CN106018587B - The detection method of her blood peace particle - Google Patents
The detection method of her blood peace particle Download PDFInfo
- Publication number
- CN106018587B CN106018587B CN201610315836.XA CN201610315836A CN106018587B CN 106018587 B CN106018587 B CN 106018587B CN 201610315836 A CN201610315836 A CN 201610315836A CN 106018587 B CN106018587 B CN 106018587B
- Authority
- CN
- China
- Prior art keywords
- peak
- blood
- particle
- solution
- blood peace
- 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
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of detection method of her blood peace particle, using protocatechuic acid as object of reference, the finger-print of strong medicine herba blumea riparia common pattern is obtained using high performance liquid chromatography progress gradient elution.Finger-print shares chromatographic peak 21, it can fully react the chemical composition of her blood peace particle, and informative, method precision, good reproducibility and stability, quality control and clinical efficacy to her blood peace particle are of great significance.
Description
Technical field
The present invention relates to drug tests, particularly a kind of detection method of her blood peace particle.
Background technology
Her blood peace particle is a kind of Chinese patent drug for treating gynecologic blood diseases, and main ingredient is blumea riparia, motherwort, prolongs recklessly
Rope, radix glycyrrhizae etc., have promoting blood circulation and hemostasis, promoting qi circulation and relieving pain and other effects.Clinically it is used for post partum lochiorrhea, uterus goes out after induced abortion
Blood is not net, differential diagnosis in tcm category syndrome of blood stasis person.
For guarantee her blood pacify particle quality and clinical application it is safe and effective, the research of its quality standard and establish increasingly
It is important.The quality standard of her existing blood peace particle, including blumea riparia, corydalis tuber, the thin layer of radix glycyrrhizae differentiate, with efficient liquid
Phase chromatography measures the content of protocatechuic acid, but this method lacks discriminating to motherwort, to the measure of corydalis tuber, radix glycyrrhizae also only
It is limited to observational measurement.
The content of the invention
The present invention provides a kind of detection method of her blood peace particle, the thin layer mirror to blumea riparia, motherwort is included
Other method, and the finger-print of her blood peace particle, the quality control for pacifying particle for her blood provide important references.
To achieve the above object, the technical scheme is that：
A kind of detection method of her blood peace particle, comprises the following steps：
(1) using protocatechuic acid and protocatechualdehyde as control, reflected using chloroform, ethyl acetate, formic acid mixed solution as solvent
Blumea riparia component not in her blood peace particle；
(2) using stachydrine hydrochloride as control, Yi Xuean is differentiated as solvent using acetone, ethyl acetate, hydrochloric acid mixed solution
Motherwort component in particle；
(3) using protocatechuic acid as object of reference, with high performance liquid chromatography detection, her blood pacifies particle extracting solution, obtains her blood peace
The finger-print of grain；Wherein, the extraction process of her blood peace particle extracting solution：Her blood peace particle is taken, is dissolved in water, adds dilute hydrochloric acid tune
PH is saved, is stirred evenly, is stood, filtration, adds water-saturated n-butanol to extract 3~6 times, divides and takes out n-butanol extracting liquid, merges n-butanol and carries
Liquid is taken, is stood, divides and takes acquisition n-butanol liquid, recycle n-butanol, residue adds methanol to dissolve, and centrifuges, takes supernatant, obtains her blood peace
Grain extracting solution.The dilute hydrochloric acid for adjusting pH is the hydrochloric acid that mass fraction is 9.5%~10.5%.
Further, the concrete operation method of the step (1) is：
(11) blumea riparia thin layer differentiates the preparation of test solution：Her blood peace particle is taken, adds water, makes her blood peace particle
Dissolving, adds dilute hydrochloric acid, stirs evenly, and filters, and add diethyl ether extraction 2~3 times, obtains ether extracted liquid, merges ether extracted liquid, adds
After water washing 2 times, aqueous is discarded, divides and takes ether extracted liquid, be evaporated, residue adds methanol to be allowed to dissolve, thin as blumea riparia
Layer differentiates test solution；Wherein, dilute hydrochloric acid is the hydrochloric acid that mass fraction is 9.5%~10.5%；
(12) protocatechuic acid reference substance solution：Protocatechuic acid reference substance adds methanol that protocatechuic acid reference substance solution is made；
(13) protocatechualdehyde reference substance solution：Protocatechualdehyde reference substance adds methanol that protocatechualdehyde reference substance solution is made；
(14) above-mentioned blumea riparia thin layer is taken to differentiate test solution, protocatechuic acid reference substance solution, protocatechualdehyde pair
According to product solution point on same silica gel thin-layer plate, using volume ratio as 6:3:5 chloroform, ethyl acetate, formic acid mixed solution are exhibition
Agent expansion is opened, is taken out, is dried, inspect in the UV lamp.
Further, the concrete operation method of the step (2) is：
(21) motherwort thin layer differentiates the preparation of test solution：Her blood peace particle is taken, adds absolute ethyl alcohol, shaking is extracted,
Filtration, the filtrate obtained are evaporated, and residue adds absolute ethyl alcohol dissolving, differentiate test solution as motherwort thin layer；
(22) stachydrine hydrochloride reference substance solution：Stachydrine hydrochloride reference substance adds absolute ethyl alcohol that stachydrine hydrochloride control is made
Product solution；
(23) take above-mentioned motherwort thin layer to differentiate test solution, stachydrine hydrochloride reference substance solution, put in same silica gel
On lamellae, using volume ratio as 6:6:1 acetone, ethyl acetate, hydrochloric acid mixed solution are solvent expansion, take out, dry, and are sprayed
To improve bismuth potassium iodide test solution, heating makes spot development clear, motherwort thin layer differentiate test solution chromatography with hydrochloric acid water
On the corresponding position of alkali reference substance solution chromatography of reviving, the spot of same color is shown.
Further, the concrete operation method of the step (3) is：
(31) preparation of reference solution：Protocatechuic acid standard items, add methanol constant volume, and reference solution is made.
(32) preparation of her blood peace particle extracting solution；
(33) chromatographic condition of high performance liquid chromatograph：
Mobile phase：The glacial acetic acid of methanol -0.1%；
Gradient elution program：Time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；
Corresponding gradient eluent is made of by volumes below ratio mobile phase A methanol and 0.1% glacial acetic acid of Mobile phase B, the body of mobile phase A
Product percentage gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70%, Mobile phase B
Percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55% → 55% → 30%；
Ultraviolet detection wavelength：256nm；
Flow velocity：1.0mL·min-1；
Column temperature：25℃.
(34) measure：Her blood peace particle extracting solution injection high performance liquid chromatograph is drawn, according to the chromatostrip of step (33)
Part measures, and obtains finger-print.
Preferably, her concrete operation method of blood peace particle extracting solution extraction is：Her blood peace 3~8g of particle is weighed,
Add water 50mL dissolve, add 0.2~0.4mL of dilute hydrochloric acid, stir evenly, stand 15~30min, filtration, add water-saturated n-butanol extraction 5~
6 times, 15~20mL, divides and takes n-butanol extracting liquid every time, merges, and stands 1~1.5h, divides and takes acquisition n-butanol liquid, recycles positive fourth
Alcohol, residue add 4~6mL of methanol, and centrifugation, takes supernatant, obtain her blood peace particle extracting solution.It is furthermore preferred that her blood pacifies particle
Extracting solution extraction concrete operation method be：Her blood peace particle 5g is weighed, adds water 50mL to dissolve, adds dilute hydrochloric acid 0.3mL, stir evenly,
20min is stood, filtration, adds water-saturated n-butanol to extract 5 times, each 15mL, divides and takes n-butanol extracting liquid, merges, and stands 1h, point
Take and obtain n-butanol liquid, recycle n-butanol, residue adds methanol 5mL, centrifuges, takes supernatant, obtains her blood peace particle extracting solution.
Further, the finger-print shares chromatographic peak 21, its Average residence time is respectively：
No. 1 peak 3.234min, No. 2 peak 5.962min, No. 3 peak 11.023min, No. 4 peak 15.353min, No. 5 peaks
16.521min, No. 6 peak 24.498min, No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peak 32.29min, No. 10 peaks
34.752min, No. 11 peak 36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, No. 14 peak 56.509min, 15
Number peak 58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18 peak 68.358min, No. 19 peaks
69.319min, No. 20 peak 85.217min, No. 21 peak 91.016min.Further, her component of blood peace particle includes
Blumea riparia, motherwort, corydalis tuber and radix glycyrrhizae, wherein No. 1 peak~No. 21 peak derives from blumea riparia, No. 9 peaks derive from
Motherwort, 10, No. 11 peaks derive from corydalis tuber, and 6,14,15, No. 16 peaks derive from radix glycyrrhizae.
Preferably, in the step (33), chromatographic column is Phenomenex Gemini C18 columns (4.6mm × 250mm, 5 μ
M), guard column is Phenomenex C18 (4 × 3.0mm).
Preferably, in the step (33), theoretical cam curve is in terms of protocatechuic acid peak, not less than 5000.
The detection method of her above-described blood peace particle, optimizes the TLC Identification of blumea riparia, mends
The TLC Identification of motherwort has been filled, while the fingerprint of her blood peace particle is determined.Her blood peace
In the fingerprint of grain, using protocatechuic acid as object of reference, by high performance liquid chromatograph, using the ice of methanol -0.1%
Acetic acid carries out gradient elution for mobile phase, obtains finger-print, chromatographic peak 21 is shared in finger-print, wherein 21 shared
Peak derives from blumea riparia, and 1 shared peak derives from motherwort, and 2 shared peaks derive from corydalis tuber, 4 shared peak sources
In radix glycyrrhizae, the chemical composition that her blood pacifies four kinds of bulk pharmaceutical chemicals in particle can be fully reacted, available for four kinds of bulk pharmaceutical chemicals of the overall evaluation
Quality and the quality for differentiating four kinds of bulk pharmaceutical chemicals, in order to control the quality of her blood peace particle provide strong theoretical foundation.The present invention
The method being combined using indentification by TLC and finger-print, quickly analyzes her blood peace particle, method reappearance, surely
Qualitative good, quality control and clinical efficacy to her blood peace particle are of great significance.
Brief description of the drawings
Fig. 1 is the chromatogram obtained in embodiment 1 using Welchrom C18 columns (4.6mm × 250mm, 5 μm)；
Fig. 2 is the chromatogram obtained in embodiment 1 using Hypersil C18 columns (4.6mm × 250mm, 5 μm)；
Fig. 3 is the chromatography obtained in embodiment 1 using Phenomenex Gemini C18 columns (4.6mm × 250mm, 5 μm)
Figure；
Fig. 4 is the chromatogram for using methanol-water to be obtained for mobile phase in embodiment 1；
Fig. 5 is the chromatogram for using acetonitrile-water to be obtained for mobile phase in embodiment 1；
Fig. 6 is the chromatogram for using the phosphoric acid of methanol -0.1% to be obtained for mobile phase in embodiment 1；
Fig. 7 is the chromatogram for using the glacial acetic acid of methanol -0.05% to be obtained for mobile phase in embodiment 1；
Fig. 8 is the chromatogram for using the glacial acetic acid of methanol -0.1% to be obtained for mobile phase in embodiment 1；
Fig. 9 is the chromatogram for using the glacial acetic acid of acetonitrile -0.1% to be obtained for mobile phase in embodiment 1；
Figure 10 is the chromatogram that a length of 230nm of 1 medium wave of embodiment is obtained；
Figure 11 is the chromatogram that a length of 256nm of 1 medium wave of embodiment is obtained；
Figure 12 is the chromatogram that 1 medium wave of embodiment a length of 280nm (0~20min) -279nm (20~30min) is obtained；
Figure 13 is the chromatogram that a length of 320nm of 1 medium wave of embodiment is obtained；
Figure 14 is the chromatogram that a length of 360nm of 1 medium wave of embodiment is obtained；
Figure 15 is that column temperature is 20 DEG C of obtained chromatograms in embodiment 1；
Figure 16 is that column temperature is 25 DEG C of obtained chromatograms in embodiment 1；
Figure 17 is that column temperature is 30 DEG C of obtained chromatograms in embodiment 1；
Figure 18 is that column temperature is 35 DEG C of obtained chromatograms in embodiment 1；
Figure 19 is that flow velocity is 0.8mLmin in embodiment 1-1Obtained chromatogram；
Figure 20 is that flow velocity is 1.0mLmin in embodiment 1-1Obtained chromatogram；
Figure 21 is that flow velocity is 1.2mLmin in embodiment 1-1Obtained chromatogram；
Figure 22 is the chromatogram detected after being extracted in embodiment 2 using water as Extraction solvent；
Figure 23 is the chromatogram detected after being extracted in embodiment 2 using methanol as Extraction solvent；
Figure 24 is the chromatogram detected after being extracted in embodiment 2 using ethanol as Extraction solvent；
Figure 25 is the chromatogram detected after being extracted in embodiment 2 using water-saturated n-butanol as extractant；
Figure 26 is the chromatogram detected after being extracted in embodiment 2 using chloroform as extractant；
Figure 27 is the chromatogram detected after being extracted in embodiment 2 using ether as extractant；
Figure 28 is the chromatogram detected after being extracted in embodiment 2 using ethyl acetate as extractant；
Figure 29 is the finger-print of her blood peace particle in embodiment 3；
Figure 30 is the chromatogram of object of reference in embodiment 3；
Figure 31 is herba blumea riparia chromatogram in embodiment 3；
Figure 32 is motherwort medicinal wood color spectrogram in embodiment 3；
Figure 33 is corydalis tuber medicinal material chromatogram in embodiment 3；
Figure 34 is licorice medicinal materials chromatogram in embodiment 3；
Figure 35 is the chromatogram that blumea riparia TLC Identification obtains；
Figure 36 is the chromatogram that motherwort TLC Identification obtains；
Figure 37 is the matching figure of her blood peace particle of 10 lot numbers in embodiment 5.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to following reality
Apply example：
Experiment material, instrument and the reagent of following embodiments prepare as follows：
Experiment material：Protocatechuic acid standard items (Nat'l Pharmaceutical ＆ Biological Products Control Institute, for assay, lot number：
110809-200503)。
Instrument：Agilent Agilent1260 high performance liquid chromatographs, are contained in line vacuum degassing machine (G-1311C), quaternary
Gradient pump (G-1311C), standard autosampler (G-1329B), intelligent column oven (G-1316A), variable-wavelenght detector
(G-1314B), Agilent1260 Infinity chromatographic work stations (Anjelen Sci. ＆ Tech. Inc of the U.S.)；SB3200T ultrasounds
Ripple cleaning device (Shanghai must can believe ultrasonic Co., Ltd)；Millipore Simplicity-185 ultra-pure waters instrument (U.S. Mi Libo
Company)；LG16-W high speed micros centrifuge (Beijing Medical Centrifugal Machine Factory)；BT224S electronic analytical balances (Beijing Sai Duolisi
Instrument system Co., Ltd).
Reagent：Methanol, acetonitrile (chromatographically pure, Fisher Scientific companies of the U.S.)；Glacial acetic acid (HPLC, Tianjin section
Mi Ou chemical reagent Co., Ltd)；Water is ultra-pure water；Other reagents are that analysis is pure.
1 finger-print of embodiment：The optimization of chromatographic condition
The preparation method of her blood peace particle extracting solution：Her blood peace particle 5g is weighed, adds water 50mL to dissolve, adds dilute hydrochloric acid
0.3mL, stirs evenly, and stands 20min, and filtration, adds water-saturated n-butanol to extract 5 times, each 15mL, divides and takes n-butanol extracting liquid, closes
And 1h is stood, divide and take acquisition n-butanol liquid, recycle n-butanol, residue adds methanol 5mL, centrifuges, takes supernatant, obtains her blood peace
Grain extracting solution.
The selection of 1-1 chromatographic columns
Other determination conditions of high performance liquid chromatograph are identical, investigated Welchrom C18 columns (4.6mm × 250mm, 5 μ
M), Hypersil C18 columns (4.6mm × 250mm, 5 μm) and Phenomenex Gemini C18 columns (4.6mm × 250mm, 5 μ
M) three kinds of chromatographic columns, obtained chromatogram are divided into as shown in Figure 1, Figure 2, Figure 3 shows, as seen from Figure 3, Phenomenex
Gemini C18 columns (4.6mm × 250mm, 5 μm) separating degree is best, and peak shape is symmetrically and sharp, and baseline is more steady.
The selection of 1-2 mobile phases
Other determination conditions of high performance liquid chromatograph are identical, investigated methanol-water, acetonitrile-water, the phosphorus of methanol -0.1%
Acid, the glacial acetic acid of methanol -0.05%, the glacial acetic acid of methanol -0.1%, the glacial acetic acid of acetonitrile -0.1% carry out gradient elution, institute for mobile phase
Obtained chromatogram as shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, can be seen that with the ice second of methanol -0.1% through comparing respectively
Acid system is preferable for the chromatogram absworption peak separation of mobile phase, and baseline is more steady.
The selection of 1-3 Detection wavelengths
Other determination conditions of high performance liquid chromatograph are identical, investigated 230nm, 256nm, 280nm, 320nm, 360nm
Chromatogram under absorbing wavelength, chromatogram as shown in Figure 10, Figure 11, Figure 12, Figure 13 and Figure 14, can be seen that through comparing respectively
Chromatogram shows more peak information at 256nm, and signal is preferable, and each peak shape is preferable in gained chromatogram, baseline is steady.
The selection of 1-4 column temperatures
Other determination conditions of high performance liquid chromatograph are identical, investigated 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C of different column temperatures,
Chromatogram is respectively as shown in Figure 15, Figure 16, Figure 17 and Figure 18, and each peak separating effect is best when can be seen that 25 DEG C through comparing.
The selection of 1-5 flow velocitys
Other determination conditions of high performance liquid chromatograph are identical, investigated 0.8mLmin-1、1.0mL·min-1、
1.2mL·min-1Different in flow rate, chromatogram is respectively as shown in Figure 19, Figure 20 and Figure 21, through comparing as can be seen that flow velocity is
1.0mL·min-1When each peak separating effect it is best.
2 finger-print of embodiment：The optimization of sample-pretreating method
The chromatographic condition of high performance liquid chromatograph：Chromatographic column using Phenomenex Gemini C18 columns (4.6mm ×
250mm, 5 μm)；Guard column uses Phenomenex C18 (4 × 3.0mm)；Mobile phase：The glacial acetic acid of methanol -0.1%；Gradient is washed
De- program：Time gradient 0min → 11min → 16min → 29min → 30min → 35min → 45min → 55min → 70min →
95min；Corresponding gradient eluent is made of by volumes below ratio mobile phase A methanol and 0.1% glacial acetic acid of Mobile phase B, flowing
The percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70% of phase A,
The percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55% → 55% of Mobile phase B →
30%；；Detection wavelength：256nm；Flow velocity：1.0mL·min-1；Column temperature：25℃；Sample size：5μL.
The selection of 2-1 Extraction solvents
Her blood peace particle 5g is weighed, three kinds of water, methanol and ethanol Extraction solvents are selected, using identical extracting method to her
Blood peace particle is extracted, and extraction is completed after same extraction, concentration, dissolution process, is carried out by high performance liquid chromatograph
Detection, obtained chromatogram is respectively as shown in Figure 22, Figure 23, Figure 24, when can be seen that through comparing using water as Extraction solvent,
More peak information is shown, the active principle of sample is completely dissolved, and peak separating degree is good.
The selection of 2-2 extractants
Her blood peace particle 5g is weighed, after obtaining extracting solution as Extraction solvent using water, selects water-saturated n-butanol, chloroform, second
Ether, ethyl acetate are extracted for extractant, and extracting process is identical, and extraction is completed after same concentration, dissolution process, are led to
Cross high performance liquid chromatograph to be detected, obtained chromatogram is respectively as shown in Figure 25, Figure 26, Figure 27 and Figure 28, through comparing
As can be seen that the sample of chloroform extraction, peak information is seldom, and the sample of ether extraction, peak information is less and peak area is smaller, acetic acid
Ethyl ester and water-saturated n-butanol extraction gained collection of illustrative plates are more similar, but peak area is smaller, and are easier to emulsify during ethyl acetate extraction,
Therefore water-saturated n-butanol effect of extracting is optimal.
The selection of 2-3 extraction times
Her blood peace particle 5g is weighed, using water as Extraction solvent, extracts by extractant of water-saturated n-butanol, investigates respectively
Extraction 3 times, 4 times, 5 times, 6 times, other extracting process are identical, and extraction is completed after same concentration, dissolution process, passes through height
Effect liquid phase chromatogram instrument is detected, and the protocatechuic acid that experiment shows extraction 5 times with extracting 6 times is on peak area without larger difference；
It is indicated above that extracting 5 times can completely extract her blood peace particle water soluble ingredient substantially, therefore extraction is advisable with 5 times.
The selection of 2-4 extraction quantities
Her blood peace particle 5g is weighed, using water as Extraction solvent, is extracted 5 times by extractant of water-saturated n-butanol, other
Extracting process is identical, investigates the effect of extracting that extraction quantity is respectively 10mL, 15mL, 20mL, and extraction is completed concentrate through same, is molten
After solution processing, it is detected by high performance liquid chromatograph, experiment shows that extraction quantity is the former youngster that 15mL is 20mL with extraction quantity
Boheic acid is on peak area without larger difference.It is indicated above that extraction quantity her blood can have been pacified substantially for 15mL particle water solubility into
Divide extraction completely.
The quality testing of her the blood peace particle of embodiment 3
The preparation of 3-1 reference solutions：Weigh protocatechuic acid standard items 0.81mg to put in 10mL volumetric flasks, add methanol appropriate
Make dissolving and be settled to scale, shake up, up to the protocatechuic acid standard solution that concentration is 0.081mgmL-1
The preparation of her blood peace particle extracting solutions of 3-2：Her blood peace particle 5g is weighed, adds water 50mL to dissolve, adds dilute hydrochloric acid
0.3mL, stirs evenly, and stands 20min, and filtration, adds water-saturated n-butanol to extract 5 times, each 15mL, divides and takes n-butanol extracting liquid, closes
And 1h is stood, divide and take acquisition n-butanol liquid, recycle n-butanol, residue adds methanol 5mL, centrifuges, takes supernatant, obtains her blood peace
Grain extracting solution.
The chromatographic condition of 3-3 high performance liquid chromatographs：
Chromatographic column：Phenomenex Gemini C18 columns (4.6mm × 250mm, 5 μm)；
Guard column：Phenomenex C18(4×3.0mm)；
Mobile phase：The glacial acetic acid of methanol -0.1%；
Gradient elution program：Time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；
Corresponding gradient eluent is made of by volumes below ratio mobile phase A methanol and 0.1% glacial acetic acid of Mobile phase B, the body of mobile phase A
Product percentage gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70%, Mobile phase B
Percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55% → 55% → 30%；
Ultraviolet detection wavelength：256nm；
Flow velocity：1.0mL·min-1；
Column temperature：25℃.
The measure of her blood peace particle extracting solutions of 3-4：Her 10 μ L injection high performance liquid chromatographs of blood peace particle extracting solution are drawn,
Measured according to the chromatographic condition of step 3-3, obtain finger-print, as shown in figure 29.By《Chromatographic fingerprints of Chinese materia medica similarity
Evaluation system A editions》Software, Auto-matching is carried out to the relevant parameter of finger-print, is demarcated her 21 fingerprints of blood peace particle and is total to
There is peak, its Average residence time is respectively：No. 1 peak 3.234min, No. 2 peak 5.962min, No. 3 peak 11.023min, No. 4 peaks
15.353min, No. 5 peak 16.521min, No. 6 peak 24.498min, No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peaks
32.29min, No. 10 peak 34.752min, No. 11 peak 36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, No. 14
Peak 56.509min, No. 15 peak 58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18 peak 68.358min,
No. 19 peak 69.319min, No. 20 peak 85.217min, No. 21 peak 91.016min.
The measure of 3-5 reference solutions；Precision draws 5 μ L reference solutions injection high performance liquid chromatograph, according to step
The chromatographic condition measure of 3-3, obtains the collection of illustrative plates of object of reference, as shown in figure 30, protocatechuic acid chromatographic peak and in test sample finger-print 5
The retention time at number peak is consistent, as with reference to peak.
3-6 fingerprints share the calibration at peak
The test solution of blumea riparia, motherwort, corydalis tuber and licorice medicinal materials is made by 3-2 respectively, by 3-3 chromatographies
Condition sample introduction is analyzed, and shared peak is assert according to each chromatographic peak retention time and ultra-violet absorption spectrum feature, with reference to Figure 31
Shown, No. 1 peak~No. 21 peak derives from blumea riparia, and with reference to shown in Figure 32, No. 9 peaks derive from motherwort, with reference to Figure 33 institutes
Show, 10, No. 11 peaks derive from corydalis tuber, and with reference to shown in Figure 34,6,14,15, No. 16 peaks derive from radix glycyrrhizae.
The TLC Identification of 3-7 blumea riparias：
Blumea riparia thin layer differentiates the preparation of test solution：Take her blood of three lot numbers to pacify each 5g of particle, add water
30mL, makes her blood peace grain dissolution, adds dilute hydrochloric acid 0.25mL, stir evenly, filter, add diethyl ether extraction 3 times, each 15mL, closes
And ether extracted liquid, add water washing 2 times, each 15mL, discards aqueous, divides and takes ether extracted liquid, is evaporated, residue adds methanol 1mL
It is allowed to dissolve, differentiates test solution as blumea riparia thin layer；
Protocatechuic acid reference substance solution：Protocatechuic acid reference substance adds methanol that 0.30mgmL-1 solution is made；
Protocatechualdehyde reference substance solution：Protocatechualdehyde reference substance adds methanol that 0.30mgmL-1 solution is made；
Negative control solution：Take her the negative blood peace particulate samples of scarce blumea riparia by blumea riparia thin layer to differentiate to supply
Test sample solution preparation method is made in the same way of negative control solution.
Tested according to thin-layered chromatography (one annex VI B of Chinese Pharmacopoeia version in 2010), draw above-mentioned blumea riparia thin layer
Differentiate test solution, protocatechuic acid reference substance solution, each 15 μ L points of protocatechualdehyde reference substance solution in same silica gel thin-layer plate
On, using volume ratio as 6:3:5 chloroform, ethyl acetate, formic acid mixed solution are solvent expansion, and it is pre-saturated to be placed in solvent
In expansion cylinder, it is unfolded, takes out, dry, inspected under ultraviolet lamp (254nm), inspect that result is as shown in figure 35, A is 3-7
Negative control solution, B are protocatechuic acid reference substance solution, and C is protocatechualdehyde reference substance solution, and D, E, F are the Yunnan of three lot numbers
Osmanthus Blumea balsamifera thin layer differentiates test solution, it is seen that blumea riparia thin layer differentiate test solution chromatography with protocatechuic acid pair
According to product, protocatechualdehyde reference substance chromatography on relevant position, the spot of same color, and good separating effect, reappearance are shown
It is good.
The TLC Identification of 3-8 motherworts：
Motherwort thin layer differentiates the preparation of test solution：Take her blood of three lot numbers to pacify each 15g of particle, be ground to 100
~300 mesh, add absolute ethyl alcohol 50ml, shaking extraction 30 minutes, filtration, filtrate is evaporated, and it is molten that residue adds absolute ethyl alcohol 1ml to make
Solution, differentiates test solution as motherwort thin layer；
The preparation of stachydrine hydrochloride reference substance solution：Hydrochloric acid water is made per 1ml absolute ethyl alcohols dissolving stachydrine hydrochloride 0.1mg
Soviet Union's alkali reference substance solution；
The preparation of negative control sample solution：Take her blood of scarce motherwort to pacify particle, differentiate according to motherwort thin layer for examination
The preparation method of product solution carries out that negative control sample solution is made.
Tested according to thin-layered chromatography (one annex VI B of Chinese Pharmacopoeia version in 2010), take above-mentioned motherwort thin layer to differentiate and supply
Each 10 μ L of test sample solution, stachydrine hydrochloride reference substance solution, put on same silica gel thin-layer plate, using volume ratio as 6:6:The third of 1
Ketone, ethyl acetate, hydrochloric acid mixed solution are unfolded for solvent, are placed in the pre-saturated expansion cylinder of solvent, take out, dry, and spray
To improve bismuth potassium iodide test solution, heating makes spot development clear, and as shown in figure 36, G, H, I are three batches of the present embodiment
Motherwort thin layer differentiates test solution, and J is the stachydrine hydrochloride reference substance solution of the present embodiment, and K is the negative control of 3-8
Sample solution, motherwort thin layer differentiate test solution chromatography in position corresponding with stachydrine hydrochloride reference substance solution chromatography
On, show the spot of same color, and good separating effect, favorable reproducibility.
The methodological study of 4 fingerprint of embodiment
The preparation of 4-1 reference solutions：Weigh protocatechuic acid standard items 0.81mg to put in 10mL volumetric flasks, add methanol appropriate
Make dissolving and be settled to scale, shake up, up to the protocatechuic acid standard solution that concentration is 0.081mgmL-1
The preparation of her blood peace particle extracting solutions of 4-2：Her blood peace particle 5g is weighed, adds water 50mL to dissolve, adds dilute hydrochloric acid
0.3mL, stirs evenly, and stands 20min, and filtration, adds water-saturated n-butanol to extract 5 times, each 15mL, divides and takes n-butanol extracting liquid, closes
And 1h is stood, divide and take acquisition n-butanol liquid, recycle n-butanol, residue adds methanol 5mL, centrifuges, takes supernatant, obtains her blood peace
Grain extracting solution.
The chromatographic condition of 4-3 high performance liquid chromatographs：
Chromatographic column：Phenomenex Gemini C18 columns (4.6mm × 250mm, 5 μm)；
Guard column：Phenomenex C18(4×3.0mm)；
Mobile phase：The glacial acetic acid of methanol -0.1%；
Gradient elution program：Time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；
Corresponding gradient eluent is made of by volumes below ratio mobile phase A methanol and 0.1% glacial acetic acid of Mobile phase B, the body of mobile phase A
Product percentage gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70%, Mobile phase B
Percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55% → 55% → 30%；
Ultraviolet detection wavelength：256nm；
Flow velocity：1.0mL·min-1；
Column temperature：25℃.
4-4 Precision Experiments
Her blood peace particle (lot number 120303) is taken, preparing her same blood by the preparation of her the blood peace particle extracting solution of 4-2 pacifies
Particle extracting solution, is measured by the chromatographic condition of 4-3, and continuous sample introduction 6 times, records chromatogram, investigates the precision of instrument.The result is shown in
Table 1, table 2, the results showed that for the relative retention time RSD values of each chromatographic peak between 0.02%~1.13%, each chromatographic peak is opposite
Peak area RSD values show that the precision of instrument is good between 1.02%~2.92%.
Her blood peace particle finger-print of table 1 Precision Experiment-relative retention time
Her blood peace particle finger-print of table 2 Precision Experiment-relative peak area
4-5 repeated experiments
6 parts of her blood peace particles (lot number 120303) are taken, her same blood is prepared by the preparation of her the blood peace particle extracting solution of 4-2
Pacify particle extracting solution, measured by the chromatographic condition of 4-3, record chromatogram, investigate the repeatability of test method.It the results are shown in Table 3, table
The relative retention time RSD values of each chromatographic peak are between 1.03%~1.64% in 4,6 parts of samples, each chromatographic peak relative peak area
RSD values show that the repeatability of experimental method is good between 1.04%~3.09%.
Her blood peace particle finger-print of table 3 repeated experiment-relative retention time
Her blood peace particle finger-print of table 4 repeated experiment-relative peak area
4-6 stability experiments
Her blood peace particle (lot number 120303) is taken, preparing her same blood by the preparation of her the blood peace particle extracting solution of 4-2 pacifies
Particle extracting solution, is measured by the chromatographic condition of 4-3, is detected respectively in 0,2,4,8,16,24h, is investigated her blood peace particle and is carried
Take the stability of liquid.5, table 6 is the results are shown in Table, the relative retention time RSD values of each chromatographic peak are between 0.07%~1.76%, respectively
The relative peak area RSD values of chromatographic peak show that her blood peace particle extracting solution 24h internal stabilities are good between 1.45%~3.11%
It is good.
Her blood peace particle finger-print of table 5 stability experiment-relative retention time
Her blood peace particle finger-print of table 6 stability experiment-relative peak area
The collection of her blood peace particle finger-print of 5 10 batch of embodiment
Take her blood of 10 lot numbers to pacify particle, prepared by the preparation of her the blood peace particle extracting solution of 4-2 in embodiment 4 same
She pacifies particle extracting solution by blood, is measured by the chromatographic condition of 4-3, she pacifies particle HPLC chromatogram by blood to 10 lot numbers of record, with《Chinese medicine
The technical requirements (provisional) of injection finger-print research》Regulation, establishes her blood peace particle finger-print, her blood of 10 lot numbers
The matching figure of peace particle is shown in Figure 37.
Similarity evaluation：The similarity evaluation software recommended using the Chinese Pharmacopoeia committee
2004A editions, overall merit is carried out to HPLC collection of illustrative plates, similarity evaluation the results are shown in Table 7.
Her blood peace particle HPLC fingerprint similarity result of calculations of table 7
Determine with reference to peak (S) by the method for 3-5 in embodiment 3, using the retention time with reference to peak and peak area as 1, divide
Not Ji Suan fingerprint share the relative retention time at peak and relative peak area is shown in Table 8, table 9.
Her blood of 8 10 lot numbers of table pacifies particulate chromatography figure and shares peak relative retention time
Her blood of 9 10 lot numbers of table pacifies particulate chromatography figure and shares peak relative peak area
Experimental data shows, it is universal higher that test sample shares peak relative peak area RSD% values, and 10 batches of her blood peace particles
The similarity of HPLC finger-prints is all only more than 0.82, not up to《The technical requirements of traditional Chinese medicine finger-print research are (temporarily
OK)》It is more than 0.9 requirement in regulation.It is probably that raw medicinal material receives region, weather, environment, collecting time, storage etc.
Influence or the reasons such as product formulation technique is unstable cause each component content ratio difference.Should further study its influence because
Element, standardized planting, keeps quality of medicinal material to stablize, and strengthens control of product quality in production technology, is just effective to ensure that preparation
Stable quality.
Claims (9)
1. a kind of detection method of her blood peace particle, it is characterised in that comprise the following steps：
（1）Using protocatechuic acid and protocatechualdehyde as control, she is differentiated as solvent using chloroform, ethyl acetate, formic acid mixed solution
Blumea riparia component in blood peace particle；
（2）Using stachydrine hydrochloride as control, differentiate that she pacifies particle by blood as solvent using acetone, ethyl acetate, hydrochloric acid mixed solution
In motherwort component：
（21）Motherwort thin layer differentiates the preparation of test solution：Her blood peace particle is taken, adds absolute ethyl alcohol, shaking extraction, filters,
The filtrate obtained is evaporated, and residue adds absolute ethyl alcohol dissolving, differentiates test solution as motherwort thin layer；
（22）Stachydrine hydrochloride reference substance solution：It is molten that stachydrine hydrochloride reference substance adds absolute ethyl alcohol that stachydrine hydrochloride reference substance is made
Liquid；
（23）Above-mentioned motherwort thin layer is taken to differentiate test solution, stachydrine hydrochloride reference substance solution, point in same silica gel thin-layer
On plate, using volume ratio as 6:6:1 acetone, ethyl acetate, hydrochloric acid mixed solution are solvent expansion, take out, dry, spray to change
Good bismuth potassium iodide test solution, heating make spot development clear, motherwort thin layer differentiate test solution chromatography with stachydrine hydrochloride
On the corresponding position of reference substance solution chromatography, the spot of same color is shown；
（3）Using protocatechuic acid as object of reference, with high performance liquid chromatography detection, her blood pacifies particle extracting solution, obtains her blood and pacifies particle
Finger-print；Wherein, the extraction process of her blood peace particle extracting solution：Her blood peace particle is taken, is dissolved in water, adds dilute hydrochloric acid to adjust
PH, stirs evenly, and stands, and filtration, adds water-saturated n-butanol to extract 3 ~ 6 times, and point taking-up n-butanol extracting liquid, merges n-butanol extraction
Liquid, stands, and divides and takes acquisition n-butanol liquid, recycles n-butanol, and residue adds methanol to dissolve, and centrifuges, takes supernatant, obtains her blood peace particle
Extracting solution.
2. the detection method of her blood peace particle according to claim 1, it is characterised in that：
The step（1）Concrete operation method be：
（11）Blumea riparia thin layer differentiates the preparation of test solution：Her blood peace particle is taken, adds water, makes her blood peace particle molten
Solution, adds dilute hydrochloric acid, stirs evenly, and filters, and add diethyl ether extraction 2 ~ 3 times, obtains ether extracted liquid, merges ether extracted liquid, adds water
After washing 2 times, aqueous is discarded, divides and takes ether extracted liquid, be evaporated, residue adds methanol to be allowed to dissolve, as blumea riparia thin layer
Differentiate test solution；
（12）Protocatechuic acid reference substance solution：Protocatechuic acid reference substance adds methanol that protocatechuic acid reference substance solution is made；
（13）Protocatechualdehyde reference substance solution：Protocatechualdehyde reference substance adds methanol that protocatechualdehyde reference substance solution is made；
（14）Above-mentioned blumea riparia thin layer is taken to differentiate test solution, protocatechuic acid reference substance solution, protocatechualdehyde reference substance
Solution point is on same silica gel thin-layer plate, using volume ratio as 6:3:5 chloroform, ethyl acetate, formic acid mixed solution are solvent
Expansion, takes out, dries, inspect in the UV lamp.
3. the detection method of her blood peace particle according to claim 1, it is characterised in that：
The step（3）Concrete operation method be：
（31）The preparation of reference solution：Protocatechuic acid standard items, add methanol constant volume, and reference solution is made；
（32）The preparation of her blood peace particle extracting solution；
（33）The chromatographic condition of high performance liquid chromatograph：
Mobile phase：The glacial acetic acid of methanol -0.1%；
Gradient elution program：Time gradient 0min → 11min → 16min → 29min → 30min → 35min → 45min →
55min→70min→95min；Corresponding gradient eluent is by volumes below ratio by 0.1% ice of mobile phase A methanol and Mobile phase B
Acetic acid forms, the percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% of mobile phase A
→ 45% → 70%, the percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% of Mobile phase B
→ 55% → 55% → 30%；
Ultraviolet detection wavelength：256nm；
Flow velocity：1.0mL·min-1；
Column temperature：25℃；
（34）Measure：Her blood peace particle extracting solution injection high performance liquid chromatograph is drawn, according to step（33）Chromatographic condition survey
It is fixed, obtain finger-print.
4. the detection method of her the blood peace particle according to claim 1 or 3, it is characterised in that：
Her blood pacifies the concrete operation method that particle extracting solution extracts and is：Her blood peace 3 ~ 8g of particle is weighed, adds water 50mL to dissolve,
Adding 0.2 ~ 0.4mL of dilute hydrochloric acid, stir evenly, stand 15 ~ 30min, filtration, adds water-saturated n-butanol extraction 5 ~ 6 times, every time 15 ~ 20mL,
Dividing and take n-butanol extracting liquid, merge, stand 1 ~ 1.5h, divide and take acquisition n-butanol liquid, recycling n-butanol, residue adds 4 ~ 6mL of methanol,
Centrifugation, takes supernatant, obtains her blood peace particle extracting solution.
5. the detection method of her blood peace particle according to claim 1, it is characterised in that：
The finger-print shares chromatographic peak 21, its Average residence time is respectively：
No. 1 peak 3.234min, No. 2 peak 5.962min, No. 3 peak 11.023min, No. 4 peak 15.353min, No. 5 peak 16.521min,
No. 6 peak 24.498min, No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peak 32.29min, No. 10 peak 34.752min, 11
Number peak 36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, No. 14 peak 56.509min, No. 15 peaks
58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18 peak 68.358min, No. 19 peak 69.319min, 20
Number peak 85.217min, No. 21 peak 91.016min.
6. the detection method of her blood peace particle according to claim 5, it is characterised in that：
Her component of blood peace particle includes blumea riparia, motherwort, corydalis tuber and radix glycyrrhizae, wherein No. 1 peak ~ No. 21 peak comes
Blumea riparia is come from, No. 9 peaks derive from motherwort, and 10, No. 11 peaks derive from corydalis tuber, and 6,14,15, No. 16 peaks are from sweet
Grass.
7. the detection method of her blood peace particle according to claim 3, it is characterised in that：
The step（33）In, chromatographic column is Phenomenex Gemini C18 columns, specification：4.6mm × 250mm, 5 μm, protection
Column is Phenomenex C18, specification：4×3.0mm.
8. the detection method of her blood peace particle according to claim 3, it is characterised in that：
The step（33）In, theoretical cam curve is in terms of protocatechuic acid peak, not less than 5000.
9. the detection method of her the blood peace particle according to claim 1 or 3, it is characterised in that：
Her blood pacifies the concrete operation method that particle extracting solution extracts and is：Her blood peace particle 5g is weighed, adds water 50mL to dissolve, adds
Dilute hydrochloric acid 0.3mL, stirs evenly, and stands 20min, and filtration, adds water-saturated n-butanol to extract 5 times, each 15mL, divides and takes n-butanol to extract
Liquid, merges, and stands 1h, divides and takes acquisition n-butanol liquid, recycles n-butanol, and residue adds methanol 5mL, centrifuges, takes supernatant, obtain her blood
Pacify particle extracting solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610315836.XA CN106018587B (en) | 2016-05-12 | 2016-05-12 | The detection method of her blood peace particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610315836.XA CN106018587B (en) | 2016-05-12 | 2016-05-12 | The detection method of her blood peace particle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106018587A CN106018587A (en) | 2016-10-12 |
| CN106018587B true CN106018587B (en) | 2018-05-04 |
Family
ID=57100647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610315836.XA Active CN106018587B (en) | 2016-05-12 | 2016-05-12 | The detection method of her blood peace particle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106018587B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024879B (en) * | 2020-01-08 | 2021-08-31 | 河北中医学院 | Rapid multi-information thin-layer identification method for fresh motherwort herb freeze-dried powder, particles and standard decoction dry powder |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539588A (en) * | 2011-11-11 | 2012-07-04 | 云南良方制药有限公司 | Method for preparing test solution for quality detection of safe stagnation removing preparation |
| CN102937635B (en) * | 2012-11-27 | 2015-01-14 | 广西万寿堂药业有限公司 | Thin-layer identification method of leonurus in Yi Xue An particles |
-
2016
- 2016-05-12 CN CN201610315836.XA patent/CN106018587B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106018587A (en) | 2016-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104458993B (en) | The method for building up of strong medicinal material blumea riparia HPLC finger-print | |
| CN103197027A (en) | Quality control method of astragalus-leech capsules capable of regulating collaterals | |
| CN108226313A (en) | In glutinous rehmannia while methods of glycosides measure and fingerprint map construction method | |
| CN101028388B (en) | Quality inspection of Chinese-medicinal preparation for treating shortsighness and asthenopia | |
| CN109164178B (en) | Quality detection and identification method for gardenia and processed products thereof | |
| CN113466355A (en) | Construction method of high performance liquid phase characteristic spectrum of caulis sinomenii | |
| CN100437112C (en) | Method for inspecting Chinese medicinal preparation quality in treatment of old man eyes dieases | |
| CN101721475B (en) | Detection method for Yaotongning capsules | |
| CN108037222A (en) | Radix Paeoniae Alba reference extract and its preparation method and application | |
| CN108254470A (en) | In glutinous rehmannia while carbohydrate content measure and its fingerprint map construction method | |
| CN102309531B (en) | Detection method of American ginseng fingerprint | |
| CN101926889B (en) | Method for detecting white paeony root-medlar particles | |
| CN106018587B (en) | The detection method of her blood peace particle | |
| CN107643343B (en) | HPLC fingerprint spectrum determination method of Yunv Jian standard soup | |
| CN103344738B (en) | Detection method of nine-component heart-calming particle | |
| CN108088715A (en) | Moutan bark reference extract and its preparation method and application | |
| CN108663440A (en) | Callicarpa nudiflora medicinal material UPLC fingerprint map constructions method and standard finger-print | |
| CN105784911B (en) | Her blood pacifies the method for building up of particle HPLC finger-prints | |
| CN110274970A (en) | The method for building up for melting poor finger-print and its application in Yixiesheng capsule Quality Control | |
| CN109633035A (en) | The HPLC finger-print and its construction method of a kind of hawthorn broken wall medicine materical crude slice and application | |
| CN110632198B (en) | HPLC fingerprint of inflammation diminishing and cough relieving tablets and construction method and application thereof | |
| CN112858526B (en) | Centipeda minima fingerprint spectrum and construction method thereof | |
| CN101953978B (en) | Heart-soothing and lipid-lowering tablet medicine quality detecting method | |
| CN104034823B (en) | The detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material | |
| CN109061024B (en) | Construction method and application of standard fingerprint spectrum of infant spleen-supporting granules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Detection method of Yixuean granules Effective date of registration: 20190428 Granted publication date: 20180504 Pledgee: Bank of Communications Ltd the Guangxi Zhuang Autonomous Region branch Pledgor: Guangxi Medictop Pharmaceutical Co., Ltd. Registration number: 2019450000016 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210604 Granted publication date: 20180504 Pledgee: Bank of Communications Ltd. the Guangxi Zhuang Autonomous Region branch Pledgor: GUANGXI MEDICTOP PHARMACEUTICAL Co.,Ltd. Registration number: 2019450000016 |