CN103869010A - HPLC (High performance liquid chromatography) detection method for distinguishing different cultivars of uncaria - Google Patents
HPLC (High performance liquid chromatography) detection method for distinguishing different cultivars of uncaria Download PDFInfo
- Publication number
- CN103869010A CN103869010A CN201410065339.XA CN201410065339A CN103869010A CN 103869010 A CN103869010 A CN 103869010A CN 201410065339 A CN201410065339 A CN 201410065339A CN 103869010 A CN103869010 A CN 103869010A
- Authority
- CN
- China
- Prior art keywords
- hooks
- yncaria stem
- stem
- uncaria
- yncaria
- 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.)
- Granted
Links
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of analysis of traditional Chinese medicinal materials, and particularly discloses an HPLC (High performance liquid chromatography) method for distinguishing different cultivars of uncaria. All chromatographic peaks are separated effectively when the uncaria traditional Chinese medicinal materials are subjected to HPLC detection by optimizing each experimental parameter; the phenomena of mutual interference of the chromatographic peaks, trailing and the like are reduced; mutual interference of the chromatographic peaks of different ingredients in different cultivars of uncaria in HPLC detection is basically avoided, so as to provide basis for authenticating different cultivars of uncaria and constructing a uncaria HPLC fingerprint spectrum.
Description
Technical field
The invention belongs to Chinese crude drug analysis field, be specifically related to the not high-efficiency liquid chromatography method for detecting of yncaria stem with hooks of the same race of a kind of differentiation.
Background technology
Yncaria stem with hooks is the conventional Chinese medicine of tcm clinical practice, and its cold nature taste is sweet, returns liver, pericardium channel, can the flat liver of heat-clearing, the arresting convulsion that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., angiocardiopathy, the nervous system disease are had to certain curative effect.
The chemical composition of yncaria stem with hooks is many, and its chemical composition of the yncaria stem with hooks of different cultivars is also not quite similar.In yncaria stem with hooks contained main chemical compositions have indole alkaloids, triterpenes, flavonoids, Coumarins, quinoline sour saponins, phenols and organic acid etc., and the main active chemical of yncaria stem with hooks is biological alkali components at present.
Within 2010, version " Chinese Pharmacopoeia " regulation yncaria stem with hooks is madder wort yncaria stem with hooks
uncaria rhynchophylla(Miq.) Miq.ex Havil., largeleaf gambirplant branchlet
uncaria macrophyllawall., uncaria hirsuta
uncaria hirsutehavil., Uncaria sinensis
uncaria sinensis(Oliv.) Havil. and stockless fruit yncaria stem with hooks
uncaria sessilifructusroxb. dry buckle stem branch.But yncaria stem with hooks medicinal material is far above that these are several on the market at present, the yncaria stem with hooks of different cultivars, even the yncaria stem with hooks of same because growing environment, collecting season, job operation and storage requirement are different, its contained chemical composition, content, curative effect have larger difference.Yncaria stem with hooks medicinal material kind is many, and source is wide, and complicated component, is kind confusion, differentiates difficult medicinal material.At present, be mainly proterties and microscopic features and the thin-layer chromatography discriminating based on medicinal material for the discriminating of different cultivars yncaria stem with hooks medicinal material, but proterties and microscopic features are differentiated difficulty, and also subjective, and thin-layered chromatography also can not be differentiated yncaria stem with hooks kind effectively.Set up the conventional discrimination method of different cultivars yncaria stem with hooks, can more effectively carry out symptomatic treatment for different cultivars yncaria stem with hooks, and solve the present situation of yncaria stem with hooks kind confusion.Therefore, set up this routine discrimination method very urgently and there is realistic meaning.
Yncaria stem with hooks alkaloid is yncaria stem with hooks main pharmacodynamics composition, is also to set up the not composition that mainly will separate of yncaria stem with hooks of the same race of difference.At present, the main yncaria stem with hooks kind of China has now found that tens yncaria stem with hooks alkaloid components, although the composition that different cultivars yncaria stem with hooks contains is scarcely same, but because yncaria stem with hooks alkaloid is indole alkaloid, precursor structure is similar, and there are more homolog, isomers, some is spatial isomerism, application separating effect far above the HPLC technical point of TLC from, and distinguish also difficulty rather of different cultivars yncaria stem with hooks composition, affect the discriminating of follow-up yncaria stem with hooks kind and the foundation of finger-print thereof.Prior art urgently a kind of for different cultivars yncaria stem with hooks be all suitable for, the high-efficiency liquid chromatography method for detecting that heterogeneity chromatographic peak does not disturb each other, so that set up a kind of method of simple, practical evaluation different cultivars yncaria stem with hooks.
Summary of the invention
Goal of the invention of the present invention is to overcome prior art different cultivars yncaria stem with hooks medicinal material proximate component liquid chromatography overlap of peaks, be difficult to the technical barrier that separates or distinguish, a kind of different cultivars yncaria stem with hooks efficient liquid-phase chromatography method of distinguishing is provided, by optimizing each experiment parameter, effective separation of each chromatographic peak when realizing yncaria stem with hooks medicinal material HPLC in 1 hour and detecting, and substantially not the disturbing each other of heterogeneity chromatographic peak of realizing multiple yncaria stem with hooks HPLC, facilitate for differentiating the yncaria stem with hooks of different cultivars and building yncaria stem with hooks HPLC finger-print.
Above-mentioned purpose of the present invention is achieved by following technical solution:
A kind of different cultivars yncaria stem with hooks high-efficiency liquid chromatography method for detecting of distinguishing, comprises the following steps:
(1) prepare yncaria stem with hooks and detect solution:
(2) high performance liquid chromatography detects: by 5 μ m Platisil ODS-RP chromatographic columns, methyl alcohol-acetonitrile-damping fluid is mobile phase eluent, flow velocity: 1.0ml/min; Detect wavelength: 245nm and 226nm; Sample size: 20 μ l; Column temperature: 35 ℃; Gradient elution program is as follows:
Time 0min eluent components is methyl alcohol 12%, acetonitrile 24%, damping fluid 64%;
Time 15min eluent components is methyl alcohol 18%, acetonitrile 26%, damping fluid 56%;
Time 30min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
Time 60min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
Described damping fluid is that pH is greater than 7 phosphate buffer or concentration and is not less than 0.1% ammonia spirit;
(3) distinguish different cultivars yncaria stem with hooks: distinguish different cultivars yncaria stem with hooks according to the relative retention time of Characteristic chromatographic peak in the high-efficient liquid phase chromatogram of yncaria stem with hooks and characteristic absorption wavelength.
Those skilled in the art all know, the yncaria stem with hooks of different cultivars, even the yncaria stem with hooks of same is due to growing environment, collecting season, job operation is different with storage requirement, its contained chemical composition, content, curative effect has larger difference, thereby in prior art, not yet there is an a kind of method of the separation that the feature liquid chromatography peak of different cultivars yncaria stem with hooks all can or not be disturbed each other, but the method disclosed in the present is applicable to different cultivars yncaria stem with hooks sample, yncaria stem with hooks of the present invention comprises certified products yncaria stem with hooks (Uncaria rhynchophylla (Miq.) Jacks.), largeleaf gambirplant branchlet (U. macrophylla Wall.), Uncaria sinensis (U. sinensis (Oliv.) Havil.), stockless fruit yncaria stem with hooks (Uncaria sessilifructus Roxb.), uncaria hirsuta (Uncaria hirsute Havil.) or climb stem yncaria stem with hooks (U.scandens (Smith) Hutch).
As a kind of preferred version, yncaria stem with hooks detects solution preparation process and is specially: take yncaria stem with hooks medicinal material powder 0.5 ~ 1g, add chloroform: methyl alcohol: ammoniacal liquor mixed solvent 25ml, ultrasonic 40 ~ 50min, get filtrate, after low temperature is concentrated, residue is diluted to 20 ~ 25ml with 70% methanol aqueous solution, filter, get filtrate as detecting solution; Described chloroform: methyl alcohol: in ammonia mixed solvent, the volume ratio of chloroform, methyl alcohol and ammoniacal liquor is 15:5:0.5.Adopt mixed solvent ultrasonic extraction to be conducive to the preparation of yncaria stem with hooks detection solution.
As another kind of preferred version, yncaria stem with hooks detects solution preparation process and is specially: take yncaria stem with hooks medicinal material powder 0.5 ~ 1g, add the ultrasonic 40 ~ 50min of 70% methanol aqueous solution 20 ~ 25ml, filter, get filtrate as detecting solution.Directly adopt the ultrasonic yncaria stem with hooks of preparing of 70% methanol aqueous solution to detect solution, can omit the steps such as the concentrated dilution again of solvent evaporates, be conducive to simplify technique, cost-saving.
Preferably, described yncaria stem with hooks medicinal material powder is crossed 40 mesh sieves, is more conducive to ultrasonic extraction.
The composition of mobile phase eluent of the present invention and ratio unconventional selection, composition and ratio that those skilled in the art all know mobile phase eluent are one of key factors affecting the separation of following liquid-phase chromatographic peak, determining of flow phase eluent components and ratio need to could be optimized out by great many of experiments, different samples, the difference of its contained substance classes and content, its high performance liquid chromatography detected parameters also must corresponding optimizing and revising, otherwise easily occur the adverse consequencess such as chromatographic peak interference, hangover, distortion.Because the main separated component indole alkaloid of yncaria stem with hooks medicinal material composition is more, there is more isomers, steric isomer, the present invention not only requires the component separating in each sample good, and to make substantially not the disturbing each other of heterogeneity chromatographic peak of different cultivars yncaria stem with hooks, this is quite difficult, needs a large amount of experimental studies.
Preferably, the concentration of described phosphate buffer is 5 ~ 10 mmol/L.Inventor found through experiments, and mobile phase eluent is selected the phosphate buffer of variable concentrations, and on chromatogram, summit has larger impact, in the time that phosphate buffer density is lower, the peak width of chromatographic peak increases, and there will be peak shape hangover when serious, in the time of excessive concentration, can shorten the serviceable life of chromatographic column.
Differentiation different cultivars yncaria stem with hooks high-efficiency liquid chromatography method for detecting disclosed by the invention, can realize effective separation of chromatographic peak, do not occur that chromatographic peak is overlapping, the phenomenon such as hangover and distortion, and make substantially not the disturbing each other of heterogeneity chromatographic peak of different cultivars yncaria stem with hooks, utilize the main absorption peak position of the HPLC chromatogram of yncaria stem with hooks sample can distinguish the different cultivars of yncaria stem with hooks in conjunction with chromatographic peak characteristic absorption, concrete detection method is as follows simultaneously:
One, in the time that yncaria stem with hooks is certified products yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 9, retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (2) 0.55min/226nm, (3) 0.58min/226nm, (4) 0.83min/246nm, (isorhynchophylline, be called for short A) 0.87min/245nm, (5) 0.89min/245nm, (rhynchophyllin, be called for short B) 1.00min/246nm, (6) 1.02min/226nm, (7) 1.04min/226nm, (8) 1.09min/226.2nm; (chromatographic peak is numbered corresponding Figure of description 1).
Two, in the time that yncaria stem with hooks is largeleaf gambirplant branchlet, in HPLC chromatogram, Characteristic chromatographic peak has 4, and retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (A) 0.87min/245nm, (B) 1.00min/246nm, (9) 1.02min/245nm, (10) 1.09min/246nm; (chromatographic peak is numbered corresponding Figure of description 2).
Three, in the time that yncaria stem with hooks is uncaria hirsuta, in HPLC chromatogram, Characteristic chromatographic peak has 6, and retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (14) 0.77min/234nm, (15) 0.82min/244nm, (16) 0.84min/239nm, (17) 0.91min/245nm, (18) 0.98min/243nm, (19) 1.08min/246nm; (chromatographic peak is numbered corresponding Figure of description 3).
Four, in the time that yncaria stem with hooks is Uncaria sinensis, in HPLC chromatogram, Characteristic chromatographic peak has 3, and retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (11) 0.69min/242nm, (12) 0.85min/243nm, (13) 0.99min/245nm; (chromatographic peak is numbered corresponding Figure of description 4).
Five, in the time that yncaria stem with hooks is stockless fruit yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 2, and retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (A) 0.87min/245nm, (B) 1.00min/246nm; (chromatographic peak is numbered corresponding Figure of description 5).
Six, when yncaria stem with hooks is when climbing stem yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 4, and retention time and the characteristic absorption wavelength of its relative rhynchophyllin is respectively (11) 0.694min/242nm, (12) 0.85min/243nm, (13) 0.99min/245nm, (20) 0.49min/243nm; (chromatographic peak is numbered corresponding Figure of description 6).
Because all knowing in high-efficiency liquid chromatography method for detecting of the prior art, the technician of yncaria stem with hooks detection field can separate the composition chromatographic peak of certified products yncaria stem with hooks or a certain yncaria stem with hooks sample, but the composition of different cultivars is many, and the composition degree of approximation is high, existing efficient liquid-phase chromatography method is difficult for realizing not disturbing each other of heterogeneity chromatographic peak between different cultivars yncaria stem with hooks, thereby not yet occurs so far the high efficiency liquid phase method of relevant discriminating different cultivars yncaria stem with hooks.The present invention has just in time filled up the blank of prior art this respect, for the finger-print of identifying different cultivars yncaria stem with hooks or building other kind yncaria stems with hooks provides strong condition in the future.
Compared with prior art, the present invention has following beneficial effect:
(1) effective separation of each chromatographic peak when method of the present invention can be that realizing different cultivars yncaria stem with hooks medicinal material HPLC in 1 hour detects, reduce the phenomenons such as the mutual interference of chromatographic peak phase, hangover and distortion, and make substantially not the disturbing each other of heterogeneity chromatographic peak of different cultivars yncaria stem with hooks, for building yncaria stem with hooks HPLC finger-print and differentiating that the yncaria stem with hooks of different cultivars facilitates.This method has gathered the clearly each kind sample of Ji Yuan dozens of and has verified (each kind is only enumerated severally, sees Fig. 1 ~ 6) below, and identification result is all accurate.
(2) adopt methyl alcohol-acetonitrile-phosphate buffer (pH is greater than 7) or 0.1% ammoniacal liquor as mobile phase eluent, durability is good, does not disturb chromatographic peak absorption spectrum, can better protect pillar; In addition, adopt 0.1% ammoniacal liquor as mobile phase eluent, go for the liquid-phase sensors such as mass spectrum.
(3) methyl alcohol-acetonitrile-phosphate buffer (pH is greater than 7) or 0.1% ammoniacal liquor mobile phase ternary gradient can be converted into binary gradient, be convenient to different instrument application.Binary gradient is as follows:
Time 0min eluent components be A20%, B80%,
Time 15min eluent components be A30%, B70%,
Time 30min eluent components be A50%, B50%,
Time 50min eluent components be A50%, B50%,
Wherein A is 40% acetonitrile+60% methyl alcohol; B is 20% acetonitrile+80% water (damping fluid).
Accompanying drawing explanation
Fig. 1 is the HPLC comparison diagram of the certified products yncaria stem with hooks of separate sources; Wherein, S1 is Tianzhu County, Guizhou certified products yncaria stem with hooks, S2 is Jiangxi certified products yncaria stem with hooks, S3 is MeiZhou,GuangDong city certified products yncaria stem with hooks, S4 is MeiZhou,GuangDong great Pu county certified products yncaria stem with hooks, and S5 is Yangshan County, Shaoguan certified products yncaria stem with hooks, and S6 is Wuzhou, Guangxi certified products yncaria stem with hooks, S7 is Kweiyang certified products yncaria stem with hooks, and S8 is Nanning certified products yncaria stem with hooks.
Fig. 2 is the HPLC comparison diagram of the largeleaf gambirplant branchlet of separate sources; Wherein, S1 is Zhaoqing Guangdong Dinghu Hill largeleaf gambirplant branchlet, and S2 is GUIHAIA garden largeleaf gambirplant branchlet, and S3 is Mt.Luofushan's largeleaf gambirplant branchlet.
Fig. 3 is the HPLC comparison diagram of the uncaria hirsuta of separate sources; Wherein, S1 is that Zhaoqing Guangdong Dinghu Hill uncaria hirsuta hook stem, S2 are that Zhaoqing Guangdong Dinghu Hill uncaria hirsuta leaf, S3 are Guangxi uncaria hirsuta.
Fig. 4 is the HPLC comparison diagram of the Uncaria sinensis of separate sources; Wherein, S1 is that From Anshun of Guizhou Uncaria sinensis, S2 are that Chongqing Sichuan Jinfo Shan Mountain Uncaria sinensis, S3 are Guizhou Jianhe Uncaria sinensis.
Fig. 5 is the HPLC comparison diagram of the stockless fruit yncaria stem with hooks of separate sources; Wherein, S1 is that GUIHAIA garden stockless fruit yncaria stem with hooks, S2 are Colleges Of Traditional Chinese Medicine Of Guangxi's medicine garden stockless fruit yncaria stem with hooks.
Fig. 6 is the HPLC comparison diagram of climbing stem yncaria stem with hooks of separate sources; Wherein, S1 is that stem yncaria stem with hooks is climbed in southern medical science institute medicine garden, Guangzhou, S2 is that stem yncaria stem with hooks hook is climbed in South China Botanical Garden, S3 is that stem yncaria stem with hooks leaf is climbed in South China Botanical Garden.
Fig. 7 is the HPLC comparison diagram of different cultivars yncaria stem with hooks; Wherein, S1 is that uncaria hirsuta, S3 are that stockless fruit yncaria stem with hooks, S4 are that Uncaria sinensis, S5 are that largeleaf gambirplant branchlet, S6 are certified products yncaria stem with hooks for climbing stem yncaria stem with hooks, S2.
Fig. 8 is the HPLC comparison diagram that comparative example 1 changes certified products yncaria stem with hooks after mobile phase ratio; Wherein, S1 is collection of illustrative plates, for a change collection of illustrative plates of mobile phase ratio of S2 that does not change mobile phase ratio.
Fig. 9 is the HPLC comparison diagram that comparative example 2 changes certified products yncaria stem with hooks after mobile phase composition; Wherein, S1 is collection of illustrative plates, for a change collection of illustrative plates of mobile phase composition of S2 that does not change mobile phase composition.
Figure 10 is the HPLC comparison diagram that comparative example 3 changes certified products yncaria stem with hooks after mobile phase composition; Wherein, S1 is collection of illustrative plates, for a change collection of illustrative plates of mobile phase composition of S2 that does not change mobile phase composition.
Figure 11 is the HPLC comparison diagram of comparative example 4 variable concentrations phosphate buffers.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.Unless stated otherwise, in embodiment, related reagent, method is the conventional reagent in this area and method.
one, prepare yncaria stem with hooks and detect solution
(1) precision took 40 object yncaria stem with hooks medicinal material powder 0.5g, added chloroform: methyl alcohol: ammoniacal liquor mixed solvent 25ml, and ultrasonic 45min, gets filtrate, and after low temperature is concentrated, residue is diluted to 25ml with 70% methanol aqueous solution, filters, and gets filtrate as detecting solution; Described chloroform: methyl alcohol: in ammonia mixed solvent, the volume ratio of chloroform, methyl alcohol and ammoniacal liquor is 15:5:0.5.
two, high performance liquid chromatography detects
Select Platisil ODS-RP chromatographic column (250mm × 4.6mm, 5 μ m, ser#5032395), and the general guard column of Phenomenex SecurityGuard (4mm × 3mm, 5 μ are m); Methyl alcohol-acetonitrile-0.1% ammoniacal liquor buffer solution is mobile phase eluent, flow velocity: 1.0ml/min; Detect wavelength: 245nm and 226nm; Sample size: 20 μ l; Column temperature: 35 ℃; Gradient elution program is as follows:
Time 0min eluent components is methyl alcohol 12%, acetonitrile 24%, damping fluid 64%;
Time 15min eluent components is methyl alcohol 18%, acetonitrile 26%, damping fluid 56%;
Time 30min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
Time 60min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
The pH value of described damping fluid is greater than 7, and concentration is 5 mmol/L phosphate buffers.
embodiment 1
Adopt the method for above-mentioned steps one, preparation source place is respectively MeiZhou,GuangDong great Pu county, Tianzhu County, Guizhou, MeiZhou,GuangDong city, Guiyang City, Guizhou, Yangshan County, Shaoguan, Wuzhou, Guangxi, certified products yncaria stem with hooks (all differentiate the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) the stem hook in Nanning and Jiangxi detects 7 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 1.
Adopt the method for above-mentioned steps one, preparation source place is respectively Zhaoqing Guangdong Dinghu Hill, GUIHAIA garden, the emerging largeleaf gambirplant branchlet of Yunfu, guangdong Province (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) stem hook detects 3 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 2.
Adopt the method for above-mentioned steps one, preparation source place is respectively that uncaria hirsuta stem hook and leaf, the Nanning uncaria hirsuta of Dinghushan, guangdong (all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) detect 3 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 3.
Adopt the method for above-mentioned steps one, preparation source place is respectively From Anshun of Guizhou, Jinfo of Chongqing, the Uncaria sinensis of zunyi, guizhou (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) stem hook detects 3 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 4.
Adopt the method for above-mentioned steps one, preparation source place is respectively that the stockless fruit yncaria stem with hooks in GUIHAIA garden, Colleges Of Traditional Chinese Medicine Of Guangxi's medicine garden (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) stem hook and leaf detect 3 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 5.
Adopt the method for above-mentioned steps one, preparation source place is respectively Guangzhou Nanfang Medical Univ medicine garden, the stem yncaria stem with hooks of climbing of South China Botanical Garden (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) stem hook and leaf detect 3 parts of solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, chromatogram as shown in Figure 6.
embodiment comparative analysis:
(1) S1 in Fig. 1 ~ 6 being reconfigured is that a figure contrasts relatively, as shown in Figure 7, as can be seen from Figure 7, adopt the inventive method to detect eluting peak in the high-efficient liquid phase chromatogram that the yncaria stem with hooks of six kinds of different cultivars obtains and separate obviously, do not have that chromatographic peak is overlapping, a phenomenon such as hangover and distortion.
(2) from Fig. 1 ~ 6 and Fig. 7 we it can also be seen that the quantity at different cultivars yncaria stem with hooks spectrum peak and go out peak position and all have difference just can be distinguished the kind of yncaria stem with hooks in conjunction with chromatographic peak spectrogram.Be to detect in conjunction with LC-MS the diagnostic characteristics composition of determining different cultivars yncaria stem with hooks below, concrete outcome is as follows:
In Fig. 1 all there are 9 characteristic peaks in the certified products yncaria stem with hooks on separate sources ground, concrete composition is respectively 3 α-cadambine alkali (2), yohimbine (3), isocorynoxeine (4), rhynchophyllin (A), corynoxeine (5), isorhynchophylline (B), hirsutine (6) seam seed piperazine methyl ether, (7) hirsuteine (8), is the common characteristic composition of certified products yncaria stem with hooks.
In Fig. 2 all there are 4 characteristic peaks in the largeleaf gambirplant branchlet on separate sources ground, and concrete composition is rhynchophyllin (A), and isorhynchophylline (B), ocrynoxine (9) and corynoxine B (10), be the common characteristic composition of largeleaf gambirplant branchlet.
In Fig. 3 all there are 6 characteristic peaks in the uncaria hirsuta of separate sources, concrete composition is respectively different mitragynine (14), isoformosanine (15), mitragynine (16), formosamine (17), the iso-19-table-ajmalicine of 3-(18), 19-epi-ajmalicine(19), be uncaria hirsuta common characteristic composition.
In Fig. 4 all there are 3 characteristic peaks in the Uncaria sinensis on separate sources ground, concrete composition is respectively different wing handle rhynchophyllin (11), wing handle rhynchophyllin (12), unacarine F(13), these characteristic components also have climbing stem yncaria stem with hooks, also have glabratine(20 but climb stem yncaria stem with hooks) composition chromatographic peak.
In Fig. 5 only there is rhynchophyllin (A) and isorhynchophylline (B) in the stockless fruit yncaria stem with hooks on separate sources ground, but other composition is not obvious, therefore this two composition is the common characteristic composition of stockless fruit yncaria stem with hooks.
In Fig. 6 all there are 4 characteristic peaks in the stem yncaria stem with hooks of climbing of separate sources, and concrete composition is different wing handle rhynchophyllin (11), wing handle rhynchophyllin (12), unacarine F(13), glabratine(20), be the common characteristic composition of climbing stem yncaria stem with hooks.
comparative example 1
Adopt the method for above-mentioned steps one, preparation source place is respectively that the certified products yncaria stem with hooks in Jiangxi (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) detection solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, but step 2 gradient elution program is made the corresponding changes, be specially: when elution time 30min changes time 37min into, eluent components is methyl alcohol 48%, acetonitrile 20%, 0.1% ammoniacal liquor 32%; When elution time 50min changes 53min into, eluent components is methyl alcohol 56%, acetonitrile 20%, 0.1% ammoniacal liquor 24%; Other conditions are constant, liquid chromatogram as shown in Figure 8, as can be seen from Figure 8, due to the variation of mobile phase, cause rhynchophyllin (B) and (different) Conan below because of the peak of alkali (6) inseparable; If change 53min into methyl alcohol 48%, acetonitrile 20%, 0.1% ammoniacal liquor 32%; Separation has certain improvement, but rhynchophyllin (B) still can not be with below (different) Conan separate because of the peak of alkali (6).
comparative example 2
Adopt the method for above-mentioned steps one, preparation source place is respectively that the certified products yncaria stem with hooks in Jiangxi (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) detection solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, but acetonitrile part in step 2 mobile phase eluent is replaced with to methyl alcohol, other conditions are constant, liquid chromatogram as shown in Figure 9, as can be seen from Figure 9, rhynchophyllin and three chromatographic peak compositions below thereof only separate two peaks, and the separation of chromatographic peak does not obviously reach requirement; Continue repeatedly to optimize on this basis, be also difficult in 80min to separate completely these chromatographic peaks below, especially rhynchophyllin and the one-tenth swarming that closes on thereof.
comparative example 3
Adopt the method for above-mentioned steps one, preparation source place is respectively that the certified products yncaria stem with hooks in Jiangxi (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) detection solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, but methyl alcohol part in step 2 mobile phase eluent is replaced with to acetonitrile, other conditions are constant, liquid chromatogram as shown in figure 10, as can be seen from Figure 10, in 1 hour, rhynchophyllin three chromatographic peak compositions below only separate two peaks, in addition before certified products yncaria stem with hooks, comprise that 1～No. 3 peak can not get good separation, therefore the separation of chromatographic peak does not reach requirement.
comparative example 4
Adopt the method for above-mentioned steps one, preparation source place is respectively that the certified products yncaria stem with hooks in Jiangxi (is all differentiated the discriminating of expert's proterties through plant, and identify through DNA molecular authentication technique) detection solution, adopt the method for step 2 to carry out high performance liquid chromatography detection, but change the concentration of phosphate buffer in step 2 mobile phase eluent into 2.5 mmol/L, 5 mmol/L, 10 mmol/L, 20 mmol/L, other conditions are constant, liquid chromatogram as shown in figure 11, as can be seen from Figure 11, when the concentration of phosphate buffer is 2.5 mmol/L, in the chromatogram that detection obtains, peak width increases, peak shape has hangover, and the concentration of phosphate buffer is while being 20 mmol/L, easily shorten the life-span of chromatographic column, thereby optimal conditions is that the concentration of phosphate buffer is 5 ~ 10 mmol/L, chromatographic peak is sharp-pointed, chromatographic peak does not trail.
comparative example interpretation of result:
In comparative example 1 ~ 4, changed respectively the composition of mobile phase or proportioning, high performance liquid chromatography detected parameters, the high efficiency liquid phase of yncaria stem with hooks detects and has all occurred overlap of peaks or conditions of streaking, and chromatographic peak cannot fully separate; This separation that can affect subsequent characteristics composition is identified, causes difficulty to the structure of yncaria stem with hooks finger-print or the evaluation of different cultivars yncaria stem with hooks.The not alkaloid component difference of yncaria stem with hooks of the same race, but there is more isomers, even steric isomer, above-mentioned comparative example is mainly the separation for the each chromatographic peak of certified products yncaria stem with hooks, also to guarantee every kind of each component separating of yncaria stem with hooks below, main is on the basis of the good every kind of each chromatographic peak of yncaria stem with hooks sample of same gradient separations in fact, also will optimize gradient through a large amount of research experiments does not disturb yncaria stem with hooks chromatographic peak not of the same race each other, differentiate completing yncaria stem with hooks not of the same race within a short period of time (1 hour), so the composition of mobile phase of the present invention and proportioning are through great many of experiments, test just can find out.
Claims (7)
1. distinguish a different cultivars yncaria stem with hooks high-efficiency liquid chromatography method for detecting, it is characterized in that, comprise the following steps:
(1) prepare yncaria stem with hooks and detect solution;
(2) high performance liquid chromatography detects: by 5 μ m ODS-RP chromatographic columns, methyl alcohol-acetonitrile-damping fluid is mobile phase eluent, flow velocity: 1.0ml/min; Detect wavelength: 245nm and 226nm; Sample size: 20 μ l; Column temperature: 35 ℃; Gradient elution program is as follows:
Time 0min eluent components is methyl alcohol 12%, acetonitrile 24%, damping fluid 64%;
Time 15min eluent components is methyl alcohol 18%, acetonitrile 26%, damping fluid 56%;
Time 30min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
Time 60min eluent components is methyl alcohol 30%, acetonitrile 30%, damping fluid 40%;
Described damping fluid is that pH is greater than 7 phosphate buffer or concentration and is not less than 0.1% ammonia spirit;
(3) distinguish different cultivars yncaria stem with hooks: distinguish different cultivars yncaria stem with hooks according to the relative retention time of Characteristic chromatographic peak in the high-efficient liquid phase chromatogram of yncaria stem with hooks and characteristic absorption wavelength.
2. method according to claim 1, it is characterized in that, yncaria stem with hooks detects solution preparation and is specially: take yncaria stem with hooks medicinal material powder 0.5 ~ 1g, add chloroform: methyl alcohol: ammoniacal liquor mixed solvent 25ml, ultrasonic 40 ~ 50min, gets filtrate, after low temperature is concentrated, residue is diluted to 20 ~ 25ml with 70% methanol aqueous solution, filters, and gets filtrate as detecting solution; Described chloroform: methyl alcohol: in ammonia mixed solvent, the volume ratio of chloroform, methyl alcohol and ammoniacal liquor is 15:5:0.5.
3. method according to claim 1, is characterized in that, yncaria stem with hooks detects solution preparation and is specially: take yncaria stem with hooks medicinal material powder 0.5 ~ 1g, add the ultrasonic 40 ~ 50min of 70% methanol aqueous solution 20 ~ 25ml, filter, get filtrate as detecting solution.
4. method according to claim 1, is characterized in that, described yncaria stem with hooks comprises certified products yncaria stem with hooks, largeleaf gambirplant branchlet largeleaf gambirplant branchlet, Uncaria sinensis Uncaria sinensis, stockless fruit yncaria stem with hooks, uncaria hirsuta or climbs stem yncaria stem with hooks.
5. method according to claim 1, is characterized in that, the concentration of described phosphate buffer is 5 ~ 10 mmol/L.
6. method according to claim 1, is characterized in that, the concrete grammar of the described differentiation different cultivars of step (3) yncaria stem with hooks is:
In the time that yncaria stem with hooks is certified products yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 9, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.55min/226nm, 0.58min/226nm, 0.83min/246nm, 0.87min/245nm, 0.89min/245nm, 1.00min/246nm, 1.02min/226nm, 1.04min/226nm, 1.09min/226.2nm;
In the time that yncaria stem with hooks is largeleaf gambirplant branchlet, in HPLC chromatogram, Characteristic chromatographic peak has 4, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.87min/245nm, 1.00min/246nm, 1.02min/245nm, 1.09min/246nm;
In the time that yncaria stem with hooks is uncaria hirsuta, in HPLC chromatogram, Characteristic chromatographic peak has 6, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.77min/234nm, 0.82min/244nm, 0.84min/239nm, 0.91min/245nm, 0.98min/243nm, 1.08min/246nm;
In the time that yncaria stem with hooks is Uncaria sinensis, in HPLC chromatogram, Characteristic chromatographic peak has 3, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.69min/242nm, 0.85min/243nm, 0.99min/245nm;
In the time that yncaria stem with hooks is stockless fruit yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 2, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.87min/245nm, 1.00min/246nm;
When yncaria stem with hooks is when climbing stem yncaria stem with hooks, in HPLC chromatogram, Characteristic chromatographic peak has 4, and the retention time of its relative rhynchophyllin and characteristic absorption wavelength are respectively 0.694min/242nm, 0.85min/243nm, 0.99min/245nm, 0.49min/243nm.
7. method according to claim 1, is characterized in that, described yncaria stem with hooks pulverizing medicinal materials is crossed 40 mesh sieves.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410065339.XA CN103869010B (en) | 2014-02-25 | 2014-02-25 | A kind of high-efficiency liquid chromatography method for detecting distinguishing different cultivars yncaria stem with hooks |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410065339.XA CN103869010B (en) | 2014-02-25 | 2014-02-25 | A kind of high-efficiency liquid chromatography method for detecting distinguishing different cultivars yncaria stem with hooks |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103869010A true CN103869010A (en) | 2014-06-18 |
| CN103869010B CN103869010B (en) | 2015-12-09 |
Family
ID=50907771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410065339.XA Active CN103869010B (en) | 2014-02-25 | 2014-02-25 | A kind of high-efficiency liquid chromatography method for detecting distinguishing different cultivars yncaria stem with hooks |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103869010B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359853A (en) * | 2014-11-10 | 2015-02-18 | 华润三九医药股份有限公司 | Method for quickly detecting ramulus uncariae cum uncis by utilizing near-infrared spectrometry and application of method |
| CN105301136A (en) * | 2015-11-23 | 2016-02-03 | 江苏康缘药业股份有限公司 | Nine-element wind-extinguishing particle component quantitative detection method and fingerprint construction method |
| CN105588890A (en) * | 2015-11-19 | 2016-05-18 | 贵阳中医学院 | Fingerprint spectrum detection method of uncaria hirsuta |
| CN107764908A (en) * | 2016-08-23 | 2018-03-06 | 天津天士力现代中药资源有限公司 | A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste |
| CN108627581A (en) * | 2018-03-01 | 2018-10-09 | 广州王老吉药业股份有限公司 | Rhynchophyllin and isorhynchophylline content assaying method in a kind of Xiao ' erqixingcha particle |
| CN109725098A (en) * | 2019-03-04 | 2019-05-07 | 陕西中烟工业有限责任公司 | A kind of flue gas rhynchophyllin rate of transform and filter tip rhynchophyllin rejection measuring method |
| CN110196293A (en) * | 2019-05-23 | 2019-09-03 | 广西壮族自治区中医药研究院 | A kind of content assaying method of largeleaf gambirplant branchlet extract total alkaloid |
| CN110927313A (en) * | 2019-12-04 | 2020-03-27 | 广东一方制药有限公司 | Construction method and identification method of UPLC characteristic maps of different primitive ligusticum medicinal materials |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013056868A (en) * | 2011-09-09 | 2013-03-28 | Asia Kankyo Kenkyusho:Kk | Physiologically active liquid composition and method for producing the same |
-
2014
- 2014-02-25 CN CN201410065339.XA patent/CN103869010B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013056868A (en) * | 2011-09-09 | 2013-03-28 | Asia Kankyo Kenkyusho:Kk | Physiologically active liquid composition and method for producing the same |
Non-Patent Citations (4)
| Title |
|---|
| RADOSŁAW PILARSKI ET AL.: "Antioxidant activity of ethanolic and aqueous extracts of Uncaria tomentosa (Willd.) DC.", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| 杨秀娟 等: "HPLC测定钩藤中钩藤碱和异钩藤碱的方法学探讨", 《中国中药杂志》 * |
| 郭星 等: "HPLC法测定不同产地钩藤中异钩藤碱和钩藤碱的含量", 《河南中医》 * |
| 黄艳萍 等: "钩藤的HPLC指纹图谱研究", 《中药新药与临床药理》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359853A (en) * | 2014-11-10 | 2015-02-18 | 华润三九医药股份有限公司 | Method for quickly detecting ramulus uncariae cum uncis by utilizing near-infrared spectrometry and application of method |
| CN105588890A (en) * | 2015-11-19 | 2016-05-18 | 贵阳中医学院 | Fingerprint spectrum detection method of uncaria hirsuta |
| CN105301136A (en) * | 2015-11-23 | 2016-02-03 | 江苏康缘药业股份有限公司 | Nine-element wind-extinguishing particle component quantitative detection method and fingerprint construction method |
| CN105301136B (en) * | 2015-11-23 | 2017-05-03 | 江苏康缘药业股份有限公司 | Nine-element wind-extinguishing particle component quantitative detection method and fingerprint construction method |
| CN107764908A (en) * | 2016-08-23 | 2018-03-06 | 天津天士力现代中药资源有限公司 | A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste |
| CN107764908B (en) * | 2016-08-23 | 2021-09-21 | 天津天士力现代中药资源有限公司 | Method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract |
| CN108627581A (en) * | 2018-03-01 | 2018-10-09 | 广州王老吉药业股份有限公司 | Rhynchophyllin and isorhynchophylline content assaying method in a kind of Xiao ' erqixingcha particle |
| CN109725098A (en) * | 2019-03-04 | 2019-05-07 | 陕西中烟工业有限责任公司 | A kind of flue gas rhynchophyllin rate of transform and filter tip rhynchophyllin rejection measuring method |
| CN110196293A (en) * | 2019-05-23 | 2019-09-03 | 广西壮族自治区中医药研究院 | A kind of content assaying method of largeleaf gambirplant branchlet extract total alkaloid |
| CN110927313A (en) * | 2019-12-04 | 2020-03-27 | 广东一方制药有限公司 | Construction method and identification method of UPLC characteristic maps of different primitive ligusticum medicinal materials |
| CN110927313B (en) * | 2019-12-04 | 2021-09-07 | 广东一方制药有限公司 | Construction method and identification method of UPLC characteristic maps of different primitive ligusticum medicinal materials |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103869010B (en) | 2015-12-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103869010B (en) | A kind of high-efficiency liquid chromatography method for detecting distinguishing different cultivars yncaria stem with hooks | |
| CN103776926B (en) | The foundation of rabdosia lophanthide HPLC-FPS | |
| CN102579612B (en) | Method for extracting total alkaloid of aconitum soongaricum | |
| CN102058641B (en) | Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract | |
| CN102370891A (en) | Method for authenticating dendrobium officinale by using HPLC fingerprint | |
| CN104833749A (en) | Finger-print spectrum construction method and quality detection method of chrysanthemum cell-disruption decoction pieces | |
| CN103558329B (en) | Method for establishing fingerprint of leech and standard fingerprint of leech | |
| CN103604877B (en) | A kind of efficient liquid-phase chromatograph finger print atlas construction method of stem of noble dendrobium alkaloids composition and application | |
| CN101637567A (en) | Rhizoma ligustici wallichii quality control method in Chinese medicine preparation | |
| CN103288846A (en) | Method for extracting and purifying total physalin from physalis plants | |
| CN101078713B (en) | Fingerprint detection method of gynostemma pentaphylla medicine added with internal standard | |
| CN104849363A (en) | Cordate houttuynia wall-breaking decoction pieces fingerprinting construction and quality detection method thereof | |
| CN102967670A (en) | Method for measuring cordycepin, adenosine and mannitol in cordyceps sinensis mycelium powder | |
| CN103575821A (en) | Detection method of 14 chemical components in Tangminling preparation | |
| CN107807184B (en) | Application of triptolide as biomarker of toxic honey | |
| CN104849362A (en) | Erigeron breviscapus wall-breaking decoction pieces fingerprinting common model construction and quality detection method thereof | |
| CN101266233B (en) | Zanthoxylum dissitum Hemsl medicinal materials discrimination method | |
| CN110003352A (en) | A kind of extracting method that moringa seeds selenium active constituent is effectively kept | |
| CN102028760A (en) | Quality detecting method for polygonum perfoliatum | |
| CN111337592B (en) | Method for simultaneously measuring multicomponent content in rhodiola sachalinensis | |
| CN101270118B (en) | Technique for spherosinin purification with continuous column chromatography | |
| CN104007220A (en) | Method for simultaneously detecting main components of compound Danlou tablet in plasma | |
| CN102419350B (en) | Method for carrying out simultaneous quantitative analysis on four lignan components in Chinese magnoliavine raw material and Chinese magnoliavine extract | |
| CN103630637B (en) | Identification method for Tibetan herb Saibei Corydalis | |
| CN106568856A (en) | Fingerprint detection method for detecting effective part of lithocarpus polysachyus rehd leaf |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 510240 40 Haizhuqu District, Guangdong, Guangzhou. Patentee after: Guangdong Pharmaceutical University Address before: 510006 No. 280 East Ring Road, Guangzhou City University, Guangdong Patentee before: Guangdong Pharmaceutical University |