CN102175629A - Biological activity detection-based evaluation method of quality of prepared radix rehmanniae - Google Patents
Biological activity detection-based evaluation method of quality of prepared radix rehmanniae Download PDFInfo
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Abstract
The invention relates to a biological activity detection-based evaluation method of the quality of prepared radix rehmanniae, effectively solving the problems of evaluating the quality of the prepared radix rehmanniae from multiple angles and then ensuring the medicinal quality of the prepared radix rehmanniae. The evaluation method comprises the following process steps of: taking medicinal materials; pretreating the medicinal materials; preparing a tested substance; carrying out a free radical elimination reaction experiment; measuring free radical elimination rate; comparing the tested substance with a reference substance; calculating valence; carrying out methodology investigation; measuring the valence value of batched samples; and evaluating the quality. By means of the method, a medical effect-based biological test method of the quality of traditional Chinese medicines by taking the prepared radix rehmanniae as an object is established and is used as the supplement and the enhancement of the conventional and chemical measurement of a pharmacopeia, the inner quality can be checked on together from multiple angles, the traditional quality control and quality evaluation method and standard are perfected, and a new technical condition is provided for the quality control and the quality evaluation research of other traditional Chinese medicines with undefined medical effect substances.
Description
One, technical field
The present invention relates to medicine, particularly a kind of prepared rhizome of rehmannia method for evaluating quality based on biological activity assay.
Two, background technology
The pattern of part index number composition qualitative detection and/or assay is taked in evaluation of existing Chinese medicine interior quality and quality control, because present most herbal medicine efficacy materials are indeterminate, this pattern both had been difficult to guarantee the consistance and the stability of such traditional Chinese medicine quality, also was difficult to association or reflected the security and the validity of its clinical use." Chinese pharmacopoeia version establishment in 2010 outline spells out, set up the quality standard system that meets the traditional Chinese medicine characteristics, progressively by of the comprehensive detection transition of single index composition qualitative, quantitative, to multicomponent and and fingerprint or the conversion of characteristic spectrum total quality control model to activity, effective constituent and biologicall test." the Chinese traditional medicine biology determination of activity governing principle " of Zhi Dinging is by " Chinese pharmacopoeia (an one) version (in January, 2010 publication date) in 2010 appendix records therefrom.
The Chinese medicine cultivated land is one of clinical the most frequently used help class Chinese medicine, current quality standard mainly is to measure the content of single index acteoside, and the chemical background of glutinous rehmannia is comparatively complicated, martynoside, Dihuang polysaccharide, acteoside or the like all are its effective constituent, and the correlativity of its quality control index and drug effect awaits further research.
The biological activity determination method is introduced traditional Chinese medicine quality control and quality evaluation system, and, complex structure various for composition, physico-chemical method can not characterize its content, physical and chemical determination can not reflect that the Chinese medicine of biologically active and curative effect can highlight its superiority.Therefore, with the glutinous rehmannia is research object, foundation is based on the traditional Chinese medicine quality bioassay method of drug effect, as replenishing and raising of pharmacopeia routine and chemical assay, can be from multiple angle its inherent quality of checking on jointly, improving existing quality control and method for evaluating quality and standard, and can provide new thinking and reference with quality evaluation research for the indefinite traditional Chinese medicine quality control of other effective substance, is the technical matters that need conscientiously solve.
Three, summary of the invention
At above-mentioned situation, for overcoming the defective of prior art, the present invention's purpose just provides a kind of prepared rhizome of rehmannia method for evaluating quality based on biological activity assay, can effectively solve from multiple angle and estimate the quality of prepared rhizome of rehmannia and then the problem of assurance prepared rhizome of rehmannia pharmaceutical quality.
The technical scheme that the present invention solves is, processing step (flow process) is: medicinal material → pre-treatment → preparation test sample → removing free radical reaction experiment → mensuration free radical scavenging activity → test sample compares → calculates with the work reference substance and tires → methodological study → applied research (measuring multiple batches of sample valence value) → carry out quality evaluation, being about to prepared rhizome of rehmannia pulverizes dry, get powder in container, use ethanol ultrasonic extraction, ethanol is flung in water-bath, dry, again dry thing is dissolved in and makes the gradient ethanolic solution in the ethanol, become test sample, standby; Choose many batches of prepared rhizome of rehmannia uniform samplings again, pulverize respectively, equivalent is chosen powder, is dissolved in making the work reference substance in the ethanol, and is standby; Then with hexichol for bitter taste phthalidyl free radical (DPPH
*) be dissolved in the absolute ethyl alcohol, keep in Dark Place; Carry out application of sample and detection after above-mentioned three kinds of formulations prepared from solutions, measure each solution absorbency respectively and calculate test sample to hexichol for bitter taste phthalidyl free radical (DPPH
*) clearance rate, carry out the contrast calibrating of sample and reference substance then, calculate tiring of test sample, to realize quality evaluation to prepared rhizome of rehmannia.
The present invention is object with the prepared rhizome of rehmannia, foundation is based on the traditional Chinese medicine quality bioassay method of drug effect, as replenishing and raising of pharmacopeia routine and chemical assay, can be from multiple angle its inherent quality of checking on jointly, improve existing quality control and method for evaluating quality and standard, and can provide new technical conditions with quality evaluation research for the indefinite traditional Chinese medicine quality control of other effective substance.
Four, description of drawings
Fig. 1 removes DPPH for prepared rhizome of rehmannia among the present invention
*Live vol effect relationship figure.
Fig. 2 removes DPPH for prepared rhizome of rehmannia among the present invention
*Live vol effect relationship linear analysis figure.
Fig. 3 is the trend comparison diagram as a result of sample determination among the present invention.
Five, embodiment
Below in conjunction with concrete condition, the specific embodiment of the present invention is elaborated.
At first it is to be noted, anti-oxidant, removing free radical effect that prepared rhizome of rehmannia has, be one of clinical the most frequently used traditional help class Chinese medicine, that modern pharmacological research shows is anti-oxidant, remove the body peroxy radical is that it builds up health, regulates one of immune main mechanism.The different extracts that studies show that prepared rhizome of rehmannia have the effect that inside and outside significantly is anti-oxidant and remove peroxy radical.
Hexichol is for bitter taste phthalidyl free radical (DPPH
*) chemical constitution is: 1,1-diphenyl-2-trinitrophenyl-hydrazine (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl) molecular formula: C
18H
12N
5O
6Be a kind of free radical, have single electron, have the last one to absorb at the 517nm place, its alcoholic solution is purple.When composition exists, can its absorption be faded away as anti-oxidant (removing free radical) with its single electron pairing, its fading extent have shown Green Tea Extract composition and DPPH
*The quantity of single electron combination or ability, thereby available spectrophotometer carries out the variation of assaying reaction system absorbance, and the oxidation resistance of quantitatively characterizing antioxidant content or material, as measuring the oxidation resistance of Biosample, chemical substance and food etc.
Use following instrument and reagent in the inventive method: analytical balance, Shanghai precision electronic device company limited, FA200KA type; Hydro-extractor, Beijing Medical Centrifugal Machine Factory, LD4-2 type; The UV-2201 spectrophotometer, day island proper Tianjin company; SY-360 Extraction by Ultrasound instrument, last Haining merchant's ultrasonic instrument company limited; The XDW-6B Lowtemperaturepulverizer; 101-1A electric heating air blast drying case.
Radix rehmanniae recen (place of production is gathered and is combined with the medicinal material market purchasing, totally 20 batches, is 1 year 11---and gather in the crops Dec, through the old piece root that is accredited as scrophulariaceae rehmannia glutinosa plant Rehmannia glutinosa Libosch. of awarding with Puritanism of Henan College Of Traditional Chinese Medicine).By " Chinese pharmacopoeia (in January, 2010 version. one one) institute record " cultivated land " down prescriptive procedure make prepared rhizome of rehmannia.
DPPH
*(U.S. Sigma company); Absolute ethyl alcohol (analyze pure, chemical industry company limited of Zhengzhou Lianbang); It is pure that all the other reagent are analysis.
The present invention is realized by following steps in the specific implementation:
The preparation of A, test sample (claiming testing sample again, as follows):
1) prepared rhizome of rehmannia is put in the Lowtemperaturepulverizer, under-40 ℃ of states, made powder, cross 40 mesh sieves;
2) made sample powder is dried to water cut≤3% in 60 ℃ of heated-air circulation ovens, puts airtight preservation in the brown bottle;
3) take by weighing sample powder 2g adds powder weight in conical flask 10 times of ethanol, normal temperature ultrasonic Extraction 30 minutes, the gained extract filters with filter paper, discards residue, puts in the surface plate, and ethanol is flung in water-bath, and dry thing is preserved in sealing behind the calculate recovery rate;
4) dry thing is dissolved in the ethanol, making concentration respectively is Xg (crude drug) mL
-1Solution (X equals 1.0000,0.5000,0.2500,0.1250,0.0625 respectively ...);
The preparation of B, work reference substance;
The powder that the uniform sampling of 20 batches of prepared rhizome of rehmannia medicinal materials fully mixes is the work object of reference, during mensuration, also by step 3)～4 of A) be prepared into solution, as the work reference substance;
C, DPPH
*The preparation of solution
Precision takes by weighing 40mg DPPH
*, be dissolved in the 480mL absolute ethyl alcohol, add absolute ethyl alcohol again and be settled to 500mL.Gained DPPH
*The concentration of solution is 20mM, and refrigerator keeps in Dark Place;
D, application of sample and detection
Press table 1 application of sample in tool plug test tube, abundant mixing, room temperature leaves standstill 30min, measures absorbance (doing blank with absolute ethyl alcohol) in 517nm wavelength place;
Table 1 adds quadrat method and application of sample amount
Table?1?The?amount?of?samples
Provide from last table, the said need testing solution that do not add is meant 2mL DPPH
*The solution of solution+2mL absolute ethyl alcohol, so Ac is 2mL DPPH
*The solution of solution+2mL absolute ethyl alcohol is in the absorbance of measuring the wavelength place; The said test solution that adds is meant 2mL DPPH
*+ 2mL need testing solution, so Ai is 2mLDPPH
*+ 2mL need testing solution is in the absorbance of measuring the wavelength place; Said blank solution is meant 2mL absolute ethyl alcohol+2mL need testing solution, so Aj is that 2mL absolute ethyl alcohol+2mL need testing solution is in the absorbance of measuring the wavelength place.
The adjustment of E, application of sample amount
Be the convenience of tiring and calculating, need the estimation of tiring according to preliminary experiment and test sample, application of sample amount to test sample is carried out necessary adjustment, the assurance response intensity centers on clearance rate (P) (± 50%) about in the of 50% (see data processing for details and tire calculating), and guarantees the amount effect relation curve parallel (seeing interpretation of result for details) that test sample and work reference substance react.
F, data processing and the calculating of tiring
1) calculates mensuration liquid to DPPH according to following formula
*Clearance rate (P):
P＝[1-(Ai-Aj)/Ac]×100％.
Said Ai, Aj, Ac are respectively the absorbance that above-mentioned D step provides.
2) calculating of tiring
Calculate for convenient, set (the P that tires of work reference substance
S) be 1.0Umg
-1(crude drug), according to the parallel line assay principle adopt quantitative response parallel line assay method (22) method (according to " and Chinese pharmacopoeia in January, 2010 version. two appendix XI V biological standardization statistic laws) carry out the contrast calibrating that test sample and work reference substance are tired.
The present invention has especially also carried out following checking in order to guarantee accuracy and practicality:
Basic demand according to the medicine biological standardization is right: the 1. precision of detection method 2. repeatability is 3. stable, verifies, to determine the science and the rationality of institute's method for building up.
As the basis of prepared rhizome of rehmannia method for evaluating quality (anti-oxidant titration method) research, at first carried out prepared rhizome of rehmannia and removed DPPH
*Linearity such as Fig. 1 of active detection and dose-effect relationship are shown in Figure 2.
As can be seen from Figure 1 be good symmetrical serpentine curve between reaction rate and the log10 dose, meet conventional pharmacological reaction rule.
From Fig. 2 to 0.125gmL
-1～0.500gmL
-1Sample is removed DPPH in the dosage range
*Dose-effect relationship carry out linear analysis, can clearly find out at 0.125gmL
-1～0.500gmL
-1Prepared rhizome of rehmannia is removed DPPH in the dosage range
*Dose-effect relationship present favorable linearity (r 〉=99%).Testing sample is good parallel relation (meeting the basic demand of the described biological standardization statistic law of 2.4.2) with the work reference substance at range of linearity internal reaction curve.
According to the requirement of medicine biological standardization, (prepared rhizome of rehmannia is removed DPPH in above-mentioned research
*Active detection research) on the basis,, carry out the methodology checking by representative sample and 20 batch samples are repeatedly detected repeatedly, consequently,
1) precision
According to the inventive method, get test liquid (0.250gmL with a prepared rhizome of rehmannia sample
-1), carry out 5 absorbance measurements continuously and also calculate DPPH
*Clearance rate, be respectively: 70.1%, 71.2%, 68.6%, 67.7%, 65.4%, result's coefficient of variation RSD is 3.26%, illustrates that precision is good.
2) repeatability
Same batch sample is got 5 parts, extracts respectively, makes 5 parts of need testing solutions, compares calibrating with the work reference substance solution respectively by last method, and calculating tires is respectively: 0.87Umg
-1, 0.83Umg
-1, 0.94Umg
-1, 0.86Umg
-1, 0.79Umg
-1The RSD that tires that measures for 5 times is 6.46%, shows that this method has better repeatability.
3) stability
Respectively at 0,1,2,3 and 4h will be stored in the detection of tiring of 4 ℃ same need testing solution, the result is respectively: 0.86Umg
-1, 0.84Umg
-1, 0.92Umg
-1, 0.85Umg
-1, 0.80Umg
-1Its RSD is 5.08%, shows that need testing solution is preserved 4h under 4 ℃ of conditions be stable.
According to said method to 20 batches of cultivated land samples with work reference substance compare detection, according to above-mentioned data processing with tire computing method (" Chinese pharmacopoeia in January, 2010 version. two appendix XI V biological standardization statistic laws) calculating of tiring.Result such as following table:
Above-mentioned different batches sample demonstrates different valence values.If remove DPPH with cultivated land
*Active be the index calculating of tiring, and, can carry out quantitative evaluation different places of production different batches cultivated land sample in order to sign cultivated land quality.The applicability of illustration method is better, method is feasible.Can be effective to the quality evaluation of Chinese medicine cultivated land,, select and excellently wash in a pan badly, guarantee prepared rhizome of rehmannia quality and drug effect to determine the quality of prepared rhizome of rehmannia quality.The present invention compared with prior art has notable attribute, first with DPPH
*The determination method of removing ability is used for the antioxidation activity in vitro of cultivated land is measured, and proposes and work out the biological activity determination method of prepared rhizome of rehmannia quality evaluation first, has solved first in the mode of tiring and has characterized the problem that prepared rhizome of rehmannia is removed the free radical ability.Be that one of prepared rhizome of rehmannia method for evaluating quality research is created greatly.
Claims (1)
1. the prepared rhizome of rehmannia method for evaluating quality based on biological activity assay is characterized in that, is realized by following steps:
The preparation of A, test sample:
1) prepared rhizome of rehmannia is placed comminutor, under-40 ℃ of states, make powder, cross 40 mesh sieves;
2) made powder is dried to water cut≤3% in 60 ℃ of heated-air circulation ovens, puts airtight preservation in the brown bottle;
3) take by weighing powder 2g adds powder weight in conical flask 10 times of ethanol, normal temperature ultrasonic Extraction 30 minutes, the gained extract filters with filter paper, discards residue, puts in the surface plate, and ethanol is flung in water-bath, and dry thing is preserved in sealing;
4) dry thing is dissolved in the ethanol, making concentration respectively and be every mL, to contain crude drug be 1.0000g, 0.5000g, 0.2500g, 0.1250g, 0.0625g, becomes test sample;
The preparation of B, work reference substance:
With 20 batches of prepared rhizome of rehmannia uniform samplings, to pulverize respectively, the powder of choosing equivalent fully mixes, and as the work object of reference, A is prepared into solution set by step, as the work reference substance;
C, DPPH
*The preparation of solution:
Take by weighing 40mg DPPH
*, be dissolved in the 480mL absolute ethyl alcohol earlier, add absolute ethyl alcohol again to 500mL, put in the refrigerator and keep in Dark Place;
D, application of sample and detection:
With 2mL DPPH
*Solution+2mL absolute ethyl alcohol is with 2mL DPPH
*+ 2mL need testing solution with 2mL absolute ethyl alcohol+2mL need testing solution, places test tube respectively, abundant mixing, and room temperature leaves standstill 30min, measures absorbance in 517nm wavelength place, does blank with absolute ethyl alcohol;
The adjustment of E, application of sample amount:
Be the convenience of tiring and calculating, need the estimation of tiring, the application of sample amount of test sample is carried out necessary adjustment, guarantee that response intensity centers on clearance rate P ± 50%, and guarantee that test sample is parallel with the amount effect relation curve of work reference substance reaction according to preliminary experiment and test sample;
F, data processing and the calculating of tiring:
1) calculates mensuration liquid to DPPH according to following formula
*Clearance rate P:
P=[1-(Ai-Aj)/Ac] * 100%, wherein Ac is 2mL DPPH
*The solution of solution+2mL absolute ethyl alcohol is in the absorbance of measuring the wavelength place; Ai is 2mL DPPH
*+ 2mL need testing solution is in the absorbance of measuring the wavelength place; Aj is that 2mL absolute ethyl alcohol+2mL need testing solution is in the absorbance of measuring the wavelength place;
2) calculating of tiring
The P that tires of setting work reference substance
SCrude drug is 1.0Umg
-1, according to parallel line assay method (22) method of parallel line assay principle employing quantitative response, according to " Chinese pharmacopoeia in January, 2010 version. two appendix XI V biological standardization statistic laws, carry out test sample and examine and determine with the contrast that the work reference substance is tired.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102914506A (en) * | 2012-10-09 | 2013-02-06 | 福建省农业科学院食用菌研究所 | Novel method for evaluating lucid ganoderma extract quality |
| CN104833643A (en) * | 2015-05-27 | 2015-08-12 | 河南中医学院 | Bioactivity detection based hemp seed quality control and evaluation method |
| CN108090669A (en) * | 2017-12-15 | 2018-05-29 | 江西汇仁药业股份有限公司 | A kind of Quality Evaluation of Chinese Medicinal evaluation method |
| CN108918798A (en) * | 2018-07-06 | 2018-11-30 | 多多药业有限公司 | A kind of Shuanhuanglian injection quality control evaluation method based on bioactivity detection |
| CN110702809A (en) * | 2019-10-12 | 2020-01-17 | 辽宁中医药大学 | Compound chicken granule quality control and evaluation method based on anti-hepatic fibrosis bioactivity |
Citations (1)
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| CN101274036A (en) * | 2008-05-12 | 2008-10-01 | 美晨集团股份有限公司 | Method for testing quality criteria of conghuang preparation for tonifying kidney |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101274036A (en) * | 2008-05-12 | 2008-10-01 | 美晨集团股份有限公司 | Method for testing quality criteria of conghuang preparation for tonifying kidney |
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| 《上海中医药大学学报》 20071130 郭威等 《熟地黄中活性成分甘露三糖提取工艺研究》 全文 1 第21卷, 第6期 * |
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| CN102914506A (en) * | 2012-10-09 | 2013-02-06 | 福建省农业科学院食用菌研究所 | Novel method for evaluating lucid ganoderma extract quality |
| CN104833643A (en) * | 2015-05-27 | 2015-08-12 | 河南中医学院 | Bioactivity detection based hemp seed quality control and evaluation method |
| CN104833643B (en) * | 2015-05-27 | 2017-08-11 | 河南中医学院 | A kind of fructus cannabis quality control evaluation method detected based on bioactivity |
| CN108090669A (en) * | 2017-12-15 | 2018-05-29 | 江西汇仁药业股份有限公司 | A kind of Quality Evaluation of Chinese Medicinal evaluation method |
| CN108090669B (en) * | 2017-12-15 | 2021-12-28 | 江西汇仁药业股份有限公司 | Traditional Chinese medicine quality evaluation method |
| CN108918798A (en) * | 2018-07-06 | 2018-11-30 | 多多药业有限公司 | A kind of Shuanhuanglian injection quality control evaluation method based on bioactivity detection |
| CN108918798B (en) * | 2018-07-06 | 2021-03-30 | 多多药业有限公司 | Method for quality control evaluation of Shuanghuanglian injection based on biological activity detection |
| CN110702809A (en) * | 2019-10-12 | 2020-01-17 | 辽宁中医药大学 | Compound chicken granule quality control and evaluation method based on anti-hepatic fibrosis bioactivity |
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