the aqueous dissolution of cyclohexane or more organic solvent variable concentrations, through the little chromatographic column pre-service of SPE of filling dodecyl bonded silica gel, water or the removal of impurities of low-concentration organic solvent wash-out, continue with acetonitrile, methyl alcohol, ethyl acetate, acetone, chloroform, sherwood oil, the solution washing of cyclohexane or more organic solvent variable concentrations, merge eluent, methyl alcohol dissolves, constant volume, filter, obtain, iii. the collocation method 2 of sample solution: it is in right amount each that precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or the herbal mixture containing Asian puccoon, by the extraction with aqueous solution of methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane or more organic solvent variable concentrations, reclaim extract, methyl alcohol dissolves, constant volume, filter, to obtain final product, iv. the collocation method 3 of sample solution: it is in right amount each that precision takes the medicinal material of radix macrotomiae or gromwell root, extract, chemical composition monomer or the herbal mixture containing Asian puccoon, by the extraction with aqueous solution of methyl alcohol, chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, cyclohexane or more organic solvent variable concentrations, filter, to obtain final product, v. method of testing: carry out efficient liquid phase chromatographic analysis is detect with circular dichroism spectra, record chromatogram, radix macrotomiae is different with the symbol of chromatographic peak in liquid phase-circular dichroism spectra chromatogram that gromwell root sample each wavelength place measures, specific features is as follows: at 220nm, 274nm, liquid phase-circular dichroism spectra chromatogram that 360nm and 550nm place measures, in radix macrotomiae, each chromatographic peak is negative peak, and in gromwell root, each chromatographic peak is posivtive spike, liquid phase-circular dichroism spectra the chromatogram measured at 245nm, 310nm and 430nm place, in radix macrotomiae, each chromatographic peak is posivtive spike, and in gromwell root, each chromatographic peak is negative peak, wavelength is that 274nm place carries out UV detect, and records chromatogram, according to the calculated by peak area enantiomter total content of ultraviolet chromatographic peak, utilize the positive and negative of the peak in circular dichroism chromatogram, qualification radix macrotomiae and gromwell root.
3. authentication method according to claim 1 and 2, is characterized in that the method can also be used to qualification radix macrotomiae and gromwell root extract, the herbal mixture containing Asian puccoon, naphthoquinones constituents monomer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110285363.0A CN103175928B (en) | 2011-09-23 | 2011-09-23 | Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110285363.0A CN103175928B (en) | 2011-09-23 | 2011-09-23 | Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103175928A CN103175928A (en) | 2013-06-26 |
| CN103175928B true CN103175928B (en) | 2016-04-20 |
Family
ID=48635927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110285363.0A Active CN103175928B (en) | 2011-09-23 | 2011-09-23 | Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103175928B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106872595B (en) * | 2017-02-20 | 2019-04-16 | 中国计量科学研究院 | The measuring method of chiral molecules content based on circular dichroism technology |
| CN107412311A (en) * | 2017-07-19 | 2017-12-01 | 深圳弘汇生物医药有限公司 | The medical usage and preparation method of Asian puccoon α glucoside inhibiting activity extracts and combinations thereof |
| CN107561192B (en) * | 2017-10-19 | 2019-12-13 | 上海中医药大学 | Method for determining contents of R-goitrin and S-goitrin in isatis root medicinal material and product thereof |
| CN108802220A (en) * | 2018-06-12 | 2018-11-13 | 江苏颐海药业有限责任公司 | HPLC methods measure 3 kinds of naphthoquinones constituents contents and quality standard detecting method in Asian puccoon medicinal material |
| CN112432906B (en) * | 2020-09-17 | 2022-03-29 | 宁波大学 | Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372709A (en) * | 2007-08-24 | 2009-02-25 | 中国科学院大连化学物理研究所 | Method for rapid quantitatively evaluating interaction of medicament nucleic acids |
| CN101744880A (en) * | 2008-12-05 | 2010-06-23 | 天津天士力之骄药业有限公司 | Quality control method for total salvianolic acid injection |
-
2011
- 2011-09-23 CN CN201110285363.0A patent/CN103175928B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 密花滇紫草萘醌成分研究及紫草素含量测定;艾克蕙等;《植物学报》;19891231;第31卷(第7期);549-553 * |
| 软紫草与硬紫草萘醌类化学成分的研究;冯文文等;《中国现代中药》;20100731;第12卷(第7期);第15页左栏，第15-16页第1-3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103175928A (en) | 2013-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105203657B (en) | Semen Ziziphi Spinosae reference extract and its preparation method and application | |
| CN103175928B (en) | Liquid phase-circular dichroism spectra (LC-CD) authentication method of a kind of radix macrotomiae and gromwell root | |
| CN102998411B (en) | A kind of detection method of ageratum dripping pill | |
| CN104614456A (en) | Method for simultaneously detecting main components of Naoxintong capsule in plasma | |
| CN104297026A (en) | Method for extracting effective flavonoid components in traditional Chinese medicine pericarpium citri reticulatae | |
| CN102579612A (en) | Method for extracting total alkaloid of aconitum soongaricum | |
| CN105181847A (en) | Sculellaria barbata medicinal material and multi-information gradient thin-layer identification method of aqueous extract thereof | |
| CN105920096B (en) | Composition of red sage root and safflower, preparation method and application thereof | |
| CN104569192A (en) | Method for detecting quality of moghania macrophylla | |
| CN104090036B (en) | A kind of enrichment and the method detecting low concentration Anthraquinones effective constituent | |
| CN106680403A (en) | Method for detecting dendrophenol in Dendrobium officinale | |
| CN101791366A (en) | Method for testing quality of Discorea nipponica Makino in different places and medicinal materials of same genera | |
| CN104817445B (en) | A kind of isolated and purified physcione and method of rheum emodin from Rhizoma Polygoni Cuspidati | |
| CN103323558A (en) | Ginkgo biloba preparation analysis sample rapid preparation method based on solid phase extraction technology | |
| CN103245737A (en) | Detection method of anti-viral oral liquid | |
| CN103822888B (en) | Catch up with the quality determining method of yellow grass | |
| CN103145775B (en) | The preparation of high purity Herba Cleidion brevipetiolae glycosides A and quality controlling means thereof | |
| CN101703610A (en) | Quality detection method of Qingnao antihypertensive tablet | |
| CN101181339A (en) | Mass control method of aidi injection preparations | |
| CN104007220B (en) | A kind of method simultaneously detecting compound red beach wormwood sheet principal ingredient in blood plasma | |
| CN103709013B (en) | Separate purification method of unique ingredients in alamo gum and application thereof | |
| CN104807944A (en) | Preparing method of scorzonera general flavone and finger-print map thereof | |
| CN101991636A (en) | Detection method of Aidi injection preparation | |
| CN104892620B (en) | A kind of preparation method of high-purity karanjin | |
| CN104133028B (en) | A kind of method for building up of madder granule efficient liquid-phase chromatograph finger print atlas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160105 Address after: 215123 No. 18, Huashan Road, Xinbei District, Jiangsu, Changzhou Applicant after: CHANGZHOU BADIAN MEDICINE TECHNOLOGY CO., LTD. Address before: 264003 No. 1 Longevity Hill Road, Yantai Development Zone, Shandong Applicant before: Shandong Target Drug Research Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210706 Address after: Room C, unit 2F, C29 / F, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province, 215024 Patentee after: SUZHOU NANOMEDICINE R&D Co.,Ltd. Address before: 215123 No.18 Huashan Road, Xinbei District, Changzhou City, Jiangsu Province Patentee before: CHANGZHOU BADIAN MEDICINE TECHNOLOGY Co.,Ltd. |