CN109521110A - A method of based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus - Google Patents
A method of based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus Download PDFInfo
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Abstract
The invention discloses a kind of methods for evaluating the rigid degree of bombyx batryticatus based on HPLC finger-print, method includes the following steps: the HPLC finger-print that (1) establishes different rigid degree bombyx batryticatuss is used as and compares map；The different rigid degree bombyx batryticatuss include at least the bombyx batryticatus to ossify 5 days or more；(2) the HPLC finger-print of bombyx batryticatus sample to be measured is established with condition in the same way；(3) the HPLC finger-print of bombyx batryticatus sample to be measured from the different degree bombyx batryticatuss that ossify is compared using similarity evaluation system, bombyx batryticatus sample to be measured possesses chromatographic peak mostly identical with the bombyx batryticatus to ossify 5 days or more, then the rigid degree of bombyx batryticatus sample to be measured is higher.Method of the invention considers Chinese medicine comprehensively and is generally this factor of multi-component complex system, the bombyx batryticatus finger-print of foundation more will comprehensively reflect the type and quantity of chemical component contained by bombyx batryticatus, and then the bombyx batryticatus degree that ossifys is evaluated on the whole, it is a kind of scientific, objective, effective, efficient method.
Description
Technical field
The invention belongs to traditional Chinese medicine quality control fields, and in particular to one kind is based on the rigid journey of HPLC finger-print evaluation bombyx batryticatus
The method of degree.
Background technique
Bombyx batryticatus is that the larva in Bombycidae insect silkworm (Bombyx mori L.) 4~5 ages infects (or artificial infection) white deadlock
Bacterium (Beauveria bassiana (Bals.) Vuillant) and lethal hirudo leech have Inflammation Zhijing, wind-expelling pain-stopping, change
The effect of phlegm dissipating bind, presss from both sides phlegm, convulsion, acute infantile convulsion, tetanus, middle air port for liver windHeadache due to pathogenic wind-heat, hot eyes pharynx
Bitterly, the treatment of the diseases such as rubella itch, hair chin or cheek mumps.
Bombyx batryticatus clinical application is extensive, has 49 kinds to contain the larva of a silkworm with batrytis in 513 kinds of people's patent medicine, contains white deadlock in 64 kinds of paediatrics patent medicine
Silkworm has 29 kinds.It is recorded altogether in " Chinese Pharmacopoeia ", " national standard for traditional Chinese medicines ", " Traditional Chinese medicine historical preparation " and " new drug become a full member standard "
175 Chinese medicine prescriptions containing bombyx batryticatus such as cure mainly " the seven precious balls " of arresting convulsion slit phlegm, disperse accumulation defaecation, and heat-clearing is relieving convulsion, relieve heat
" Infantile Niuhuang Powder " of phlegm, dispelling pathogenic wind and eliminating phlegm determine epilepsy and stop " Yixian pill " etc. jerked.To sum up, bombyx batryticatus is main as calming the liver to stop the wind medicinal material
Wanting effect is " dispelling wind and relieving convulsion, reducing phlegm and resolving masses ".
Chinese medicine standardization is the modernization of Chinese medicine and international prerequisite.The artificial standardization production of bombyx batryticatus is that bombyx batryticatus produces
The main trend and inevitable choice of industry development.However, bombyx batryticatus quality standard is lower in current " Chinese Pharmacopoeia ", it can not be in the market
True and false bombyx batryticatus effectively identified, also can not to bombyx batryticatus carry out section histological grading, cause manually standardize production bombyx batryticatus can not
The income for obtaining high quality and favourable price, affects the standardized production of bombyx batryticatus.
Summary of the invention
For currently being not possible to effectively to identify that bombyx batryticatus ossifys this status of degree, it is an object of the invention to propose a kind of base
In the fingerprint image that the method for the rigid degree of HPLC finger-print evaluation bombyx batryticatus, this method pass through the different rigid degree bombyx batryticatuss of foundation
Spectrum realizes the purpose for identifying the bombyx batryticatus true and false and evaluating bombyx batryticatus quality.
The purpose of the invention is achieved by the following technical solution:
A method of based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus, comprising the following steps:
(1) the HPLC finger-print of different rigid degree bombyx batryticatuss is established as control map；
The different rigid degree bombyx batryticatuss at least should include the bombyx batryticatus to ossify 5 days or more；It is described ossify 5 days or more be from
Silkworm is susceptible to be started to calculate at the point of death；Preferably, the bombyx batryticatus including ossifing 5-9 days；
In step (1), the preparation method of test solution is: bombyx batryticatus powder is weighed, with the ethanol solution of 80% (V/V), with
Extract 60min under the solid-liquid ratio ultrasonic wave of 1:20,60 DEG C of Extracting temperature；It filters, is evaporated after extraction, with methanol constant volume, 0.45 μm
Filtering with microporous membrane obtains test solution；
The step of efficient liquid phase chromatographic analysis, is: drawing 10 μ L of test solution, injects in high performance liquid chromatograph and carry out
Detection records the finger-print in 60min；
The chromatographic condition of efficient liquid phase chromatographic analysis is: 25 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength: and 360nm, into
10 μ L of sample amount, mobile phase A: 0.4% glacial acetic acid solution, Mobile phase B: 100% acetonitrile, using gradient elution mode: from 0-
The volumn concentration of 40min, mobile phase A are reduced to 75% by 95%, and the volumn concentration of Mobile phase B is improved extremely by 5%
25%；From 40-45min, the volumn concentration of mobile phase A is reduced to 65% by 75%, the volumn concentration of Mobile phase B by
25% improves to 35%；From 45-50min, the volumn concentration of mobile phase A is reduced to 50% by 65%, the volume of Mobile phase B
Percentage composition is improved by 35% to 50%；
(2) the HPLC finger-print of bombyx batryticatus sample to be measured is established with condition in the same way；
(3) the HPLC fingerprint image of bombyx batryticatus sample to be measured from the different degree bombyx batryticatuss that ossify is compared using similarity evaluation system
Spectrum, bombyx batryticatus sample to be measured possess higher similarity identical with bombyx batryticatus that is ossifing 5 days or more, then the rigid journey of bombyx batryticatus sample to be measured
It spends higher.
The similarity evaluation system is preferably " similarity evaluation ".
The forming process of bombyx batryticatus mainly includes two stages.The previous stage is the interaction of silkworm and muscardine, muscardine
The defense response of silkworm is induced after infected silkworm, muscardine also by nutrient competition, secretion mycotoxin and can destroy silkworm oxygen
Changing reduction balance causes silkworm dead.
Latter stage is the biotransformation of muscardine after silkworm is dead, that is, " rigid " stage being commonly called as, white
Stiff bacterium is bred using the continued growth of silkworm nutrition substance, and the endemic element of bombyx batryticatus is generated by bioconversion, to constitute deadlock
The effective substance of silkworm.
The present invention has found that the anticonvulsant efficacy of bombyx batryticatus water extract and rigid degree are obviously related by zoopery, ossifys
Degree is higher, and the medicinal quality of bombyx batryticatus is better.Therefore, the identification that rigid degree is carried out to bombyx batryticatus (powder) raw material, facilitates bombyx batryticatus
Quality control.
The present invention has the following advantages and effects with respect to the prior art:
1, the invention proposes a kind of method based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus, this method is examined comprehensively
Chinese medicine is considered and has been generally this factor of multi-component complex system, the bombyx batryticatus finger-print of foundation more will comprehensively reflect bombyx batryticatus
The type and quantity of contained chemical component, and then the bombyx batryticatus degree that ossifys is evaluated on the whole, be it is a kind of it is scientific, objective,
Effectively, efficient method.
2, method of the invention is not necessarily to determine that bombyx batryticatus ossifys degree, it can be achieved that intact bombyx batryticatus or deadlock from bombyx batryticatus appearance character
The quality evaluation of the various forms bombyx batryticatus raw material such as silkworm powder.
Detailed description of the invention
Fig. 1 is the influence column that bombyx batryticatus water extract and alcohol extract cause epilepsy death prevention rate rate to strychnine in embodiment 1
Figure；Wherein, S: model group, W1: water extract low dose group, W2: water extract middle dose group, W3: water extract high dose group, E1: alcohol
Extract low dose group, E2: alcohol extract middle dose group, E3: alcohol extract high dose group.
Fig. 2 is different rigid its cosmetic variation of the bombyx batryticatus of time.
Fig. 3 is that the bombyx batryticatus water extract of different rigid times in embodiment 2 causes the shadow of epilepsy death prevention rate rate to strychnine
Ring histogram；Wherein, S: model group, SW: normal silkworm, D1: ossify 1d bombyx batryticatus, D5: ossify 5d bombyx batryticatus, D9: ossify 9d bombyx batryticatus.
Fig. 4 is the HPLC chromatogram of normal silkworm with the time bombyx batryticatus that Bu Tong ossifys.
Fig. 5 is the characteristic fingerprint pattern of normal silkworm with the time bombyx batryticatus that Bu Tong ossifys；Wherein, the normal silkworm of SW-, D1- rigid 1
It, D5- ossifys 5 days, and D9- ossifys 9 days.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The anticonvulsant efficacy of bombyx batryticatus water extract and alcohol extract compares:
The effective substance that epilepsy mouse is caused to understand bombyx batryticatus to strychnine, by zoopery to bombyx batryticatus water extract and alcohol
The anticonvulsant action of extract is determined.
The preparation of bombyx batryticatus water extract: weighing bombyx batryticatus powder, and solid-liquid ratio 1:5-1:10 adds water refluxing extraction 2 times, and each 1h is mentioned
It is concentrated in vacuo after taking liquid to filter, freeze-dried acquisition bombyx batryticatus water extract.
The preparation of bombyx batryticatus alcohol extract: weighing bombyx batryticatus powder 150g, with ethanol solution refluxing extraction 2 times of 65%, solid-liquid ratio 1:
5-1:10, each 1h, extracting solution vacuum concentration, freeze-dried acquisition bombyx batryticatus alcohol extract.
Experimental method:
1, the method for breeding of experimental animal
Experimental animal is SPF grades of kunming mices of male, 18~22g.Rear adaptable fed 3d is bought, it is then that mouse is random
Grouping, every group 10.The pertinent regulations of management of laboratory animal and protection are abided by the raising of animal in experimentation.
2, modeling and medication
Water extract and alcohol extract are dissolved with sterile water.According to preset dose gastric infusion, model group is given isometric
Sterile water, continuous gavage 5d.After 30min is administered in 6d, the injection for curing of 1.6mg/kg taxi, continuous observation is injected intraperitoneally
30min, record clonic spasm incubation period (I-III grades of duration of seizure) of mouse, tetanic convulsion incubation period (V grades of breaking-outs) and
The number of elements of dead mouse in 30min.Pass through video monitoring mouse epilepsy outbreak process.
The observation of mice convulsion reaction is referring to Racine standard: 0 grade: reactionless；I grades: rhythmicity bicker, ear or facial flesh
Meat twitches clonic spasm；II grades: nodding and twitch clonic spasm with more serious facial muscles；III level: there is forelimb clonic spasm but be not accompanied by
Uprightly；IV grades: forelimb clonic spasm is with upright；V grades: generalized tonic-clonic breaks out and falls.
3, animal experimental data counts
Data carry out statistical analysis using 18.0 software of SPSS, and measurement data is with mean ± standard errorTable
Show, the comparison of multisample mean uses One-Way ANOVA variance analysis, and comparison among groups use LSD method.
Experiment sets the low, middle and high dose groups of model group, water extract and alcohol extract, and (dosage is respectively in terms of crude drug
10g/kg, 20g/kg and 30g/kg), water extract and alcohol extract are accordingly converted when dissolving according to its yield.Experimental result is shown in
Table 1 and Fig. 1:
1 bombyx batryticatus water extract of table and alcohol extract cause the influence of epilepsy mouse to strychnine
Compared with model group, p < 0.01 * p < 0.05, * *.
As can be seen from Table 1, bombyx batryticatus water extract high dose group being capable of extremely significant extension mouse clonic spasm incubation period (p < 0.01)
With tetanic incubation period (p < 0.01).Bombyx batryticatus water extract middle dose group also can significantly extend mouse tetanic incubation period (p < 0.05).
As seen from Figure 1, bombyx batryticatus water extract high dose group can also significantly improve the Death prevention rate of mouse.With model
Group is compared, and alcohol extract causes the improvement of epilepsy mouse not significant strychnine.
The above results show that bombyx batryticatus water extract causes the therapeutic effect of epilepsy mouse preferable strychnine, and apparent agent is presented
Graded effect is the anticonvulsant main pharmacodynamics material base of bombyx batryticatus.
Embodiment 2
Ossify influence of the degree to the anticonvulsion drug effect of bombyx batryticatus water extract:
Water extract preparation: bombyx batryticatus powder is weighed, solid-liquid ratio 1:5-1:10 adds water refluxing extraction 2 times, each 1h or more, extracting solution
It is concentrated in vacuo after filtering, freeze-dried acquisition bombyx batryticatus water extract, yield 25.21%.
Body surface shows typical bombyx batryticatus feature covered with white conidia powder after bombyx batryticatus ossifys 5 days.With bombyx batryticatus water extract into
The drug effect of the different rigid degree bombyx batryticatuss of row compares.Bombyx batryticatus progress hot water extraction to normal silkworm and ossify 1,5,9d, in terms of crude drug
The dosage of each sample is 25g/kg.As a result as shown in Table 2 and Fig. 3:
The bombyx batryticatus of the different rigid times of table 2 causes the influence of epilepsy mouse to strychnine
Compared with model group, p < 0.01 * p < 0.05, * *
The result shows that the drug effect of bombyx batryticatus is obvious related (table 2) to rigid degree.Compared with model group, the bombyx batryticatus for the 5d that ossifys
Also the clonic spasm incubation period (P < 0.05) of mouse can significantly be extended, the 9d bombyx batryticatus that ossifys can the extremely significant clonic spasm incubation period for extending mouse
(P < 0.01), the significant tetanic incubation period (P < 0.05) for extending mouse, moreover it is possible to significantly improve the Death prevention rate (figure of mouse
3).Each bombyx batryticatus sample drug effect is successively are as follows: D9 > D5 > D1 > SW shows that the rigid time is longer, and the degree that ossifys is higher, the medicine of bombyx batryticatus
It is better with quality.
Embodiment 3
A method of based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus, comprising the following steps:
(1) foundation of the rigid process HPLC finger-print of bombyx batryticatus
The HPLC finger-print of normal silkworm (the being uninfected by muscardine) time samples that ossify different with bombyx batryticatus is established respectively, such as
Shown in Fig. 4.
The preparation of test solution: accurately weighing 2.00g bombyx batryticatus powder, with 80% ethanol solution, with the solid-liquid ratio of 1:20
Extract 60min under ultrasonic wave, 60 DEG C of Extracting temperature.It is filtered after extraction, Rotary Evaporators evaporated under reduced pressure, with methanol constant volume to 25mL
Volumetric flask, 0.45 μm of filtering with microporous membrane are spare.
Efficient liquid phase chromatographic analysis:
Precision draws 10 μ L of test solution, injects in high performance liquid chromatograph and is detected, and records the fingerprint in 60min
Map.
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18 (4.6mm × 250nm, 2.6 μm), diode array inspection
It surveys device (DAD)；25 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength: 360nm, 10 μ L of sample volume, mobile phase A: 0.4% ice second
Acid solution, Mobile phase B: 100% acetonitrile, using gradient elution mode: from 0-40min, the volumn concentration of mobile phase A by
95% is reduced to 75%, and the volumn concentration of Mobile phase B is improved by 5% to 25%；From 40-45min, the volume of mobile phase A
Percentage composition is reduced to 65% by 75%, and the volumn concentration of Mobile phase B is improved by 25% to 35%；From 45-50min, stream
The volumn concentration of dynamic phase A is reduced to 50% by 65%, and the volumn concentration of Mobile phase B is improved by 35% to 50%.
Auto-matching, matching knot are carried out to chromatographic peak by " similarity evaluation 2012 editions "
Fruit is shown in Table 3.
The normal silkworm of table 3 and time bombyx batryticatus fingerprint image spectral peak retention time and the peak area of Bu Tong ossifing
From table 3 it can be seen that 4 groups of samples find 23 independent peaks altogether, wherein shared peak only has 6, as shown in Figure 5.
The above results show that compared with normal silkworm SW, the type and content of chemical component are had occurred in each bombyx batryticatus sample
Acute variation.In addition, the finger-print of D5 and D9 are more similar, have with D1 in the bombyx batryticatus (D1, D5 and D9) of different rigid degree
There were significant differences.
(2) similarity evaluation of HPLC finger-print
Above-mentioned similarity evaluation system is further used, the similarity of four group fingerprints is calculated, the results are shown in Table 4.
4 bombyx batryticatus medicinal materials fingerprint similarity of table
As can be seen from Table 4, the similarity of normal silkworm and 3 groups of bombyx batryticatuss is between 0.481~0.568, the bombyx batryticatus for the 1d that ossifys
And other 3 groups of similarities, between 0.467~0.568, the bombyx batryticatus similarity for the 5d and 9d that ossifys is 0.96, and similarity is higher.By
This as it can be seen that using finger-print can effectively discern the false from the genuine bombyx batryticatus and Bu Tong ossify degree bombyx batryticatus.
(3) make sample to be tested HPLC chromatogram and above-mentioned 4 kinds of controls map is compared, find and the bombyx batryticatus for the 1d that ossifys
Similarity is up to 0.95, illustrates that rigid degree is lower, inferior quality.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of method based on the rigid degree of HPLC finger-print evaluation bombyx batryticatus, it is characterised in that the following steps are included:
(1) the HPLC finger-print of different rigid degree bombyx batryticatuss is established as control map；
The different rigid degree bombyx batryticatuss include at least the bombyx batryticatus to ossify 5 days or more；
The chromatographic condition of efficient liquid phase chromatographic analysis is: 25 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength: 360nm, sample volume
10 μ L, mobile phase A: 0.4% glacial acetic acid solution, Mobile phase B: 100% acetonitrile, using gradient elution mode: from 0-40min, stream
The volumn concentration of dynamic phase A is reduced to 75% by 95%, and the volumn concentration of Mobile phase B is improved by 5% to 25%；From
The volumn concentration of 40-45min, mobile phase A are reduced to 65% by 75%, and the volumn concentration of Mobile phase B is mentioned by 25%
Up to 35%；From 45-50min, the volumn concentration of mobile phase A is reduced to 50% by 65%, and the volume basis of Mobile phase B contains
Amount is improved by 35% to 50%；
(2) the HPLC finger-print of bombyx batryticatus sample to be measured is established with condition in the same way；
(3) the HPLC finger-print that bombyx batryticatus sample to be measured from the different degree bombyx batryticatuss that ossify are compared using similarity evaluation system, to
It surveys bombyx batryticatus sample and possesses higher similarity identical with bombyx batryticatus that is ossifing 5 days or more, then the rigid degree of bombyx batryticatus sample to be measured is got over
It is high.
2. according to the method described in claim 1, it is characterized by: the different rigid degree bombyx batryticatuss include ossifing 5-9 days
Bombyx batryticatus.
3. according to the method described in claim 1, it is characterized by: the preparation method of test solution is: weighing bombyx batryticatus powder, use
80% ethanol solution, to extract 60min under the solid-liquid ratio ultrasonic wave of 1:20,60 DEG C of Extracting temperature；It filters, is evaporated after extraction,
With methanol constant volume, 0.45 μm of filtering with microporous membrane obtains test solution.
4. according to the method described in claim 1, it is characterized by: the step of efficient liquid phase chromatographic analysis be: draw test sample
10 μ L of solution is injected in high performance liquid chromatograph and is detected, and records the finger-print in 60min.
5. according to the method described in claim 1, it is characterized by: the similarity evaluation system is " Chinese medicine chromatographic fingerprint
Map similarity evaluation system ".
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023082512A1 (en) * | 2021-11-10 | 2023-05-19 | 广东一方制药有限公司 | Stiff silkworm marker polypeptide and method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products |
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| CN101953913A (en) * | 2010-09-13 | 2011-01-26 | 广州市香雪制药股份有限公司 | Quality analysis method of Zhongfenghuichun capsules |
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| WO2023082512A1 (en) * | 2021-11-10 | 2023-05-19 | 广东一方制药有限公司 | Stiff silkworm marker polypeptide and method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products |
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Application publication date: 20190326 |