CN109374785A - The construction method and detection method of lophatherum gracile medicinal material UPLC characteristic spectrum - Google Patents
The construction method and detection method of lophatherum gracile medicinal material UPLC characteristic spectrum Download PDFInfo
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Abstract
The present invention relates to a kind of lophatherum gracile medicinal material UPLC characteristic spectrum construction method and detection methods.This feature map construction method includes the following steps: to prepare reference substance reference solution using Lutonaretin and swertiajaponin as reference substance；Control medicinal material reference solution is prepared with lophatherum gracile control medicinal material；Lophatherum gracile medicinal material and standard decoction sample are taken respectively, Extraction solvent is added to extract, and are filtered, gained filtrate is as test solution and standard decoction test solution；The reference substance reference solution, test solution, standard decoction test solution are drawn respectively, inject Ultra Performance Liquid Chromatography instrument, measurement；Compare gained test sample map and standard decoction test sample map, demarcated the characteristic peak of 4 water soluble ingredients, obtains the UPLC characteristic spectrum of lophatherum gracile medicinal material.This feature map can be used for the quality of qualitative and quantitative analysis lophatherum gracile medicinal material, it can be ensured that using the quality of the lophatherum gracile traditional decoction of medicinal material preparation, be also applied for detecting other preparations containing lophatherum gracile.
Description
Technical field
The present invention relates to pharmaceutical technology fields, more particularly to a kind of construction method of lophatherum gracile medicinal material UPLC characteristic spectrum
And detection method.
Background technique
Lophatherum gracile is the drying cauline leaf of grass henon bamboo leaf LophatherumgracileBrongn..Summer does not take out
It taps, dries before flower spike.Have clearing heat-fire, relieving restlessness quench the thirst, inducing diuresis for treating strangurtia the effect of.Lophatherum gracile is bright first recorded in " the southern regions of the Yunnan Province book on Chinese herbal medicine "
Claim it in Compendium of Material Medica for Li Shizhen (1518-1593 A.D.): sweet, cold, nontoxic, go dysphoria with smothery sensation, diuresis, clear away heart-fire.Currently, chemistry known to lophatherum gracile
Ingredient additionally includes mainly volatile oil, triterpenes, phenolic acid, polysaccharide, amino acid etc. based on flavonoids.Modern pharmacology card
Bright, lophatherum gracile has antipyretic, diuresis, antibacterial action, is clinically usually used in treating pyreticosis and polydipsia, aphthae, swelling and aching of gum, children
The card such as crying with fear, cough with lung heat, stomach hot vomiting, hot urination stranguria with turbid discharge.In addition to it can be used for medicinal purpose, lophatherum gracile can also be used in health care of food product
Equal fields, in recent years with the development of herbal tea, annual consumption is consequently increased.It is needed with stronger clinical value, market
It asks wide, is developed to Chinese medicinal granule and related compound preparation is widely used in clinic.
The clinical use of Chinese medicine is mostly based on traditional decoction.The material base of traditional Chinese herbal decoction is prevented under instruction of Chinese Medicine theory
Control the basis of disease.Existing statutory standards carry out quantitative control only for single component, during dose-effect relationship cannot reflect comprehensively
The mass action of medicine ingredient.In the case where Chinese medicine overwhelming majority effective component at this stage is not known, Chinese medicine characteristic spectrum/spy
The foundation of sign map can greatly improve the technical level and scientific and technological content of traditional Chinese medicine quality control.
" Chinese Pharmacopoeia " version in 2015 does not establish the content determination item of lophatherum gracile.At present literature research report about light
The foundation of leaf of bamboo characteristic spectrum, just for medicinal raw material, index components mostly using liposoluble constituent as research object, are mainly used for
The Chinese medicine true and false, the place of production, quality discrepancy Qualitive test, it is more difficult to comprehensively reflection lophatherum gracile qualitative character, biography can not be reacted
The material base feature for traditional Chinese herbal decoction of uniting.
Summary of the invention
Based on this, the present invention provides the construction method and detection method of a kind of lophatherum gracile medicinal material UPLC characteristic spectrum.Building
Lophatherum gracile medicinal material characteristic spectrum have 4 water soluble ingredients characteristic peak, can realize quickly, comprehensively to lophatherum gracile medicinal material
The quality monitoring of multiple characteristic components, and the material base feature of the traditional Chinese medicine decoction of lophatherum gracile can be reacted.
The specific technical proposal is:
A kind of construction method of lophatherum gracile medicinal material characteristic spectrum, comprising the following steps:
The preparation of reference solution: respectively using Lutonaretin and swertiajaponin as reference substance, solubilizer dissolution, preparation pair
According to product reference solution；Extraction solvent is added into lophatherum gracile control medicinal material, is ultrasonically treated, extracting solution is obtained, by the extracting solution
Constant volume, filtration, takes subsequent filtrate as control medicinal material reference solution；
The preparation of test solution: Extraction solvent being added into lophatherum gracile medicinal material, and ultrasonic treatment obtains extracting solution I, will be described
Extracting solution I constant volume, filtration, takes subsequent filtrate as test solution I；Extraction solvent is added into lophatherum gracile standard decoction sample,
Ultrasonic treatment, obtains extracting solution II, and by the extracting solution II constant volume, filtration takes subsequent filtrate as test solution II；
Measurement: respectively by the reference substance reference solution, control medicinal material referring to reference solution, test solution I and
Test solution II injects Ultra Performance Liquid Chromatography instrument, and measurement demarcates water-soluble shared peak, obtains lophatherum gracile medicinal material UPLC feature
Map.
The chromatographic condition of the ultra performance liquid chromatography includes: in one of the embodiments,
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution that volume fraction is 0.05%-0.2%, gradient
Elution；
The gradient elution specifically:
0-2min, keeping the volume fraction of mobile phase A is 10%, keeps the volume fraction 90% of Mobile phase B；
The volume fraction of 2min-3min, mobile phase A are increased to 16% by 10%, and the volume fraction of Mobile phase B is by 90% drop
As low as 84%；
3min-7.5min, keeping the volume fraction of mobile phase A is 16%, and keeping the volume fraction of Mobile phase B is 84%；
7.5min-16min, the volume fraction of mobile phase A are increased to 52% by 16%, the volume fraction of Mobile phase B by
84% is reduced to 48%.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, further include: 30 DEG C -40 DEG C of column temperature,
Flow velocity is 0.1mL-0.5mL per minute, Detection wavelength 340nm-360nm.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, are as follows: mobile phase A is acetonitrile, flowing
Phase B is the phosphate aqueous solution that volume fraction is 0.1%, and 30 DEG C of column temperature, flow velocity is 0.25mL per minute, Detection wavelength 350nm.
The Extraction solvent is the ethanol water that volume fraction is 50%-70% in one of the embodiments,.
The Extraction solvent is the ethanol water that volume fraction is 60% in one of the embodiments,.
The time of the ultrasonic treatment is 20min-40min in one of the embodiments,.
The present invention also provides a kind of detection methods of lophatherum gracile medicinal material.
The specific technical proposal is:
A kind of detection method of lophatherum gracile medicinal material, comprising the following steps:
The preparation of reference solution: respectively using Lutonaretin and swertiajaponin as reference substance, solubilizer dissolution, preparation pair
According to product reference solution；Extraction solvent is added into lophatherum gracile control medicinal material, is ultrasonically treated, extracting solution is obtained, by the extracting solution
Constant volume, filtration, takes subsequent filtrate as control medicinal material reference solution；
The preparation of testing sample solution: Extraction solvent being added into sample to be tested, and ultrasonic treatment obtains extracting solution III, by institute
Extracting solution III constant volume is stated, filters, takes subsequent filtrate as testing sample solution；
Measurement: the reference substance reference solution, control medicinal material are injected referring to reference solution, testing sample solution and surpassed
High performance liquid chromatograph, measurement.
In one of the embodiments, in the preparation of test solution, the lophatherum gracile medicinal material includes different sources
Lophatherum gracile medicinal material, test sample raw material of the invention come from the national biggish 6 genuine or major production areas of henon bamboo leaf-making quantity, and totally 18 batches
Secondary sample has adequately representative.
The chromatographic condition of the ultra performance liquid chromatography detection includes: in one of the embodiments,
Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution that volume fraction is 0.05%-0.2%, gradient elution；
The gradient elution specifically:
0-2min, keeping the volume fraction of mobile phase A is 10%, keeps the volume fraction 90% of Mobile phase B；
The volume fraction of 2min-3min, mobile phase A are increased to 16% by 10%, and the volume fraction of Mobile phase B is by 90% drop
As low as 84%；
3min-7.5min, keeping the volume fraction of mobile phase A is 16%, and keeping the volume fraction of Mobile phase B is 84%；
7.5min-16min, the volume fraction of mobile phase A are increased to 52% by 16%, the volume fraction of Mobile phase B by
84% is reduced to 48%.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, further include: chromatographic column is with octadecane
Base silane bonded silica gel is filler, and 30 DEG C -40 DEG C of column temperature, flow velocity is 0.1mL-0.5mL per minute, Detection wavelength 340nm-
360nm。
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, are as follows: mobile phase A is acetonitrile, flowing
Phase B is the phosphate aqueous solution that volume fraction is 0.1%, and 30 DEG C of column temperature, flow velocity is 0.25mL per minute, Detection wavelength 350nm.
The Extraction solvent is the ethanol water that volume fraction is 50%-70% in one of the embodiments,.
The Extraction solvent is the ethanol water that volume fraction is 60% in one of the embodiments,.
The time of the ultrasonic treatment is 20min-40min in one of the embodiments,.
Compared with prior art, the invention has the following advantages:
The present invention introduces lophatherum gracile mark using the characteristic spectrum of ultra performance liquid chromatography (UPLC) method building lophatherum gracile medicinal material
The characteristic spectrum of quasi- decoction is studied, the water-soluble character ingredient shared for lophatherum gracile standard decoction and lophatherum gracile medicinal material into
Row research (confirmed the ingredient of 4 common characteristic peaks, respectively rhinoceros grass element 6-C- β D- six using UPLC-MS and related control product
Carbon alditol acidic group (1 → 2)-β-D-Glucose glycosides, Lutonaretin, swertiajaponin and luteolin 6-C- β D- hexose aldehydic acid
Base (1 → 2)-α-L-arabinose glycosides), and the foundation determined as lophatherum gracile medicinal material characteristic spectrum characteristic peak, it can be good at
The substance for characterizing medicinal raw material to decoction transmits, can not only good control quality of medicinal material, and can also ensure that decoction
Quality；Meanwhile reference substance, the dual control of control medicinal material are used, it can effectively overcome liquid-phase condition finger-print to be inherently present
Durability offset issue, compare more comprehensively.
Using chromatographic condition of the invention, 4 common characteristic peaks that can be realized in lophatherum gracile medicinal material characteristic spectrum are preferable
Separation, characteristic spectrum abundant information, characteristic is strong.And using assay peak (different Polygonum orientale) as reference peak, it is specified that other 3
Relative retention time and relative peak area the limitation range of characteristic peak, realize the quantification of multiple characteristic components, meet Chinese medicine
Mass action theory.
Present invention employs UPLC methods, compared with conventional H PLC method, more efficiently, quickly, environmental protection；It is constructed using the present invention
Characteristic spectrum and method, favorable reproducibility can accurately and reliably realize quickly, comprehensively to the multiple characteristic components of lophatherum gracile medicinal material
Quality monitoring, not only improved the quality control level of lophatherum gracile medicinal material, but also promote and stablize the inherent quality of lophatherum gracile medicinal material,
The raw material for meeting lophatherum gracile standard decoction requirement is provided for clinic, is provided for the relevant preparation process production process of lophatherum gracile important
Multi-index parameter foundation.
Detailed description of the invention
Fig. 1 is the characteristic spectrum of lophatherum gracile medicinal material under different wave length；
Fig. 2 is the characteristic spectrum of lophatherum gracile medicinal material under full wavelength scanner；
Fig. 3 is the characteristic spectrum of lophatherum gracile medicinal material under different solvents；
Fig. 4 is total peak area/sample weighting amount comparison column diagram in lophatherum gracile medicinal material characteristic spectrum under different solvents；
Fig. 5 is 17 batches of lophatherum gracile medicinal material characteristic spectrum (peaks 1: luteolin 6-C- β D- hexose aldehydic acid base (1 → 2)-β-
D-Glucose glycosides；Peak 2 (S): Lutonaretin；Peak 3: swertiajaponin；Peak 4: luteolin 6-C- β D- hexose aldehydic acid base (1 →
2)-α-L-arabinose glycosides)；
Fig. 6 is 17 batches of lophatherum gracile standard decoction characteristic spectrums；
Fig. 7 is lophatherum gracile standard decoction characteristic spectrum；
Fig. 8 is lophatherum gracile medicinal material, lophatherum gracile standard decoction characteristic spectrum；
Fig. 9 is lophatherum gracile medicinal material compare feature map；
Figure 10 is lophatherum gracile medicinal material, lophatherum gracile control medicinal material object of reference characteristic spectrum comparison diagram；
Figure 11 is the characteristic spectrum of each reference substance and one figure of comparison of lophatherum gracile characteristic spectrum；
Figure 12 is the characteristic spectrum of each reference substance and two figure of comparison of lophatherum gracile characteristic spectrum；
Figure 13 is the characteristic spectrum of sample to be tested.
Specific embodiment
Construction method and detection side below in conjunction with specific embodiment to lophatherum gracile medicinal material UPLC characteristic spectrum of the invention
Method is described in further detail.The invention can be realized in many different forms, however it is not limited to reality described herein
Apply mode.On the contrary, the purpose of providing these embodiments is that making to understand the disclosure of invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
The building of 1 lophatherum gracile medicinal material characteristic spectrum of embodiment
1, instrument, reagent and reagent
Instrument is as shown in table 1, and reagent is as shown in table 2, and reagent is as shown in table 3:
Table 1
Table 2
Table 3
2, research object
The present embodiment lophatherum gracile medicinal material comes from that national henon bamboo leaf-making quantity biggish 7 is genuine or major production areas totally 17 batch sample
Product are specifically shown in Table 4:
Table 4
Remarks: Lutonaretin content is the lower content assaying method measurement of lophatherum gracile by " Hong Kong Chinese medicine standard ".
3, the preparation of reference substance reference solution, control medicinal material reference solution, standard decoction
Lutonaretin reference substance 10mg is taken, is set in 50mL volumetric flask, methanol is added to dissolve and is settled to scale, precision measures
1mL is set in 20mL measuring bottle, is diluted to scale with the ethanol water that volume fraction is 30%, and every 1mL is made containing the 10 different Polygonum orientales of μ g
The solution of glycosides is to get Lutonaretin reference substance reference solution.
Swertiajaponin reference substance 10mg is taken, is set in 50mL volumetric flask, methanol is added to dissolve and is settled to scale, precision measures
1mL is set in 20mL measuring bottle, is diluted to scale with the ethanol water that volume fraction is 30%, and every 1mL is made containing 10 μ and works as medicine within g days
The solution of flavine is to get swertiajaponin reference substance reference solution.
Control medicinal material reference solution: taking 1.0g lophatherum gracile control medicinal material powder, accurately weighed, sets in 50mL measuring bottle, adds
Enter 60% (volume fraction) appropriate ethanol water, ultrasonic treatment (power 500W, frequency 40HKz) 10 minutes takes extracting solution, will
The extracting solution is let cool, and is settled to scale with 60% (volume fraction) ethanol water, is shaken up, and filtration takes subsequent filtrate as light
Leaf of bamboo control medicinal material reference solution.
Standard decoction: taking lophatherum gracile medicinal material, sorts removal of impurities, and dissection 1-2cm obtains lophatherum gracile medicine materical crude slice；Piece 100g is drunk, water is added
Secondary, first time decoction plus 15 times of amount water are decocted, after impregnating 30 minutes, mild fire keeps slightly boiled 20 points of decoction again after first boiling by intense fire
Clock is filtered while hot with 350 mesh screens, and filtrate is cooling with cold water rapidly；Second of decoction plus 13 times of amount water, after first boiling by intense fire again
Mild fire keeps slightly boiled and decocts 15 minutes, is filtered while hot with 350 mesh screens, and the rapid cold water of filtrate is cooling, merges filtrate twice.Decompression
Concentration (temperature: 65 DEG C, revolving speed: 70-90 revs/min, vacuum degree: -0.08~-0.1MPa) it is left for 15% to clear cream solid content
It is right；Packing is into cillin bottle, every bottle of packing 2ml, vacuum freeze drying, takes out, rolls aluminium lid to obtain the final product.
4, chromatographic condition
4.1 chromatographic conditions determine are as follows: mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of 0.1% (volume fraction),
Gradient elution, gradient condition is as shown in table 5, and chromatographic column is CORTECS T3 column (column length 100mm, internal diameter 2.1mm, partial size
It is 1.6 μm), column temperature is 30 DEG C, and flow velocity is 0.25mL per minute, Detection wavelength 350nm.
Under above-mentioned chromatographic condition, characteristic peak information is more complete in the characteristic spectrum of lophatherum gracile medicinal material, and separating degree is preferable.
Table 5
The determination of 4.2 Detection wavelengths
By full wavelength scanner, the peak situation of lophatherum gracile medicinal material difference chromatography wavelength is investigated, by mobile wavelength, is investigated not
Chromatographic peak information under co-wavelength, selects last Detection wavelength.
The specific method is as follows:
It takes 0.1g lophatherum gracile medicinal powder (lot number: DZY04), it is accurately weighed, it sets in 50mL measuring bottle, 60% (volume is added
Score) appropriate ethanol water, it is ultrasonically treated (power 500W, frequency 40HKz) 10 minutes, extracting solution is taken, by the extracting solution
It lets cool, is settled to scale with 60% (volume fraction) ethanol water, shakes up, filter, take subsequent filtrate as test solution, it will
Above-mentioned test solution injecting chromatograph records the absorption spectrum within the scope of 190~400nm, as depicted in figs. 1 and 2.
Fig. 1 and Fig. 2 the result shows that: wavelength be 350mm locate, the Whole Response at each peak is preferable, interfere it is less, selection 350mm
For Detection wavelength.
5, the preparation of test solution
The selection of 5.1 Extraction solvents
Methanol, 50% (volume fraction) methanol aqueous solution, 30% (volume fraction) ethanol water, 60% are chosen respectively
(volume fraction) ethanol water and water are as Extraction solvent, by temporarily pointing out 4 chromatographic peak total peak area/sample weighting amounts and color
Spectrogram compares different solvents characteristic spectrum result.Method particularly includes:
It takes 1.0g Lophatherum Herb (crossing No. four sieves, lot number: DZY04), it is parallel 5 parts, accurately weighed, it sets in 50mL measuring bottle,
It is separately added into methanol, 50% (volume fraction) methanol aqueous solution, 30% (volume fraction) ethanol water, 60% (volume fraction)
Ethanol water and appropriate amount of water, ultrasonic treatment (power 500W, frequency 40kHZ) 30 minutes, take extracting solution, the extracting solution are put
It is cold, it is settled to graduation mark with coordinative solvent, is shaken up, filters, takes subsequent filtrate to get test solution.
Using the chromatographic condition in 4.1, UPLC survey is carried out to the test solution for the lophatherum gracile medicinal material that different solvents extract
Examination, obtains the characteristic spectrum of 5 lophatherum gracile medicinal materials, as shown in figure 3, its total peak area/sample weighting amount is as shown in table 6 and Fig. 4.
Table 6
Experimental result: the characteristic spectrum of the lophatherum gracile medicinal material extracted by 5 kinds of different solvents of comparison can be found: 60% (body
Fraction) ethanol water as Extraction solvent when, total peak area/sample weighting amount be maximum, extraction efficiency better than other four kinds it is molten
Agent, and peak type is preferable, toxicity is lower, therefore chooses 60% (volume fraction) ethanol water as Extraction solvent.
5.2 determine test solutions the preparation method is as follows:
It takes 1.0g Lophatherum Herb (crossing No. four sieves), it is accurately weighed, it sets in 50mL measuring bottle, 60% (volume fraction) second is added
Appropriate alcohol solution, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, takes extracting solution, the extracting solution is let cool, and uses
60% (volume fraction) ethanol water is settled to scale, shakes up, and filtration takes subsequent filtrate to get test solution.
6, the determination of lophatherum gracile medicinal material characteristic spectrum characteristic peak
17 batches of lophatherum gracile medicinal materials, by the preparation method of the test solution in 5.2, prepare 17 batches of lophatherum gracile medicinal materials for examination
Product solution I carries out UPLC measurement to above-mentioned 17 batches of lophatherum graciles medicinal material test solution I by the chromatographic condition in 4.1, obtains 17 batches
Lophatherum gracile medicinal material characteristic spectrum.Referring to Fig. 5.
It separately takes 17 batches of lophatherum gracile standard decoction samples appropriate, by the preparation method of the test solution in 5.2, prepares 17 batches
The test solution II of lophatherum gracile standard decoction sample, by the chromatographic condition in 4.1 to above-mentioned 17 batches of lophatherum graciles standard decoction sample
The test solution II of product carries out UPLC measurement, obtains 17 batches of lophatherum gracile standard decoction characteristic spectrums.Referring to Fig. 6.
The results showed that 17 batches of lophatherum gracile medicinal material characteristic spectrums have 4 characteristic peaks, and the equal energy of 4 characteristic peaks
It is transferred in lophatherum gracile standard decoction from lophatherum gracile medicinal material stabilization.That is lophatherum gracile medicinal material characteristic spectrum and standard decoction characteristic spectrum
In 4 characteristic peaks it is corresponding, therefore, select this 4 characteristic peaks for the shared peak of radix stemonae tuberosae medicinal material, and with (the different Polygonum orientale of peak 2
Glycosides) it is to carry out research on standard referring to peak.Details are referring to Fig. 7, Fig. 8.
7, methodology validation
Study on the stability
Precision draws lophatherum gracile medicinal material (DZY04), and the preparation method of the test solution in use 5.2 prepares test sample
Solution, the chromatographic condition in use 4.1 carry out UPLC measurement, respectively in 0,4,8,12,16,20,24 hour sample introduction, with different Polygonum
Careless glycosides peak is to calculate relative retention time and relative peak area referring to peak, the results are shown in Table 7 and table 8.
7 characteristic spectrum stability result (relative retention time) of table
8 characteristic spectrum stability result (relative peak area) of table
Experimental result is shown: in 24 hours, each chromatography relative retention time relative peak area RSD of test solution is less than
2%, show that test solution is stablized in 24 hours.
8, the determination of lophatherum gracile medicinal material characteristic spectrum
(1) 17 batches of lophatherum gracile medicinal material characteristic spectrums are analyzed, is to calculate each peak referring to peak with 2 Lutonaretin peak of peak
Relative retention time and relative peak area, experimental result are shown in Table 9- table 12.
9 17 batches of lophatherum gracile medicinal material characteristic spectrums (retention time) of table
10 17 batches of lophatherum gracile medicinal material characteristic spectrums (relative retention time) of table
11 17 batches of lophatherum gracile medicinal material characteristic spectrums (peak area) of table
12 17 batches of lophatherum gracile medicinal material characteristic spectrums (relative peak area) of table
The results show that lophatherum gracile medicinal material there are 4 characteristic peaks relatively stable, peak is shared with lophatherum gracile standard decoction characteristic spectrum
Unanimously.
It is that remaining 3 characteristic peak of 17 batches of lophatherum gracile medicinal material characteristic spectrums are opposite referring to peak with 2 Lutonaretin peak of peak to retain
Time RSD value meets the standard requirements of lophatherum gracile medicinal material characteristic spectrum 0.05%~1.44%, respectively less than 2.0%；17 batches light
The RSD of 3 characteristic peak relative peak areas of leaf of bamboo medicinal material characteristic spectrum is 23.83%~57.18%.The result shows that different sources
There is some difference for each characteristic peak area of lophatherum gracile medicinal material, and 1 relative peak area ranges of peak are 1.008~2.349, and peak 3 is opposite
Peak area range is 0.085~1.882, and 4 relative peak area ranges of peak are 0.043~1.729.
It is henon bamboo leaf preparation, such as lophatherum gracile standard decoction and granule for the quality of strict control lophatherum gracile medicinal material
Preparation provides raw medicinal material of fine quality and stable, to the characteristic peak relative peak area setting limitation mark of lophatherum gracile medicinal materials fingerprint
Standard is very important.It is fluctuated according to the relative peak area at the peak 2 of 17 batch different sources lophatherum gracile medicinal materials and other 3 peaks
Range considers the representativeness of 17 batch different sources samples, takes that each characteristic peak relative peak area is minimum, peak, therefore, fixes tentatively
The standard of lophatherum gracile medicinal material characteristic spectrum are as follows: 4 characteristic peaks should be presented in sample chromatogram, and spy should be compareed with lophatherum gracile medicinal material
The 4 characteristic peak retention times levied in map are corresponding；Peak corresponding with Lutonaretin object of reference peak is the peak S, calculates each feature
The relative retention time at peak and the peak S, relative retention time should be within the scope of ± the 10% of specified value, it is specified that value are as follows: 0.866
(peak 1), 1.046 (peaks 3), 1.195 (peaks 4).The relative peak area of each characteristic peak Yu the peak S is calculated, relative peak area should advise
Determine in range, it is specified that range are as follows: 1.0~2.3 (peaks 1), 0.08~1.9 (peak 2), 0.04~1.7 (peak 3).
(2) characteristic spectrum standard is drafted
It uses " Chinese medicine chromatographic characteristics map similarity evaluation software " to match 17 batches of lophatherum gracile medicinal material characteristic spectrums, generates
Map is compareed, establishes lophatherum gracile medicinal material compare feature map, as shown in Figure 9.
The standard of tentative lophatherum gracile medicinal material characteristic spectrum are as follows: 4 characteristic peaks should be presented in sample chromatogram, and should be with henon bamboo
4 characteristic peak retention times in leaf medicinal material compare feature map are corresponding；Peak corresponding with Lutonaretin object of reference peak is S
Peak, calculates the relative retention time of each characteristic peak Yu the peak S, relative retention time should within the scope of ± the 10% of specified value,
Specified value are as follows: 0.866 (peak 1), 1.046 (peaks 3), 1.195 (peaks 4).Calculate the relative peak area of each characteristic peak Yu the peak S, phase
It should be within the specified scope, it is specified that range to peak area are as follows: 1.0~2.3 (peaks 1), 0.08~1.9 (peak 2), 0.04~1.7 (peak
3)。
9, characteristic peak is pointed out
Chemistry is carried out to the characteristic peak on lophatherum gracile characteristic spectrum using UPLC-MS/MS to point out, and determines its chemical component.
(1) instrument and reagent
Waters ACQUITY UPLCTMI-Class type Ultra Performance Liquid Chromatography instrument；When water generation ultra high efficiency liquid phase flight
Between high resolution mass spectrum combined system (Xevo G2-XS QTOF MS), data processing system be 1.8 work station of UNIFI
(Waters,Manchester,U.K.)；Cortecs T3 (2.1*100mm, 1.6 μm) chromatographic column.
T-114 a ten thousandth balance (Beijing Sai Duolisi instrument system Co., Ltd)；KQ-3000E ultrasonic cleaner
(Kunshan Ultrasonic Instruments Co., Ltd.)；Direct-Q5 type ultrapure water machine (Millipore company of the U.S.).
(2) liquid-phase condition
Chromatographic condition is the same as 4.1.
(3) Mass Spectrometry Conditions
Atomization of the nitrogen as mass ion source, taper hole gas；Electrospray ionisation positive ion mode；Capillary voltage:
2.0KV；Orifice potential: 40V；Ion source temperature: 120 DEG C；Desolvation temperature: 450 DEG C；Desolvention gas velocity: 800L/h；It sweeps
Retouch the time: 0.5s；Trace interval: 0.02s；Mass charge ratio range: 50~1200；Data acquisition scheme: it is acquired under ESI (-)
MSE；It is that Internal standard correction methods are high in quality with leucine enkephalin (Leucine-enkephalin) with sodium formate solution correction mass axis
Degree, taper hole gas flow: 50L/hr.
(4) preparation of test solution, control medicinal material reference solution
The preparation of test solution is the same as 5.2.
The preparation of control medicinal material reference solution is the same as 3.
(5) experiment purpose
Using above-mentioned chromatography and mass spectrometric analysis method, test solution is detected.Pass through the chromatographic peak of compound
Retention behavior, accurate molecular weight identify that peak 1,3,4 corresponds to compound in the ultraviolet chromatogram of lophatherum gracile.Figure 10 is lophatherum gracile medicine
The characteristic spectrum of material and the comparison diagram of control medicinal material object of reference, have pointed out multiple characteristic peaks, including peak 1: luteolin 6-C- β D-
Hexose aldehydic acid base (1 → 2)-β-D-Glucose glycosides；Peak 2: Lutonaretin；Peak 3: swertiajaponin；Peak 4: luteolin 6-C- β
D- hexose aldehydic acid base (1 → 2)-α-L-arabinose glycosides.
(6) experimental result
According to document and point out report display lophatherum gracile in deposited luteolin 6-C- β D- hexose aldehydic acid base (1 → 2)-
β-D-Glucose glycosides, swertiajaponin, Lutonaretin, orientoside, Vitexina, Saponaretin and chlorogenic acid etc., MS points out report
Show that maximum peak is luteolin 6-C- β D- hexose aldehydic acid base (1 → 2)-β-D-Glucose glycosides, but because can't buy control,
It can not be confirmed.Other reference substances that can be bought have: swertiajaponin, Lutonaretin, orientoside, Vitexina, Saponaretin
With chlorogenic acid reference substance, reference substance information is as shown in table 14, prepares each reference substance reference solution according to 3, joins to each reference substance
UPLC detection is carried out according to object solution, as a result as shown in Figure 11,12, is found in lophatherum gracile almost without chlorogenic acid；Orientoside, Vitex negundo var cannabifolia
Glycosides and Saponaretin peak response are all very weak and unstable, therefore only determining swertiajaponin and Lutonaretin peak are in characteristic spectrum
Known peak.
Table 14
(7) characteristic peak belongs to
The final common characteristic peaks ownership determined in Fig. 9 is as follows:
Peak 1: luteolin 6-C- β D- hexose aldehydic acid base (1 → 2)-β-D-Glucose glycosides；
Peak 2 (S): Lutonaretin (*)；
Peak 3: swertiajaponin (*)；
Peak 4: luteolin 6-C- β D- hexose aldehydic acid base (1 → 2)-α-L-arabinose glycosides
(* is confirmed with reference substance)
The detection method of 2 lophatherum gracile medicinal material of embodiment
The present embodiment provides a kind of detection methods of lophatherum gracile medicinal material, and steps are as follows:
(1) chromatographic condition
With 4.1 in embodiment 1.
(2) preparation of reference substance reference solution
With in embodiment 13.
(3) preparation of testing sample solution
Sample to be tested 1.0g (lot number: DZY18, qualified by Chinese Pharmacopoeia full inspection) is taken, sets in 50mL volumetric flask, adds 60%
(volume fraction) appropriate ethanol water, ultrasonic treatment (power 500W, frequency 40kHz) 30 minutes, takes extracting solution, mentions described
It takes liquid to let cool, is settled to scale with 60% (volume fraction) ethanol water, shakes up, filter, take subsequent filtrate as sample to be tested
Solution.
(3) it measures
Precision draws 1 μ l of testing sample solution, injects liquid chromatograph, measurement.
Interpretation of result and discussion
The characteristic spectrum of sample to be tested is as shown in figure 13, compares with Fig. 9 it is found that the characteristic spectrum of sample to be tested does not have feature
Peak 4 further compares the relative retention time and relative peak area of sample to be tested characteristic spectrum and Fig. 9 characteristic peak, such as table 15,16
It is shown.
15 DZY18 characteristic spectrum of table is compared with lophatherum gracile medicinal material characteristic spectrum (relative retention time)
16 DZY18 characteristic spectrum of table is compared with lophatherum gracile medicinal material characteristic spectrum (relative peak area)
By Figure 13, table 15-16 it is found that lot number is the lophatherum gracile medicinal material of DZY18 and compareing for other 17 batches of lophatherum gracile medicinal materials
Characteristic spectrum differs greatly, and is mainly reflected in the lophatherum gracile medicinal material that lot number is DZY18 and does not have peak 4.The display of table 16, the phase of the two
To peak area significant difference, the compare feature map and defined relative peak area established by 17 batch samples of preceding research are limited the quantity,
Lophatherum gracile sample can be effectively distinguished, the medicinal material that lot number is DZY18 is adulterant.
It further illustrates that the detection method of lophatherum gracile medicinal material described in the present embodiment has taste, can be controlled from source
Pharmacy material, and the particle of folk prescription preparation is identified, it provides the foundation for lophatherum gracile research, enhances lophatherum gracile granule
Raw materials used total quality control.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of construction method of lophatherum gracile medicinal material UPLC characteristic spectrum, which comprises the following steps:
The preparation of reference solution: respectively using Lutonaretin and swertiajaponin as reference substance, solubilizer dissolution prepares reference substance
Reference solution；Extraction solvent is added into lophatherum gracile control medicinal material, is ultrasonically treated, obtains extracting solution, by the extracting solution constant volume,
Filtration, takes subsequent filtrate as control medicinal material reference solution；
The preparation of test solution: Extraction solvent being added into lophatherum gracile medicinal material, and ultrasonic treatment obtains extracting solution I, by the extraction
Liquid I constant volume, filtration, takes subsequent filtrate as test solution I；Extraction solvent is added into lophatherum gracile standard decoction sample, ultrasound
Processing, obtains extracting solution II, and by the extracting solution II constant volume, filtration takes subsequent filtrate as test solution II；
Measurement: respectively by the reference substance reference solution, control medicinal material referring to reference solution, test solution I and for examination
Product solution II injects Ultra Performance Liquid Chromatography instrument, and measurement demarcates water-soluble shared peak, obtains lophatherum gracile medicinal material UPLC characteristic pattern
Spectrum.
2. construction method according to claim 1, which is characterized in that the chromatographic condition packet of the ultra performance liquid chromatography
It includes:
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution that volume fraction is 0.05%-0.2%, and gradient is washed
It is de-；
The gradient elution specifically:
0-2min, keeping the volume fraction of mobile phase A is 10%, keeps the volume fraction 90% of Mobile phase B；
The volume fraction of 2min-3min, mobile phase A are increased to 16% by 10%, and the volume fraction of Mobile phase B is reduced to by 90%
84%；
3min-7.5min, keeping the volume fraction of mobile phase A is 16%, and keeping the volume fraction of Mobile phase B is 84%；
The volume fraction of 7.5min-16min, mobile phase A are increased to 52% by 16%, and the volume fraction of Mobile phase B is by 84% drop
As low as 48%.
3. construction method according to claim 2, which is characterized in that the chromatographic condition of the ultra performance liquid chromatography also wraps
Include: 30 DEG C -40 DEG C of column temperature, flow velocity is 0.1mL-0.5mL per minute, Detection wavelength 340nm-360nm.
4. construction method according to claim 1, which is characterized in that the Extraction solvent is that volume fraction is 50%-
70% ethanol water.
5. construction method according to claim 1-4, which is characterized in that the time of the ultrasonic treatment is
20min-40min。
6. a kind of detection method of lophatherum gracile medicinal material, which comprises the following steps:
The preparation of reference solution: respectively using Lutonaretin and swertiajaponin as reference substance, solubilizer dissolution prepares reference substance
Reference solution；Extraction solvent is added into lophatherum gracile control medicinal material, is ultrasonically treated, obtains extracting solution, by the extracting solution constant volume,
Filtration, takes subsequent filtrate as control medicinal material reference solution；
The preparation of testing sample solution: Extraction solvent being added into sample to be tested, and ultrasonic treatment obtains extracting solution III, mentions described
Liquid III constant volume is taken, filters, takes subsequent filtrate as testing sample solution；
Measurement: the reference substance reference solution, control medicinal material are injected into ultra high efficiency referring to reference solution, testing sample solution
Liquid chromatograph, measurement.
7. detection method according to claim 6, which is characterized in that the chromatographic condition of the ultra performance liquid chromatography detection
Include:
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution that volume fraction is 0.05%-0.2%, and gradient is washed
It is de-；
The gradient elution specifically:
0-2min, keeping the volume fraction of mobile phase A is 10%, keeps the volume fraction 90% of Mobile phase B；
The volume fraction of 2min-3min, mobile phase A are increased to 16% by 10%, and the volume fraction of Mobile phase B is reduced to by 90%
84%；
3min-7.5min, keeping the volume fraction of mobile phase A is 16%, and keeping the volume fraction of Mobile phase B is 84%；
The volume fraction of 7.5min-16min, mobile phase A are increased to 52% by 16%, and the volume fraction of Mobile phase B is by 84% drop
As low as 48%.
8. detection method according to claim 7, which is characterized in that the chromatographic condition of the ultra performance liquid chromatography also wraps
Include: 30 DEG C -40 DEG C of column temperature, flow velocity is 0.1mL-0.5mL per minute, Detection wavelength 340nm-360nm.
9. detection method according to claim 6, which is characterized in that the Extraction solvent is that volume fraction is 50%-
70% ethanol water.
10. according to the described in any item detection methods of claim 6-9, which is characterized in that the time of the ultrasonic treatment is
20min-40min。
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