CN107621455A - The assay method of total cardiac glycoside content in strong medicinal material rattan kuh-seng - Google Patents
The assay method of total cardiac glycoside content in strong medicinal material rattan kuh-seng Download PDFInfo
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- CN107621455A CN107621455A CN201710644530.3A CN201710644530A CN107621455A CN 107621455 A CN107621455 A CN 107621455A CN 201710644530 A CN201710644530 A CN 201710644530A CN 107621455 A CN107621455 A CN 107621455A
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- kuh
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- cardiac glycoside
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Abstract
The invention discloses a kind of method that colorimetric method for determining strengthens total cardiac glycoside content in medicinal material rattan kuh-seng, after this method sample adds chromogenic reagent, the assay of total cardiac glycoside in rattan kuh-seng is carried out using ultraviolet spectrophotometry, experiment condition is for reference substance with digoxin (Digoxin), alkaline picric acid solution is developer, detected under 415nm wavelength, reference substance content and absorbance are in good linear relationship in the range of 0~0.08mg/ml;Regression equation is y=14.968x+0.008 (r=0.9969);Precision RSD=0.1%;Mean sample recovery rate is 95.51%, RSD 2.16%（n=6）；Total cardiac glycoside content is 2.07 mg/g in rattan kuh-seng.This method is simple, quick, accurate, favorable reproducibility, available for the assay of total cardiac glycoside in rattan kuh-seng, to evaluate and controlling rattan kuh-seng to provide quality foundation.
Description
Technical field
The present invention relates to a kind of active component detection method in strong medicinal material rattan kuh-seng, specifically a kind of measure rattan kuh-seng
The method of middle active component total cardiac glycoside content, belongs to pharmaceutical technology field.
Background technology
Strong medicine rattan kuh-seng (RADIX STREPTOCAULONIS) is that Asclepiadaceae plant horse connects saddle Streptocaulon
Juventas (Lour.) Merr. dry root, strong language claim " hook baa ", and the medicinal material is distributed widely in the province such as China Guangxi, Yunnan
And the ground such as India, Vietnam, Burma, Laos, Cambodia and Malaysia；Alias griffith streptocaulon root or leaf, crow nozzle, Mao Qingcai, southern kuh-seng, milk
Rattan, motor, red horse connect saddle, the cloudy rattan of tiger, crow mouth.Its wooden be used to treat cat fever, enteritis, dysentery, stomachache, bruise
The diseases such as swelling and pain, venomous snake bite.Rattan kuh-seng category Guangxi characteristic, large, strong precious jade medicinal material, be national ministry standard national medicine product and
The main prescription of well-known strong patent medicine diseases caused by external factors wind acute diseases such as cholera and sunstroke particle (piece), and cure mainly one of medicine in dai medicine classical prescription Yajiao Hadun San.Document
Report isolates 19 compounds from rattan radix sophorae ethanol extract, wherein it is triterpenoid sapogenin to have 9,8 cardiac glycosideses
Composition, also other compositions.This 19 compounds are：α-amyrin acetic acid esters, Thin Layer scanning, 11- ethyoxyls-
3- acetyl group -12- Usus alkene -3- alcohol, 11- ketone-α-amyrin acetic acid esters, α-amyrin, lupeol, 24- methylene
Ring wood POLO alkanol, the β of ring jackfruit -23- alkene -3,25- glycol, β-ancient sterol, periplogenin digoxigenin glucosides, rattan kuh-seng poison
Aglycon, ring wood POLO alkane -3,24,25- triols, nicotinic acid, digitoxigenin, 16-O- acetylgitoxins member, thick stick
Willow aglycon, 16-O- acetyl group hydroxyls periplogenin, periplogenin glucoside, rattan kuh-seng poison glycosides A.Shown according to literature research
Griffithin (16-O-acetglgitoxgenin) can induce apoptosis of tumor cells in vitro, have preferable antitumor activity.
Rattan kuh-seng record in《Guangxi Chinese medicine standard》(nineteen ninety version),《Guangxi Zhuang quality of medicinal material standard compendium (first
Volume)》Etc. in provincial standard, but only have simple microscopical characters in initial quality standard, without the detection of active component content
Method, it is difficult to the quality of accurate control medicinal material comprehensively.Total cardiac glycoside for rattan kuh-seng one of active component, establish it is a kind of it is quick,
Accurately, the method for detecting active component-total cardiac glycoside in rattan kuh-seng of favorable reproducibility, preferably to carry out quality to the medicinal material
Monitor significant.
The content of the invention
Present invention aims at provide a kind of side for determining one of active component total cardiac glycoside content in Chinese medicine rattan kuh-seng
Method, this method is simple, stably, favorable reproducibility, the quality control available for the medicinal material.
The object of the present invention is achieved like this：The assay method utilizes alkaline picric acid solution and total cardiac stimulant in rattan kuh-seng
After chromogenic reaction occurs for glycoside composition, the absorbance for the sample that developed the color using determined by ultraviolet spectrophotometry, so as to determine total cardiac stimulant
The content of glycosides, is comprised the following steps that：
(1) preparation of reference substance solution and the drafting of standard curve：Precision weighs digoxin reference substance 10mg, is placed in 25ml
Scale is settled to after being dissolved in volumetric flask with 60% ethanol, and standard liquid is made.Titer 0 is pipetted with pipette, 0.1,0.2,
0.4,0.6,0.8,1.0ml, it is respectively placed in tool plug test tube, adds 60% ethanol and be diluted to 1ml, be separately added into alkaline bitter taste
Acid solution (A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, room temperature is put
20min is put, absorbance is surveyed in wavelength 415nm.With absorbance A ordinate, concentration C is abscissa, makees standard curve.As a result
Regression equation y=14.968x+0.008, r=0.9969.
(2) preparation of need testing solution：Precision weighs rattan kuh-seng coarse powder 2g, is placed in 100ml conical flasks, adds 70% ethanol
40ml, ultrasonic extraction 2 times, 40 minutes every time, weight is mended after extraction, filters merging filtrate, volatilizes solvent, it is molten to add 60% ethanol
Solution, is settled to 50ml, 5ml sample solutions is being taken from 50ml volumetric flasks, is being put into 25ml volumetric flasks, is diluted to scale, produce
Need testing solution.
(3) sample detection：Sample solution 1.0ml is pipetted with pipette precision, is placed in tool plug test tube, adds alkaline bitter taste
Acid solution (A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, at room temperature
20min is placed, absorbance is surveyed in wavelength 415nm, calculates the content of cardiac glycoside.
The present invention has the advantages that：
1st, the method for the invention is simple, accurate, reappearance and stability are good, can be thoroughly evaluating and the strong medicinal material rattan of control
The quality of kuh-seng is provided fundamental basis and scientific basis.
2nd, the inventive method uses colorimetric method, using the content of total cardiac glycoside in determined by ultraviolet spectrophotometry rattan kuh-seng,
With digoxin (Digoxin) for reference substance, alkaline picric acid solution is developer, is detected under 415nm wavelength, 0~
Reference substance content and absorbance are in good linear relationship in the range of 0.08mg/ml；Regression equation is y=14.968x+0.008
(r=0.9969)；Precision RSD=0.1%；Mean sample recovery rate is that 95.51%, RSD is 2.16% (n=6)；Rattan kuh-seng
Middle total cardiac glycoside content is 2.07mg/g.
3rd, cardiac glycosides composition is the main active of rattan kuh-seng, and total cardiac glycoside contains in measure rattan kuh-seng of the present invention
The method of amount, theoretical foundation is provided for the extensive clinical practice of rattan Sophora flavescens and industrialization.
Brief description of the drawings
Fig. 1 is reference substance and test sample UV spectrograms of the present invention
(description of reference numerals：1- need testing solution 2- reference substance solutions)
Fig. 2 is canonical plotting of the present invention
Embodiment
The present invention is made with further elucidated above, so as to those skilled in the art below in conjunction with the drawings and specific embodiments
Member knows more about the present invention, but does not limit the present invention with this.
Material and instrument
Material rattan kuh-seng is bought in Jing Chang prepared slices of Chinese crude drugs Co., Ltd of Nanning City, product batch number 20160401.Digoxin
Reference substance is bought in Nat'l Pharmaceutical ＆ Biological Products Control Institute, product batch number 100015-200709, and agents useful for same is analysis
It is pure.
(Shun's space permanent flat scientific instrument in Shanghai are limited for Instrumental Analysis balance (ME104E), 725 type ultraviolet-uisible spectrophotometers
Company).
Comprise the following steps that：
The preparation of reference substance solution：Precision weighs digoxin reference substance 10mg, is placed in 25ml volumetric flasks with 60% ethanol
Scale is settled to after dissolving standard liquid is made.
The preparation of need testing solution：Precision weighs rattan kuh-seng coarse powder 2g, is placed in 100ml conical flasks, adds 70% ethanol
40ml, ultrasonic extraction 2 times, 40 minutes every time, weight is mended after extraction, filters merging filtrate, volatilizes solvent, it is molten to add 60% ethanol
Solution, is settled to 50ml, 5ml sample solutions is being taken from 50ml volumetric flasks, are being put into 25ml volumetric flasks, scale is being diluted to and produces confession
Test sample solution.
Determine the selection of wavelength：Precision measures above-mentioned reference substance solution respectively, need testing solution 1ml is placed in tool plug test tube
In, add alkaline picric acid solution (A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing)
4ml.Shake up, place 20min at room temperature, to be not added with the alkaline picric acid solution under sample solution operates with method as blank, carry out
Spectral scan, as a result see Fig. 1.Show a length of 415nm of maximum absorption wave, therefore it is 415nm to select measure wavelength.
Methodology is examined or check
Linear relationship：Titer 0,0.1,0.2,0.4,0.6,0.8,1.0ml is pipetted with pipette, is respectively placed in tool plug examination
Guan Zhong, add 60% ethanol and be diluted to 1ml, be separately added into alkaline picric acid solution (A liquid：2% picric acid solution：B liquid：15%
NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, room temperature places 20min, and absorbance is surveyed in wavelength 415nm.With extinction
A ordinates are spent, concentration C is abscissa, makees standard curve.As a result regression equation y=14.968x+0.008, r=0.9969 are obtained.
Standard curve is shown in Fig. 2.
Precision Experiment precision measures reference substance solution 0.5ml, is diluted to 1ml, adds alkaline picric acid solution (A liquid：
2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, develop the color 20min, in wavelength
415nm, 6 A values of replication.1 is the results are shown in Table, the RSD of absorbance is 0.1%, illustrates that instrument precision is good.
The Precision test result of table one
Stability experiment precision measures sample solution 1ml, adds alkaline picric acid solution (A liquid：2% picric acid solution：B
Liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, develop the color 20min, in wavelength 494nm, every 10min
Determine an absorbance.The RSD of absorbance is 3.29%, and room temperature is placed in 20-40min after illustrating colour developing, and its absorbance is more steady
It is fixed, it the results are shown in Table two.
The stability test result of table two
Repeated experiment precision measures 6 parts of sample solution 1ml, adds alkaline picric acid solution (A liquid：2% picric acid is molten
Liquid：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, place 20min, determined under wavelength 415nm
Absorbance.As a result the RSD of absorbance is 2.4%, the results are shown in Table three, shows that the repeatability of the method is good.
Three repeated result of the test of table
Sample-adding recovery experiment precision measures 5 parts of the rattan kuh-seng sample of known content, is separately added into a certain amount of digoxin pair
According to product, alkaline picric acid solution (A liquid is added：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 is mixed
Close) 4ml.Shake up, place 20min, survey absorbance in wavelength 415nm, calculate the rate of recovery, average recovery rate 95.51%, RSD
For 2.16%, four are the results are shown in Table.
Table four is loaded recovery experiment result
The measure of content pipettes sample solution 1.0ml with pipette precision, is placed in tool plug test tube, adds alkaline picric acid
Solution (A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml.Shake up, room temperature decentralization
20min is put, absorbance is surveyed in wavelength 415nm, calculates the content of cardiac glycoside, the results are shown in Table five.
Five or six batches of sample total cardiac glycoside contents of table
| Sample | Absorbance (A) | Cardiac glycoside content (g/mg) |
| 1 | 0.106 | 2.046 |
| 2 | 0,109 | 2.109 |
| 3 | 0.107 | 2.067 |
| 4 | 0.108 | 2.084 |
| 5 | 0.105 | 2.027 |
| 6 | 0.107 | 2.065 |
| Average value | 2.067 |
To sum up, the method that the colorimetric method for determining that the present invention is established strengthens total cardiac glycoside content in medicinal material rattan kuh-seng, has behaviour
Make the characteristics of easy, precision is good, repeatability is high, can comprehensively, more effectively control the quality of the medicinal material.
Claims (4)
1. strong medicinal material rattan kuh-seng（RADIX STREPTOCAULONIS）The assay method of middle total cardiac glycoside content, it is characterised in that：
This method utilizes sample after chromogenic reagent, using the content of total cardiac glycoside in determined by ultraviolet spectrophotometry rattan kuh-seng.
2. the preparation of reference substance solution and the drafting of standard curve：Precision weighs digoxin reference substance 10mg, is placed in 25ml capacity
With being settled to scale after the dissolving of 60% ethanol standard liquid is made in bottle, titer 0 is pipetted with pipette, 0.1,0.2,0.4,
0.6,0.8,1.0ml, it is respectively placed in tool plug test tube, adds 60% ethanol and be diluted to 1ml, be separately added into alkaline picric acid solution
（A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml, shake up, room temperature places 20min,
Wavelength 415nm survey absorbance, with absorbance A ordinate, concentration C is abscissa, makees standard curve, as a result obtain the side of recurrence y=
14.968x+0.008 r=0.9969.
3. the preparation of need testing solution：Precision weighs rattan kuh-seng coarse powder 2g, is placed in 100ml conical flasks, adds 70% ethanol 40ml,
Ultrasonic extraction 2 times, 40 minutes every time, weight is mended after extraction, filters merging filtrate, volatilizes solvent, add the dissolving of 60% ethanol, constant volume
To 50ml, 5ml sample solutions are being taken from 50ml volumetric flasks, is being put into 25ml volumetric flasks, is diluted to scale, it is standby to survey.
4. sample detection：Sample solution 1.0ml is pipetted with pipette precision, is placed in tool plug test tube, it is molten to add alkaline picric acid
Liquid（A liquid：2% picric acid solution：B liquid：15%NaOH, A is pressed using preceding:B=90:10 mixing) 4ml, shake up, place at room temperature
20min, absorbance is surveyed in wavelength 415nm, calculates the content of cardiac glycoside.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111066657A (en) * | 2020-01-17 | 2020-04-28 | 广西中医药大学制药厂 | Tissue culture method for vine radix sophorae flavescentis |
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| CN106769958A (en) * | 2016-11-24 | 2017-05-31 | 成都中医药大学 | The detection method of total saponin content in balsampear leaf |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001055701A1 (en) * | 2000-01-31 | 2001-08-02 | Board Of Regents, The University Of Texas System | System and method for the analysis of bodily fluids |
| CN106769958A (en) * | 2016-11-24 | 2017-05-31 | 成都中医药大学 | The detection method of total saponin content in balsampear leaf |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111066657A (en) * | 2020-01-17 | 2020-04-28 | 广西中医药大学制药厂 | Tissue culture method for vine radix sophorae flavescentis |
| CN111066657B (en) * | 2020-01-17 | 2022-10-14 | 广西中医药大学制药厂 | Tissue culture method for sophora flavescens |
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Application publication date: 20180123 |