CN105203486B - A kind of detection method for CAULIS MARSDENIAE TENACISSIMAE - Google Patents
A kind of detection method for CAULIS MARSDENIAE TENACISSIMAE Download PDFInfo
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- CN105203486B CN105203486B CN201510520828.4A CN201510520828A CN105203486B CN 105203486 B CN105203486 B CN 105203486B CN 201510520828 A CN201510520828 A CN 201510520828A CN 105203486 B CN105203486 B CN 105203486B
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- caulis marsdeniae
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Abstract
The invention belongs to the technical field of Chinese medicine detection, a kind of detection method for CAULIS MARSDENIAE TENACISSIMAE is specifically related to.This kind is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE, comprises the following steps：（1）The preparation of need testing solution, reference substance solution and control medicinal material solution；（2）The discriminating of chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H；（3）The content detection of chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H；（4）Set up using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as object of reference characteristic fingerprint pattern.The detection method differentiates simultaneously to chlorogenic acid in Marsdenia tenacissima, CAULIS MARSDENIAE TENACISSIMAE glycosides H, the content of two kinds of compositions is detected simultaneously, and set up using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as object of reference characteristic fingerprint pattern, medicinal material, water extract, Xiaoaiping preparation against cancers can be applied to simultaneously, and stability is good, easy to operate, disposably detect two kinds of compositions, specificity is strong, saves detection time, greatly improves detection precision and sensitivity.
Description
Technical field
The invention belongs to the technical field of Chinese medicine detection, a kind of detection method for CAULIS MARSDENIAE TENACISSIMAE is specifically related to.
Background technology
CAULIS MARSDENIAE TENACISSIMAE is Asclepiadaceae plant, also known as glaucescent fissistigma root etc., first recorded in《The southern regions of the Yunnan Province book on Chinese herbal medicine》, medicinal material standard recorded in 1974
Version《Yunnan Province's drug standards》, 1977 years versions《Chinese Pharmacopoeia》And version in 2010《Chinese Pharmacopoeia》.The side of CAULIS MARSDENIAE TENACISSIMAE is detected at present
Method mainly includes the methods such as Characters Identification, Microscopic Identification, thin layer identification, physics and chemistry identification and assay.Assay mainly leads to
Cross AAS or such as《The method for building up and application of CAULIS MARSDENIAE TENACISSIMAE medicinal material and preparation finger》(number of patent application
CN201410119474.8 contents of steroid saponin in CAULIS MARSDENIAE TENACISSIMAE) is determined using HPLC-ELSD methods in a text, also had《CAULIS MARSDENIAE TENACISSIMAE medicine
The method for building up and application of material and preparation finger》The use HPLC- that (number of patent application CN201310684459.3) is mentioned
ELSD methods determine phenolic acid class content in CAULIS MARSDENIAE TENACISSIMAE.
Patent《A kind of detection method of Glaucescent fissistigma root saponin》(number of patent application CN201110232427.0) thinks：" black bone
Rattan saponin(e does not have significant characteristic absorption in ultraviolet region, therefore is difficult that accurate accurately survey is carried out to wherein chemical composition content
It is fixed ", " UV-detector is selected, due to Glaucescent fissistigma root saponin in ultraviolet region without characteristic absorption, therefore must from this method detection
Have the shortcoming that specificity is poor, error is big.”
Defect from the above in the presence of prior art is as follows：
1st, in the prior art merely by certain single composition, one in such as chlorogenic acid or CAULIS MARSDENIAE TENACISSIMAE glycosides H it is single into
It is divided into index to differentiate and control CAULIS MARSDENIAE TENACISSIMAE content, so for the measure accuracy of CAULIS MARSDENIAE TENACISSIMAE content, stability and sensitive
Degree can not reach optimal effect, so that the quality of medicinal material and preparation can not be controlled very well；
2nd, prior art thinks there is that specificity is poor using HPLC-UV detections CAULIS MARSDENIAE TENACISSIMAE glycosides H, the big defect of error, thus
Do not use HPLC-UV detection method, but select HPLC-ELSD side of the sensitivity with detection limit much smaller than HPLC-UV
Method is detected；
3rd, the object of prior art detection is medicinal material or preparation, not yet has a kind of detection method to be applied to medicinal material, water
The detection of CAULIS MARSDENIAE TENACISSIMAE content in extract and preparation.
The content of the invention
It is an object of the invention to provide a kind of detection method for CAULIS MARSDENIAE TENACISSIMAE for above-mentioned defect, the inspection
Survey method differentiates simultaneously to chlorogenic acid in Marsdenia tenacissima, CAULIS MARSDENIAE TENACISSIMAE glycosides H, while detecting the content of two kinds of compositions, and sets up
Using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as the characteristic fingerprint pattern of object of reference, medicinal material, water extract, the flat system of the cancer that disappears can be applied to simultaneously
Agent, stability is good, easy to operate, disposably two kinds of compositions of detection, and specificity is strong, saves detection time, greatly improves detection accurate
Degree and sensitivity.
The technical scheme is that：A kind of detection method for CAULIS MARSDENIAE TENACISSIMAE, comprises the following steps：
(1) preparation of need testing solution, reference substance solution and control medicinal material solution：Prepare respectively need testing solution, 2~
6mg/ml chlorogenic acids reference substance solution, 0.1~0.5mg/ml CAULIS MARSDENIAE TENACISSIMAEs glycosides H reference substance solutions and CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution；
(2) chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H discriminating：Need testing solution, chlorogenic acid reference substance solution, CAULIS MARSDENIAE TENACISSIMAE glycosides H are taken respectively
Reference substance solution, CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution 10ul, put in same silica gel g thin-layer plate, then prepare solvent, take solvent
Upper solution be placed on lamellae；Standing treats that upper solution is deployed, and lamellae is taken out and dried；Will after lamellae dries
Lamellae is placed under ultraviolet lamp, is inspected under conditions of wavelength 365nm, first the chromatogram of need testing solution respectively with it is green
Ortho acid reference substance solution, the chromatogram of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution show the fluorescence spot or main spot of same color on relevant position
Point；Then developed the color using mass concentration for 5% sulfuric acid vanillic aldehyde test solution, be heated to clear spot, the chromatogram of need testing solution
Show the spot of same color on relevant position with CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substance solutions, the chromatogram of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution respectively
Point or principal spot；
(3) chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H content detection：Take need testing solution, chlorogenic acid reference substance solution and CAULIS MARSDENIAE TENACISSIMAE glycosides
H reference substance solutions are detected using HPLC-UV；Wherein mobile phase is acetonitrile and 0.5% phosphate aqueous solution, and Detection wavelength is
190~220nm；
(4) set up using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as object of reference characteristic fingerprint pattern.
Solvent in the step (2) is made up of butyl acetate, formic acid and water.
The butyl acetate：Formic acid：The volume ratio of water is 14:5:5.
Detection wavelength in the step (3) is 201nm.
The HPLC-UV fillers used in the step (3) is octadecylsilane chemically bonded silicas.
Using eluent gradient elution in the step (3), wherein time point of gradient change mobile phase ratio and should
The volume ratio of the acetonitrile and 0.5% phosphate aqueous solution of time point setting is as follows：During 0min, acetonitrile and 0.5% phosphate aqueous solution
Volume ratio is 15:85；During 5min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 8:92；During 50min, acetonitrile and 0.5%
The volume ratio of phosphate aqueous solution is 80:20；During 55min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:85；60min
When, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:85.
The detection method is applied to CAULIS MARSDENIAE TENACISSIMAE medicinal material, CAULIS MARSDENIAE TENACISSIMAE water extract and Xiaoaiping preparation against cancers.
Beneficial effects of the present invention are：Detection method (1) of the present invention for CAULIS MARSDENIAE TENACISSIMAE disposably detect chlorogenic acid,
Two kinds of compositions of CAULIS MARSDENIAE TENACISSIMAE glycosides H, specificity is strong, easy to detect, easy to be time saving compared with prior art, saves testing cost and people
Work, increases substantially detection and analysis ability, while detecting two kinds of index components, more can effectively control the quality of product, improve inspection
Survey the degree of accuracy；(2) using the detection of HPLC-UV methods, do not occur that specificity is poor, the big defect of error, while improving detection
Sensitivity and detection limit；(3) detection method can detect CAULIS MARSDENIAE TENACISSIMAE medicinal material, CAULIS MARSDENIAE TENACISSIMAE water extract and CAULIS MARSDENIAE TENACISSIMAE system simultaneously
Agent, can preferably control the quality of whole production link；(4) prior art Content of Chlorogenic Acid is all detected in 327nm or so, and this hair
It is bright to breach intrinsic notion, detected, worked well at 190~220nm.
Brief description of the drawings
Fig. 1 is that specific embodiment of the invention Content of Chlorogenic Acid differentiates collection of illustrative plates, wherein 1 being CAULIS MARSDENIAE TENACISSIMAE blank sample, 2 being green original
Sour reference substance, 3 be CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, 4 be CAULIS MARSDENIAE TENACISSIMAE control medicinal material, 5 be CAULIS MARSDENIAE TENACISSIMAE medicinal material sample, 6 be CAULIS MARSDENIAE TENACISSIMAE extract
Thing sample, 7 be XIAOAIPING PIAN sample, 8 be Xiaoaiping Capsules sample, 9 be the flat scattered sample of the cancer that disappears for the flat particulate samples of cancer that disappear, 10,
11 it is XIAOAIPING TANGJIANG sample for the aiping bolus for cancer sample that disappears, 12,13 be oral liquor Xiao-Ai-Ping for treating cancer sample, 14 is Xiaoaiping injection sample
Product.
Fig. 2 is CAULIS MARSDENIAE TENACISSIMAE glycosides H, CAULIS MARSDENIAE TENACISSIMAE control medicinal material discriminating collection of illustrative plates in the specific embodiment of the invention, wherein 1 is clearance
Rattan blank sample, 2 be chlorogenic acid reference substance, 3 be CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, 4 be CAULIS MARSDENIAE TENACISSIMAE control medicinal material, 5 be CAULIS MARSDENIAE TENACISSIMAE medicinal material
Sample, 6 be Marsdenia tenacissima extract sample, 7 be XIAOAIPING PIAN sample, 8 be Xiaoaiping Capsules sample, 9 for disappear the flat particulate samples of cancer,
10 it is XIAOAIPING TANGJIANG sample for the aiping bolus for cancer sample that disappears, 12 for the flat scattered sample of the cancer that disappears, 11,13 is oral liquor Xiao-Ai-Ping for treating cancer sample, 14
For Xiaoaiping injection sample.
Fig. 3 is methanol solvate peak collection of illustrative plates in the specific embodiment of the invention.
Fig. 4 is reference substance collection of illustrative plates in the specific embodiment of the invention, and S1 is that chlorogenic acid, S2 are CAULIS MARSDENIAE TENACISSIMAE glycosides H.
Fig. 5 is ortho acid reference substance collection of illustrative plates in the specific embodiment of the invention, and S1 is chlorogenic acid.
Fig. 6 is Marsdenia tenacissima extract finger-print in the specific embodiment of the invention (S1, S2 are control peak).
Fig. 7 is CAULIS MARSDENIAE TENACISSIMAE medicinal materials fingerprint in the specific embodiment of the invention.
Fig. 8 is XIAOAIPING PIAN agent finger-print in the specific embodiment of the invention.
Fig. 9 is Xiaoaiping Capsules agent finger-print in the specific embodiment of the invention.
Figure 10 is the flat granule finger-print of cancer that disappears in the specific embodiment of the invention.
Figure 11 is the flat powder finger-print of cancer that disappears in the specific embodiment of the invention.
Figure 12 is the aiping bolus for cancer agent finger-print that disappears in the specific embodiment of the invention.
Figure 13 is XIAOAIPING TANGJIANG agent finger-print in the specific embodiment of the invention.
Figure 14 is oral liquor Xiao-Ai-Ping for treating cancer finger-print in the specific embodiment of the invention.
Figure 15 is Xiaoaiping injection finger-print in the specific embodiment of the invention.
Figure 16 is specific embodiment of the invention Content of Chlorogenic Acid canonical plotting.
Figure 17 is CAULIS MARSDENIAE TENACISSIMAE glycosides H canonical plottings in the specific embodiment of the invention.
Figure 18 is that solvent is that the CAULIS MARSDENIAE TENACISSIMAE glycosides H of chloroform-acetone-methanol differentiates collection of illustrative plates in the specific embodiment of the invention,
Wherein 1 is CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, and 2 be blank test sample, and 3 be CAULIS MARSDENIAE TENACISSIMAE control medicinal material, and 4 be CAULIS MARSDENIAE TENACISSIMAE medicinal material, and 5 be clearance
Boisiana extract, 6 be XIAOAIPING PIAN.
Figure 19 is that solvent is that the CAULIS MARSDENIAE TENACISSIMAE glycosides H of chloroform-methanol differentiates collection of illustrative plates in the specific embodiment of the invention, wherein 1
It is CAULIS MARSDENIAE TENACISSIMAE medicinal material for CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, 2,3 be Marsdenia tenacissima extract, and 4 be XIAOAIPING PIAN.
Figure 20 is that solvent is that the CAULIS MARSDENIAE TENACISSIMAE glycosides H of chloroform-methanol-water differentiates collection of illustrative plates in the specific embodiment of the invention, its
In 1 be CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances (5ul), 2 be CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances (10ul), 3 be CAULIS MARSDENIAE TENACISSIMAE medicinal material, 4 carry for CAULIS MARSDENIAE TENACISSIMAE
Thing is taken, 5 be XIAOAIPING PIAN.
Figure 21 is the CAULIS MARSDENIAE TENACISSIMAE collection of illustrative plates under constant speed elution requirement in the specific embodiment of the invention.
Figure 22 is CAULIS MARSDENIAE TENACISSIMAE collection of illustrative plates in the specific embodiment of the invention.
Figure 23 is that mobile phase is acetonitrile-water 30 in the specific embodiment of the invention：Test sample under 70 constant speed elution requirements
Chromatogram.
Figure 24 is that mobile phase is acetonitrile-water 50 in the specific embodiment of the invention:Test sample under 50 constant speed elution requirements
Chromatogram.
Figure 25 is that mobile phase is the phosphate aqueous solution of acetonitrile -0.4% 13 in the specific embodiment of the invention:87 constant speed are eluted
Under the conditions of chromatogram.
Figure 26 is the collection of illustrative plates that mobile phase is chloroform in the specific embodiment of the invention.
Figure 27 is the collection of illustrative plates that mobile phase is ethanol in the specific embodiment of the invention.
Embodiment
Below by embodiment, the present invention will be described in detail.
The detection method is applied to CAULIS MARSDENIAE TENACISSIMAE medicinal material, CAULIS MARSDENIAE TENACISSIMAE water extract and Xiaoaiping preparation against cancers.Wherein CAULIS MARSDENIAE TENACISSIMAE water
The preparation method of extract：CAULIS MARSDENIAE TENACISSIMAE is taken, is crushed, 50g, add water 300ml, boils and carries 2 hours, filtering, filter residue adds 300ml water, boiled
Carry 1.5 hours, filter, merging filtrate, be concentrated under reduced pressure drying.Xiaoaiping preparation against cancers are solid pharmaceutical preparation and liquid preparation.The cancer that disappears is flat solid
Body preparation includes tablet, capsule, granule, powder, pill；Liquid preparation includes syrup, oral liquid, parenteral solution.
The detection method for CAULIS MARSDENIAE TENACISSIMAE, comprises the following steps：
(1) preparation of need testing solution, reference substance solution and control medicinal material solution：Prepare respectively need testing solution 2~
6mg/ml chlorogenic acids reference substance solution, 0.1~0.5mg/ml CAULIS MARSDENIAE TENACISSIMAEs glycosides H reference substance solutions and CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution；
A. the preparation of need testing solution：
CAULIS MARSDENIAE TENACISSIMAE medicinal material need testing solution：CAULIS MARSDENIAE TENACISSIMAE medicinal material sample, takes at 2~4g, plus 50% 10~30ml of methanol, ultrasound
15~45min, filtration are managed, filtrate, which is steamed near, does, plus methanol 1ml makes dissolving, takes supernatant molten as CAULIS MARSDENIAE TENACISSIMAE medicinal material test sample
Liquid.
Marsdenia tenacissima extract and the flat solid pharmaceutical preparation need testing solution of the cancer that disappears：Marsdenia tenacissima extract and solid pharmaceutical preparation sample take
0.1~1g, wherein extract and tablet, capsule, particle, powder sample are ground, and pill is shredded, plus 50% 10~30ml of methanol,
Ultrasonically treated 15~45min, filtration, filtrate is volatilized, and residue adds methanol 1ml to make dissolving, and supernatant is supplied as extract and preparation
Test sample solution.
The flat liquid preparation need testing solution of the cancer that disappears：Liquid preparation takes 0.5~2ml, is evaporated, plus alcohol 1ml makes dissolving, supernatant
It is used as liquid preparation need testing solution.
B. the preparation of chlorogenic acid reference substance solution and CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substance solutions：2~6mg of chlorogenic acid reference substance is taken to add
1 milliliter of methanol, takes 5 milliliters of CAULIS MARSDENIAE TENACISSIMAE glycosides 0.5~2.5mg of H reference substances plus methanol, 2~6mg/ of chlorogenic acid reference substance is prepared respectively
Ml, 0.1~0.5mg/ml of CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances.
C. CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution：3 grams of CAULIS MARSDENIAE TENACISSIMAE control medicinal material is taken, is added water 50 milliliters, refluxing extraction 2 hours, filter
Cross, filtrate, which is steamed near, does, plus 1 milliliter of methanol makes dissolving, takes supernatant as control medicinal material solution.
(2) chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H discriminating：Need testing solution, chlorogenic acid reference substance solution, CAULIS MARSDENIAE TENACISSIMAE glycosides H are taken respectively
Reference substance solution, CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution 10ul, put in same silica gel g thin-layer plate, then prepare solvent, wherein deploying
Agent is made up of butyl acetate, formic acid and water；The butyl acetate：Formic acid：The volume ratio of water is 14:5:5.Take the upper strata of solvent
Solution is placed on lamellae；Standing treats that upper solution is deployed, and lamellae is taken out and dried；By lamellae after lamellae dries
Be placed under ultraviolet lamp, inspected under conditions of wavelength 365nm, first the chromatogram of need testing solution respectively with chlorogenic acid pair
Chromatogram according to product solution, CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution shows the fluorescence spot or principal spot of same color on relevant position；So
Developed the color afterwards using mass concentration for 5% sulfuric acid vanillic aldehyde test solution, be heated to clear spot, the chromatogram of need testing solution respectively with
CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substance solutions, the chromatogram of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution show spot or the master of same color on relevant position
Spot；
(3) chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H content detection：Take need testing solution, chlorogenic acid reference substance solution and CAULIS MARSDENIAE TENACISSIMAE glycosides
H reference substance solutions are detected using HPLC-UV；Wherein filler is octadecylsilane chemically bonded silica；Mobile phase is acetonitrile
With 0.5% phosphate aqueous solution, eluted using eluent gradient, wherein the time point of gradient change mobile phase ratio and the time
The volume ratio of the acetonitrile and 0.5% phosphate aqueous solution of point setting is as follows：During 0min, the volume of acetonitrile and 0.5% phosphate aqueous solution
Than for 15:85；During 5min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 8:92；During 50min, acetonitrile and 0.5% phosphoric acid
The volume ratio of the aqueous solution is 80:20；During 55min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:85；During 60min, second
The volume ratio of nitrile and 0.5% phosphate aqueous solution is 15:85.Detection wavelength is 190~220nm；
(4) set up using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as object of reference characteristic fingerprint pattern.
Below by taking the detection process of CAULIS MARSDENIAE TENACISSIMAE water extract as an example, content assaying method checking test is carried out, specifically such as
Under.
CAULIS MARSDENIAE TENACISSIMAE medicinal material need testing solution：CAULIS MARSDENIAE TENACISSIMAE medicinal material sample comminution, takes 2g, accurately weighed, puts in conical flask with cover,
Precision adds 50% methanol 10ml, and close plug, weighed weight, ultrasonically treated 30min is let cool, then weighed weight, is mended with 50% methanol
The weight of sufficient less loss, shakes up, filtration, is used as need testing solution.
CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution：3 grams of CAULIS MARSDENIAE TENACISSIMAE control medicinal material is taken, is added water 50 milliliters, refluxing extraction 2 hours, filtration,
Filtrate, which is steamed near, does, plus 1 milliliter of methanol makes dissolving, takes supernatant as control medicinal material solution.
Marsdenia tenacissima extract and XIAOAIPING PIAN agent, capsule, granule, powder, pill need testing solution：CAULIS MARSDENIAE TENACISSIMAE is extracted
Thing and tablet, capsule, granule, powder, pill sample take 0.2g, accurately weighed, put in conical flask with cover, and precision is added
50% methanol 10ml, close plug, weighed weight, ultrasonically treated 30min is let cool, then weighed weight, and less loss is supplied with 50% methanol
Weight, shakes up, filtration, is used as extract and preparation need testing solution.
XIAOAIPING TANGJIANG, oral liquid, parenteral solution need testing solution：XIAOAIPING TANGJIANG, oral liquid, parenteral solution take 1ml, accurate
It is weighed, plus 50% methanol is used as liquid preparation need testing solution to 10ml.
The preparation of reference substance solution：Take chlorogenic acid reference substance 4mg plus 1 milliliter of methanol, CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances 1mg plus first
5 milliliters of alcohol, prepares chlorogenic acid reference substance 4mg/ml, CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances 0.2mg/ml respectively；
Chromatographic condition and system suitability：From HPLC-UV detectors, octadecylsilane chemically bonded silica is filling
Agent；Acetonitrile and 0.5% phosphate aqueous solution are mobile phase, and Detection wavelength is 201nm；
The time point of gradient change mobile phase ratio, and time point setting acetonitrile and 0.5% phosphate aqueous solution
Volume ratio is as follows：During 0min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:85；During 5min, acetonitrile and 0.5% phosphoric acid
The volume ratio of the aqueous solution is 8:92；During 50min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 80:20；During 55min, second
The volume ratio of nitrile and 0.5% phosphate aqueous solution is 15:85；During 60min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:
85。
Experiment is carried out with need testing solution, reference substance solution and testing conditions etc. according to above-mentioned condition.
(1) stability test
Extract sample test liquid of the present invention is taken, by 2, is determined within 4,6,8,12,24,48 hours, measurement result is shown in Table 1-4.
As a result show, the relative retention time at each shared peak and the relative peak area of main peaks do not have significant change substantially in test liquid
(RSD<3%) technical requirements of finger-print, are met, test liquid is stable in 48 hours.Note：tRRepresent the retention time at the peak
tR/tSRepresent the relative retention time at the peak；S1 is chlorogenic acid, and S2 is CAULIS MARSDENIAE TENACISSIMAE glycosides H.
Table 1：Using S1 as with reference to peak sample liquid study on the stability result (relative retention time at shared peak).
Table 2：Using S2 as with reference to peak sample liquid study on the stability result (relative retention time at shared peak).
Table 3：Investigated using S1 as with reference to peak sample liquid stability (relative peak area for accounting for the main peaks of the peak gross area more than 5%)
As a result.
Table 4：Investigated using S2 as with reference to peak sample liquid stability (relative peak area for accounting for the main peaks of the peak gross area more than 5%)
As a result.
(2) precision test
Same Marsdenia tenacissima extract sample test liquid, continuous sample introduction is measured for 6 times, and measurement result is shown in Table 5-8.As a result table
It is bright, the basically identical (RSD of the relative peak area of relative retention time and main peaks at each shared peak in test liquid<3%) finger, is met
The technical requirements of line collection of illustrative plates.
Table 5：Result (relative retention time at shared peak) is investigated using S1 as with reference to peak sample liquid precision.
Table 6：Result (relative retention time at shared peak) is investigated using S2 as with reference to peak sample liquid precision.
Table 7：Knot is investigated using S1 as with reference to peak sample precision (relative peak area for accounting for the main peaks of total peak area more than 5%)
Really.
Table 8：Investigated using S2 as with reference to peak sample precision (relative peak area for accounting for the main peaks of total peak area more than 5%)
As a result.
(3) replica test
Same lot number Marsdenia tenacissima extract, prepares 6 parts of sample liquid with method, determines in accordance with the law, the results are shown in Table 9-12.As a result table
Bright, the relative peak area of the relative retention time at each shared peak and main peaks (accounting for total peak area more than 5%) is basic in test liquid
Consistent (RSD<3%) technical requirements of finger-print, are met.
Table 9：Result (relative retention time at shared peak) is investigated using S1 as with reference to peak sample liquid repeatability.
Table 10：Result (relative retention time at shared peak) is investigated using S2 as with reference to peak sample liquid repeatability.
Table 11：Investigated using S1 as with reference to peak sample repeatability (relative peak area for accounting for the main peaks of total peak area more than 5%)
As a result.
Table 12：Investigated using S2 as with reference to peak sample repeatability (relative peak area for accounting for the main peaks of total peak area more than 5%)
As a result.
The measure of (4) ten batches of sample finger-prints：10 batches of Marsdenia tenacissima extract samples are taken, are prepared with method, outside object of reference
Mark method, calculates standard finger-print.It the results are shown in Table 13-16.
Table 13：Using S1 as ten batches of sample determining fingerprint pattern results of object of reference (showing all peak relative retention times).
After upper table
Table 14：Using S2 as ten batches of sample determining fingerprint pattern results of object of reference (showing all peak relative retention times).
After upper table
Table 15：Result is investigated by object of reference 10 batches (relative peak area for accounting for the main peaks of total peak area more than 5%) of S1.
After upper table
Table 16：Result is investigated by object of reference 10 batches (relative peak area for accounting for the main peaks of total peak area more than 5%) of S2.
After upper table
| 7 | 8 | 9 | 10 |
| A | A/AS | A | A/AS | A | A/AS | A | A/AS |
| 208035 | 1.000 | 400418 | 1.000 | 572915 | 1.000 | 439886 | 1.000 |
| 1651945 | 7.941 | 2437946 | 6.089 | 2359226 | 4.118 | 2107267 | 4.790 |
| 21015548 | 101.019 | 27012361 | 67.460 | 26085066 | 45.530 | 29895492 | 67.962 |
| 1149035 | 5.523 | 1202306 | 3.003 | 1828870 | 3.192 | 1238487 | 2.815 |
| 462480 | 2.223 | 710295 | 1.774 | 1102437 | 1.924 | 1361252 | 3.095 |
| 548833 | 2.638 | 886800 | 2.215 | 1142423 | 1.994 | 1148683 | 2.611 |
| 121558 | 0.584 | 209780 | 0.524 | 375416 | 0.655 | 304414 | 0.692 |
(5) linear relationship is investigated
It is initial soln that precision, which draws chlorogenic acid 150ug/ml and CAULIS MARSDENIAE TENACISSIMAE glycosides H3.07mg/ml mixing controls, is progressively diluted
Into 1.5, the mixed reference substance solution of 2,3,6,12 times of various concentrations.The mixed reference substance solution of various concentrations is taken, respectively sample introduction
20ul.Using reference substance concentration as abscissa, peak area is ordinate, draws standard curve, calculates regression equation.Chlorogenic acid：Y=
30762x+10669, r=0.9996,0.25-3ug of the range of linearity；CAULIS MARSDENIAE TENACISSIMAE glycosides H：Y=1.1 ╳ 106X+6162.4, r=
0.9990,5.126-61.4ug of the range of linearity, as a result show that linear relationship is good.Linear determination result such as table 17 below and table 18.
Table 17：Chlorogenic acid linear determination result.
| Chlorogenic acid (μ g/ml) | Peak area |
| 150 | 4580230 |
| 100 | 3171254 |
| 75 | 2297361 |
| 50 | 1555288 |
| 25 | 765311 |
| 12.5 | 384098 |
Table 18：CAULIS MARSDENIAE TENACISSIMAE glycosides H linear determination results.
| CAULIS MARSDENIAE TENACISSIMAE glycosides H (mg/ml) | Peak area |
| 3.07 | 3404801 |
| 2.05 | 2272503 |
| 1.437 | 1710879 |
| 1.025 | 1151680 |
| 0.5125 | 529301 |
| 0.25625 | 282886 |
(6) average recovery is tested
6 parts of test sample powder is taken, every part of about 0.1g is accurately weighed, every part of difference is accurate to add 93.89ug/ml chlorogenic acids
Reference substance solution 5.0ml, 0.593mg/ml CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substance solution 5ml, volatilize solvent, by need testing solution preparation side
Prepared by method, product, which are determined, in the same old way determines under item and calculate average recovery, as a result see the table below.
Table 19：Average recovery.
The selection control of solvent is as follows in chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H discrimination process：
1. solvent is used for chloroform-acetone-methanol (20-1-1)
Conclusion：In test sample chromatogram, do not occur with CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, control medicinal material chromatogram relevant position aobvious identical
The spot of color, is shown in Figure 18.
2. solvent is used for chloroform-methanol (95-5)
Conclusion：In test sample chromatogram, do not occur with CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substances, control medicinal material chromatogram relevant position aobvious identical
The spot of color, is shown in Figure 19.
3. solvent is used for chloroform-methanol-water (65-35-10)
Conclusion：Expansion failure, is shown in Figure 20.
The CAULIS MARSDENIAE TENACISSIMAE glycosides H content detection of selection analysis during chlorogenic acid of the present invention, to(for) condition is as follows：
1. the reason for present invention selects the gradient elution：Contain Multiple components in CAULIS MARSDENIAE TENACISSIMAE, using acetonitrile and 0.5% phosphorus
Aqueous acid is mobile phase, when constant speed is eluted, and composition separating degree is bad in CAULIS MARSDENIAE TENACISSIMAE, and active ingredient can not be separated, and causes detection
Effect is undesirable, sees Figure 21.
2. time point and the ratio of the present invention selection gradient elution：By verification experimental verification, the time of the application selection
Point is suitable with ratio, and selection other times point and ratio cause CAULIS MARSDENIAE TENACISSIMAE separating degree too low, and active ingredient separation is not thorough；
And the peak row of active ingredient is asymmetric, hangover or leading peak are caused, Figure 22 is seen.
3. the present invention is for the selection of mobile phase.
(1) acetonitrile-water (30 is selected：70) constant speed is eluted, and occurs, than larger miscellaneous peak, seeing Figure 23 in test sample chromatogram.
(2) acetonitrile-water (50 is selected:50), constant speed is eluted, and chlorogenic acid peak does not occur, sees Figure 24.
(3) selection acetonitrile -0.4% phosphate aqueous solution (13:87), constant speed is eluted, and chromatographic peak separating degree is not high, sees Figure 25.
(4) chloroform is selected, test sample is extracted, no shaping collection of illustrative plates is shown in Figure 26.
(5) ethanol is selected, test sample is extracted, no shaping collection of illustrative plates is shown in Figure 27.
Claims (6)
1. a kind of detection method for CAULIS MARSDENIAE TENACISSIMAE, comprises the following steps：
（1）The preparation of need testing solution, reference substance solution and control medicinal material solution：Need testing solution, 2~6mg/ml are prepared respectively
Chlorogenic acid reference substance solution, 0.1~0.5mg/ml CAULIS MARSDENIAE TENACISSIMAEs glycosides H reference substance solutions and CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution；
（2）The discriminating of chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H：Need testing solution, chlorogenic acid reference substance solution, CAULIS MARSDENIAE TENACISSIMAE glycosides H controls are taken respectively
Product solution, the μ l of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution 10, put in same silica gel g thin-layer plate, then prepare solvent, take solvent
Upper solution is placed on lamellae；Standing treats that upper solution is deployed, and lamellae is taken out and dried；Will be thin after lamellae dries
Laminate is placed under ultraviolet lamp, is inspected under conditions of wavelength 365nm, first the chromatogram of need testing solution respectively with green original
Sour reference substance solution, the chromatogram of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution show the fluorescence spot or main spot of same color on relevant position
Point；Then developed the color using mass concentration for 5% sulfuric acid vanillic aldehyde test solution, be heated to clear spot, the chromatogram point of need testing solution
The spot of same color is not shown on relevant position with CAULIS MARSDENIAE TENACISSIMAE glycosides H reference substance solutions, the chromatogram of CAULIS MARSDENIAE TENACISSIMAE control medicinal material solution
Or principal spot；
（3）The content detection of chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H：Take H pairs of need testing solution, chlorogenic acid reference substance solution and CAULIS MARSDENIAE TENACISSIMAE glycosides
Detected according to product solution using HPLC-UV；Wherein mobile phase be acetonitrile and 0.5% phosphate aqueous solution, Detection wavelength be 190~
220nm；The step（3）The elution of middle use eluent gradient, wherein the time point of gradient change mobile phase ratio and this when
Between put setting acetonitrile and 0.5% phosphate aqueous solution volume ratio it is as follows：During 0min, the volume of acetonitrile and 0.5% phosphate aqueous solution
Than for 15:85；During 5min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 8:92；During 50min, acetonitrile and 0.5% phosphoric acid water
The volume ratio of solution is 80:20；During 55min, the volume ratio of acetonitrile and 0.5% phosphate aqueous solution is 15:85；During 60min, acetonitrile
Volume ratio with 0.5% phosphate aqueous solution is 15:85；
（4）Set up using chlorogenic acid, CAULIS MARSDENIAE TENACISSIMAE glycosides H as object of reference characteristic fingerprint pattern.
2. it is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE according to claim 1, it is characterised in that the step（2）In solvent
It is made up of butyl acetate, formic acid and water.
3. it is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE according to claim 2, it is characterised in that the butyl acetate：Formic acid：Water
Volume ratio is 14:5:5.
4. it is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE according to claim 1, it is characterised in that the step（3）In detection ripple
A length of 201nm.
5. it is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE according to claim 1, it is characterised in that the step（3）Middle use
HPLC-UV fillers are octadecylsilane chemically bonded silica.
6. it is used for the detection method of CAULIS MARSDENIAE TENACISSIMAE according to claim 1, it is characterised in that the detection method is applied to clearance
Rattan medicinal material, CAULIS MARSDENIAE TENACISSIMAE water extract and Xiaoaiping preparation against cancers.
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