CN110261514A - The content assaying method of the dried venom of toads in pills for curing heart disease - Google Patents
The content assaying method of the dried venom of toads in pills for curing heart disease Download PDFInfo
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- 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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of content assaying method of the dried venom of toads in Huatuo's pills for curing heart disease, including determine chromatographic condition, the preparation of reference substance solution, the preparation of test solution and measuring method and etc..This method has simple and efficient, it is time-consuming short, consumptive material is low, and there is precision and favorable reproducibility, the advantages of good stability, it can effectively detect the content of the dried venom of toads in Huatuo's pills for curing heart disease, it can be used for the quantitative assessment of Huatuo's pills for curing heart disease quality, and it can detect simultaneously eight kinds of ingredients in the dried venom of toads under same chromatographic condition, this can not only effectively identify the presence or absence of microcomponent dried venom of toads in Huatuo's pills for curing heart disease, also the content of the dried venom of toads accurately can be detected in time, so that the quality control to Huatuo's pills for curing heart disease is more comprehensive, effectively, important leverage is provided for the quality control of Huatuo's pills for curing heart disease.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to the content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease.
Background technique
The dried venom of toads is the secretion of Bufonidae animal bufo gargarizans Cantor or black surround toad ear rear gland and epidermal gland, white " milky "
Liquid or pale yellow syrup, it is toxic.The dried venom of toads extracts slurries, dry, and colouring can be used as medicine after being sealed, be that China is common
Traditional rare Chinese medicine simply.Its is warm-natured, acrid flavour, toxic, thoughts of returning home warp, has the effect of removing toxic substances, analgesic, inducing resuscitation of having one's ideas straightened out, uses
It vomits and diarrhoea in treatment ulcer boils, abscess of throat, heatstroke coma, erputive abdominal disease.And modern pharmacology research shows, the dried venom of toads has
Enhance myocardial contractive power, boosting, inhibit platelet aggregation, increase coronary flow, it is anti-inflammatory, anti-radiation, antitumor, decompression and
The pharmacological activity such as anesthesia.The dried venom of toads is also II grade of natural crude drugs of special-protection-by-the-State, and 28 kinds of toxic traditional Chinese medicine material kinds it
One, at home and abroad it is widely used.The preparation being used as medicine with the dried venom of toads shares 88 in version in 2015 in " ministry standard " and " Chinese Pharmacopoeia "
A prescribed preparation (329 lot numbers), the quality of quality will have a direct impact on the curative effect of preparation.And dried venom of toads toxicity is also larger, uses
Medicine is improper to will appear serious toxic side effect, and 2015 editions Chinese Pharmacopoeia regulation dried venoms of toads do not enter decoction, enters ball and dissipates human body dosage 0 for oral administration
.015~0 .03g is calculated by adult's 60kg weight, and the taking dose of people is the .0005g/kg of 0 .00025~0, to the dried venom of toads
Taking dose has strict requirements.
The prescription of Huatuo's pills for curing heart disease is the dried venom of toads, ginseng stem and leave general saponin, ox courage cream powder, muscone, pearl, artificial ox
Huang, borneol, eight kinds of compatibility of drugs compositions of Radix Notoginseng cream powder.Huatuo's pills for curing heart disease executes standard and records in " the Sanitation Ministry medicine standard Chinese medicine
Prescribed preparation " the 19th copy (standard code: WS3-B-3675-98).But currently, to the evaluation criterion of the dried venom of toads in Huatuo's pills for curing heart disease
It is that its quality is characterized with the content's index of every kind of individual event ingredient or certain several ingredient mostly, there are cumbersome, inspections method
Survey the disadvantages of time is long, and detection is not comprehensive, and testing cost is high.Therefore, it is necessary to study develop simple and efficient, the Quan Mianzhun of one kind
The content assaying method of the dried venom of toads in true and time-consuming short Huatuo's pills for curing heart disease, so that the quality progress to Huatuo's pills for curing heart disease is effectively comprehensive
Detect and control.
Summary of the invention
The purpose of the present invention is to provide a kind of content assaying method of the dried venom of toads in Huatuo's pills for curing heart disease, this method has simplicity
Fast, time-consuming short, consumptive material is low, and has the advantages that precision and favorable reproducibility, good stability, is advantageously implemented and rescues to Huatuo
The quality progress of heart ball effectively comprehensively detects and controls.
The present invention is achieved through the following technical solutions:
The content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease, comprising the following steps:
(1) chromatographic condition determined: using octadecylsilane chemically bonded silica as filler；Detection wavelength is 296nm；It is stream with water
Dynamic phase A, using acetonitrile as Mobile phase B, gradient elution sequence are as follows: 0min, 20%B；2min, 38%B；5min, 56%B；19min, 66%
B；19.1min 20%B；20min, 20%B；
(2) prepared by reference substance solution: precision weighs Cinobufagin, resibufogenin, his spirit of day toadpoison, Bufalin, husky toadpoison
His clever reference substance of essence, bufotalien, remote cinobufagin and magnificent toadpoison is solute, proper amount of methanol is added, mixing reference substance standard is made
Solution to get；
(3) prepared by test solution: Huatuo's pills for curing heart disease test sample is taken, it is finely ground, and 0.3g is taken, accurately weighed, methanol 10- is added in precision
20mL, weighed weight are heated to reflux or are ultrasonically treated 20-60min, lets cool, then weighed weight, and the weight of less loss is supplied with methanol
Amount, shake up, filter, take subsequent filtrate to get；
(4) measuring method: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, it is surveyed according to above-mentioned chromatographic condition
It is fixed, it is calculated by external standard method to obtain the final product.
Preferably, in step (1), the flow velocity of determining chromatographic condition is 0.6-1mL/min, preferably 0.6 mL/min,
25-30 DEG C of column temperature, preferably 30 DEG C.
Preferably, in step (2), the mass concentration for mixing each solute in reference substance standard solution is 0.1mg/ml.
Comprehensively consider extraction efficiency and extraction time, it is preferred that in step (3), Huatuo's pills for curing heart disease test sample is taken, it is finely ground,
0.3g is taken, accurately weighed, methanol 20ml is added in precision, and weighed weight is ultrasonically treated 20min, lets cool, then weighed weight, use first
Alcohol supplies the weight of less loss, shakes up, and filtration takes subsequent filtrate to get test solution.
Compared with prior art, the present invention having the following beneficial effects:
The content assaying method of the dried venom of toads in Huatuo's pills for curing heart disease provided by the invention, has simple and efficient, and time-consuming short, consumptive material is low, and
And have the advantages that precision and favorable reproducibility, good stability, it can effectively detect the content of the dried venom of toads in Huatuo's pills for curing heart disease,
It can be used for the quantitative assessment of Huatuo's pills for curing heart disease quality.
The content assaying method of the dried venom of toads can detect in the dried venom of toads simultaneously under same chromatographic condition in Huatuo's pills for curing heart disease of the present invention
Eight kinds of ingredients, this can not only effectively identify the presence or absence of microcomponent dried venom of toads in Huatuo's pills for curing heart disease, also can be in time to the content of the dried venom of toads
It is accurately detected, so that the quality control to Huatuo's pills for curing heart disease is more comprehensive, effective, is mentioned for the quality control of Huatuo's pills for curing heart disease
For important leverage.
Detailed description of the invention
Fig. 1 is the chromatogram of mixed reference substance solution of the present invention；
Fig. 2 is the chromatogram of test solution of the present invention；
Fig. 3 is the chromatogram of method one in the selection of mobile phase and elution requirement；
Fig. 4 is the chromatogram of method two in the selection of mobile phase and elution requirement；
Fig. 5 is the chromatogram of method three in the selection of mobile phase and elution requirement.
Specific embodiment
Further illustrate that the present invention, following embodiment are the specific embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments, it is other any without departing from Spirit Essence of the invention
With changes, modifications, substitutions, combinations, simplifications made under principle, equivalent substitute mode should be, be included in of the invention
Within protection scope.
One, instrument and reagent
Reference substance: Cinobufagin, resibufogenin, his spirit of day toadpoison, Bufalin, Arenobufagin, bufotalien, remote China's toadpoison
Essence and magnificent toadpoison he clever (HPLC >=98% is purchased from Chengdu Crow Ma Biotechnology Co., Ltd).
Test sample: Huatuo's pills for curing heart disease sample of lot number 201901,201902,201903,201904,201905, by China
He traditional Chinese medicines limited liability company provides.
Instrument: Waters2489 efficient liquid phase system, electronic balance (Beijing Sai Duolisi scientific instrument Co., Ltd), from
Scheming (ten thousand elegant demeanour device Manufacturing Co., Ltd of Changzhou).
Reagent: acetonitrile (chromatographically pure), ultrapure water (PALL).
Two: experimental method
The preparation of 2.1 reference substance solutions:
Precision weighs Cinobufagin, resibufogenin, his spirit of day toadpoison, Bufalin, Arenobufagin, bufotalien, remote China's toad
Malicious his spirit each 1mg of reference substance of smart and magnificent toadpoison is solute, the obtained mixing reference substance standard solution of proper amount of methanol is added, so that mixing
In reference substance standard solution the concentration of each solute be all 0.1mg/mL to get.
The preparation of 2.2 test solutions:
Above-mentioned 5 batches of test samples are taken respectively, it is finely ground, about 0.3g is respectively taken, accurately weighed, methanol 20mL is added in precision, and weighed weight surpasses
Sonication 20min, lets cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and filtration takes subsequent filtrate to get for examination
Product solution.
2.3 chromatographic conditions determined:
Chromatographic column: Thermo Scientific C18 (250mm × 4 .6mm, 5 μm)；
Mobile phase: water (A)-acetonitrile (B), flow velocity 0.6mL/min, 30 DEG C of column temperature, Detection wavelength 296nm, sample volume is 10 μ L, ladder
Spend eluotropic series are as follows: 0min, 20%B；2min, 38%B；5min, 56%B；19min, 66%B；19.1min 20%B；20min, 20%
B。
2.4 measuring methods:
It is accurate respectively to draw reference substance solution and each 10 μ L of test solution, it is measured according to above-mentioned chromatographic condition, obtains liquid phase
Chromatogram carries out peak positioning according to relative retention time, calculates the peak area of each chromatographic peak, then in conjunction with each regression equation, obtain
His spirit of day toadpoison out, Arenobufagin, remote cinobufagin, bufotalien, his spirit of magnificent toadpoison, Bufalin, Cinobufagin and ester toad
Content of the malicious aglucon in each batch Huatuo's pills for curing heart disease sample, as a result see the table below 1.
The content of 8 kinds of ingredients in 1 Huatuo's pills for curing heart disease dried venom of toads of table
Three, methodological study:
(1) linear relationship is investigated
Recurrence processing is carried out to integrating peak areas value with compound concentration, obtain day toadpoison he spirit, Arenobufagin, remote cinobufagin,
Bufotalien, he is clever for magnificent toadpoison, Bufalin, and Cinobufagin and resibufogenin regression equation are respectively as follows: y=12456x+
1275.3(R2=0.9991),y=8716.6x+15331(R2=0.9999),y=11021x+5727.1(R2=0.9994),y=
16480x+10447(R2=0.9993),y=11639x+5340.8(R2=0.9995),y=16538x+8757.9(R2=0.9996),
y=12967x+2357.1(R2=0.9996),y=13345x+2421.9(R2=0.9998).As it can be seen that this in Huatuo's pills for curing heart disease dried venom of toads
Eight compounds are linear good within the scope of 0 .005-0 .1mg/mL.
(2) precision investigates the accurate same 10 μ L of mixed reference substance solution of absorption, continuous sample introduction 6 times, records peak area,
As a result 2 be see the table below.The result shows that instrument precision is good.
(3) stability test takes same test solution respectively at 2h, 4h, 6h, 12h, and for 24 hours, 48h is analyzed, record
As a result peak area see the table below 2, the results showed that test solution is stable within 48 hours.
(4) repetitive test weighs totally 6 parts of Huatuo's pills for curing heart disease powder about 0.3g, accurately weighed, handles by test solution
Method prepares sample, as a result measurement see the table below 2.The result shows that this method repeatability is good.
The precision of 8 kinds of ingredients, stability and repeatability in 2 Huatuo's pills for curing heart disease dried venom of toads of table
| Precision (n=6) | Stability (n=6) | Repeated (n=6) | |
| RSD% | RSD% | RSD% | |
| His spirit of day toadpoison | 0.41 | 0.54 | 2.35 |
| Arenobufagin | 0.43 | 0.52 | 3.47 |
| Remote cinobufagin | 0.42 | 0.45 | 3.59 |
| Bufotalien | 0.43 | 0.58 | 2.84 |
| Magnificent toadpoison he spirit | 0.41 | 0.46 | 1.46 |
| Bufalin | 0.44 | 0.60 | 1.67 |
| Cinobufagin | 0.52 | 0.65 | 0.27 |
| Resibufogenin | 0.51 | 0.61 | 0.49 |
Four, the optimization of chromatographic condition
It realizes under same chromatographic condition while measuring the assay of 8 kinds of ingredients in Huatuo's pills for curing heart disease dried venom of toads, mobile phase and wash
The selection of de- condition is key technology.
The selection of mobile phase of the present invention and elution requirement is compared with the following method:
Method one: mobile phase uses 0.5% formic acid (A)-acetonitrile (B), and with 0min, 20%B；4min, 35%B；7min, 45%B；
10min, 55%B；17min, 63%B；21min, 65%B；21.1min 35%B；The gradient elution program of 22min, 20%B are tried
It tests, HPLC chromatogram is as shown in figure 3, wherein his separating degree of Ling Feng Peak and surrounding peak of day toadpoison is lower, and Cinobufagin
Peak and resibufogenin appearance time spacing are too small.
Method two: the shortcomings that improve the above method one, laborsaving so that chromatogram is easier to observe, consumptive material is reduced, mobile phase
Using water (A)-acetonitrile (B), and with 0min, 20%B；3min, 30%B；4min, 37%B；8min, 57%B；22min, 65%B；
23min, 35%B；23.1min 20%B；The elution program of 24min, 20%B are tested, and HPLC chromatogram is as shown in figure 4, discovery toad
There is imperfect phenomenon in malicious Ling Feng Peak.
Method three: to advanced optimize, by second method time-program(me) it is constant in the case where, change organic phase in 8min
Shi Wei
41% acetonitrile, remaining period organic phase constant rate, HPLC chromatogram is as shown in figure 5, discovery Bufalin chromatographic peak
Do not occur splitting a point phenomenon, chromatography peak type keeps complete, but part chromatographic peak is at a distance of excessively close.
Method four: further consider the problems of the gradient elution time, select mobile phase and elution requirement of the invention: with water
For stream
Dynamic phase A, using acetonitrile as Mobile phase B, gradient elution sequence is determined are as follows: 0min, 20%B；2min, 38%B；5min, 44%B；
19min, 66%B；19.1min 20%B；20min, 20%B, HPLC chromatogram is as shown in Fig. 2, gone out result either mixes pair
According to product or test solution, his spirit of day toadpoison, the chromatographic peak separating degree of eight kinds of ingredients such as Arenobufagin is all larger than 1.5, and
Peak type is complete.
In conclusion the present invention, by optimization chromatographic condition, the content assaying method of the dried venom of toads has in the Huatuo's pills for curing heart disease provided
Simple and efficient, time-consuming short, consumptive material is low, and has the advantages that precision and favorable reproducibility, good stability, can be in same chromatography
Under the conditions of simultaneously detect 8 kinds of ingredients in the dried venom of toads, this can not only effectively identify the presence or absence of microcomponent dried venom of toads in Huatuo's pills for curing heart disease,
The content of the dried venom of toads can accurately be detected in time, so that the quality control to Huatuo's pills for curing heart disease is more comprehensive, effective, it is Huatuo
The quality control of pills for curing heart disease provides important leverage.
Claims (4)
1. the content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease, which comprises the following steps:
(1) chromatographic condition determined: using octadecylsilane chemically bonded silica as filler；Detection wavelength is 296nm；It is stream with water
Dynamic phase A, using acetonitrile as Mobile phase B, gradient elution sequence are as follows: 0min, 20%B；2min, 38%B；5min, 56%B；19min, 66%
B；19.1min 20%B；20min, 20%B；
(2) prepared by reference substance solution: precision weighs Cinobufagin, resibufogenin, his spirit of day toadpoison, Bufalin, husky toadpoison
His clever reference substance of essence, bufotalien, remote cinobufagin and magnificent toadpoison is solute, proper amount of methanol is added, mixing reference substance standard is made
Solution to get；
(3) prepared by test solution: Huatuo's pills for curing heart disease test sample is taken, it is finely ground, and 0.3g is taken, accurately weighed, methanol 10- is added in precision
20mL, weighed weight are heated to reflux or are ultrasonically treated 20-60min, lets cool, then weighed weight, and the weight of less loss is supplied with methanol
Amount, shake up, filter, take subsequent filtrate to get；
(4) measuring method: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, it is surveyed according to above-mentioned chromatographic condition
It is fixed, it is calculated by external standard method to obtain the final product.
2. the content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease according to claim 1, which is characterized in that step
(1) in, the flow velocity of determining chromatographic condition is 0.6-1mL/min, 25-30 DEG C of column temperature.
3. the content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease according to claim 1, which is characterized in that step
(2) in, the mass concentration for mixing each solute in reference substance standard solution is 0.1mg/mL.
4. the content assaying method of the dried venom of toads in a kind of Huatuo's pills for curing heart disease according to claim 1, which is characterized in that step
(3) in, Huatuo's pills for curing heart disease test sample is taken, it is finely ground, 0.3g is taken, accurately weighed, methanol 20mL, weighed weight, ultrasound is added in precision
20min is handled, lets cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, filters, takes subsequent filtrate to get test sample
Solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986443A (en) * | 2021-03-16 | 2021-06-18 | 上海和黄药业有限公司 | Method for evaluating main active ingredient content of toad venom in cardiovascular medicine by one test |
| CN114894915A (en) * | 2022-03-31 | 2022-08-12 | 安徽中医药大学 | Method for establishing fingerprint spectrum of heart-saving pill and application |
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| CN106706812A (en) * | 2017-01-22 | 2017-05-24 | 雷允上药业集团有限公司 | Method for detecting bufadienolide components in Liushen pill |
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| CN102512453A (en) * | 2011-12-29 | 2012-06-27 | 北京协和制药二厂 | Traditional Chinese medicine venenum bufonis extract and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112986443A (en) * | 2021-03-16 | 2021-06-18 | 上海和黄药业有限公司 | Method for evaluating main active ingredient content of toad venom in cardiovascular medicine by one test |
| CN114894915A (en) * | 2022-03-31 | 2022-08-12 | 安徽中医药大学 | Method for establishing fingerprint spectrum of heart-saving pill and application |
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