CN102175629B - 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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- CN102175629B CN102175629B CN201010554359.5A CN201010554359A CN102175629B CN 102175629 B CN102175629 B CN 102175629B CN 201010554359 A CN201010554359 A CN 201010554359A CN 102175629 B CN102175629 B CN 102175629B
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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 detecting based on biologically active.
Two, background technology
The pattern of part index number composition qualitative detection and/or assay is taked in the evaluation of existing Chinese medicine interior quality and quality control, because current most Chinese drugs are indefinite, this pattern had both been difficult to guarantee consistance and the stability of such traditional Chinese medicine quality, was also difficult to association or reflected security and the validity of its clinical use." Chinese Pharmacopoeia " version establishment outline in 2010 explicitly points out, set up the quality standard system that meets traditional Chinese medicine feature, the progressively comprehensive detection transition to activity, effective constituent and biologicall test by single index composition qualitative, quantitative, to multicomponent and and fingerprint or characteristic spectrum global quality control mode-conversion." the Chinese traditional medicine biology determination of activity governing principle " formulated therefrom recorded by " Chinese Pharmacopoeia " (one) version (in January, 2010 publication date) appendix in 2010.
Chinese medicine cultivated land is one of clinical the most frequently used help class Chinese medicine, current quality standard is mainly to measure the content of single index acteoside, and the chemical background of glutinous rehmannia is comparatively complicated, martynoside, Dihuang polysaccharide, acteoside etc. are all its effective constituent, and the correlativity of its quality control index and drug effect awaits further research.
Biological activity determination method is introduced traditional Chinese medicine quality and is controlled 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, take glutinous rehmannia as research object, foundation be take drug effect as basic traditional Chinese medicine quality bioassay method, supplementing and improving as pharmacopeia routine and chemical assay, can be from multiple angle its inherent quality of jointly checking on, improving existing quality control and method for evaluating quality and standard, and can provide new thinking and reference for the indefinite traditional Chinese medicine quality control of other effective substance and quality evaluation research, is the technical matters that need to conscientiously solve.
Three, summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the present invention's object is just to provide a kind of prepared rhizome of rehmannia method for evaluating quality detecting based on biologically active, can effectively solve from multiple angle and evaluate 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 → prepare test sample → removing free radical reaction is tested → measured free radical scavenging activity → test sample and work reference substance and compares → calculate tire → 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, with the ultrasonic extraction of ethanol, ethanol is flung in water-bath, dry, to be dried thing is dissolved in and in ethanol, makes gradient ethanolic solution again, become test sample, standby; Choose many batches of prepared rhizome of rehmannia uniform samplings again, pulverize respectively, equivalent is chosen powder mixing, is dissolved in ethanol and makes work reference substance, standby; Then by hexichol for bitter taste phthalidyl free radical (DPPH
*) be dissolved in absolute ethyl alcohol, keep in Dark Place; After above-mentioned three kinds of solution preparation, carry out application of sample and detection, measure respectively the absorbance of each solution and calculate test sample to hexichol for bitter taste phthalidyl free radical (DPPH
*) clearance rate, then carry out the contrast calibrating of sample and reference substance, calculate tiring of test sample, to realize the quality evaluation to prepared rhizome of rehmannia.
The present invention be take prepared rhizome of rehmannia as object, foundation be take drug effect as basic traditional Chinese medicine quality bioassay method, supplementing and improving as pharmacopeia routine and chemical assay, can be from multiple angle its inherent quality of jointly checking on, improve existing quality control and method for evaluating quality and standard, and can provide new technical conditions for the indefinite traditional Chinese medicine quality control of other effective substance and quality evaluation research.
Four, accompanying drawing explanation
Fig. 1 is that in the present invention, prepared rhizome of rehmannia is removed DPPH
*live vol effect relationship figure.
Fig. 2 is that in the present invention, prepared rhizome of rehmannia is removed DPPH
*live vol effect relationship linear analysis figure.
Fig. 3 is sample determination result trend comparison diagram in the present invention.
Five, embodiment
Below in conjunction with concrete condition, the specific embodiment of the present invention is elaborated.
First it is to be noted, prepared rhizome of rehmannia has anti-oxidant, effect of scavenging radical, be one of clinical the most frequently used traditional help class Chinese medicine, that modern pharmacological research shows is anti-oxidant, remove body peroxy radical is that it builds up health, regulates one of immune main mechanism.Research shows that the different extracts of prepared rhizome of rehmannia have the anti-oxidant effect with removing peroxy radical in inside and outside significantly.
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, with single electron, have the last one to absorb at 517nm place, its alcoholic solution is purple.When anti-oxidant when having (removing free radical) composition exists, can its absorption be faded away 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 measured the oxidation resistance of Biosample, chemical substance and food etc.
In the inventive method, use following instrument and reagent: analytical balance, Shanghai precision electronic device company limited, FA200KA type; Hydro-extractor, Beijing Medical Centrifugal Machine Factory, LD4-2 type; UV-2201 spectrophotometer, Japanese Shimadzu company; SY-360 ultrasound wave extraction apparatus ，Shang Haining business's ultrasonic instrument company limited; XDW-6B Lowtemperaturepulverizer; 101-1A electric heating air blast drying case.
Radix rehmanniae recen (place of production gathers and combines with 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) institute records " cultivated land " lower prescriptive procedure and makes prepared rhizome of rehmannia.
DPPH
*(U.S. Sigma company); Absolute ethyl alcohol (analyzing Chun， Zhengzhou Chemical Co., Ltd. of Lianbang); It is pure that all the other reagent are analysis.
The present invention in the specific implementation, is realized by following steps:
The preparation of A, test sample (claiming again testing sample, as follows):
1) prepared rhizome of rehmannia is put in 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 brown bottle;
3) take 10 times of ethanol that sample powder 2g adds powder weight in conical flask, the ultrasonic extraction of normal temperature 30 minutes, gained extract filters with filter paper, discards residue, puts in surface plate, and ethanol is flung in water-bath, and after calculate recovery rate, dry thing is preserved in sealing;
4) will be dried thing and be dissolved in ethanol, making respectively concentration is Xg (crude drug) mL
-1solution (X equals respectively 1.0000,0.5000,0.2500,0.1250,0.0625 ...);
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 work object of reference, during mensuration, also by the step 3 of the A)～4) be prepared into solution, as work reference substance;
C, DPPH
*the preparation of solution
Precision takes 40mg DPPH
*, be dissolved in 480mL absolute ethyl alcohol, then add absolute ethyl alcohol 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, fully mix, the standing 30min of room temperature, 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
From upper table, provide, the said need testing solution that do not add refers to 2mL DPPH
*the solution of solution+2mL absolute ethyl alcohol, so Ac is 2mL DPPH
*the solution of solution+2mL absolute ethyl alcohol is being measured the absorbance at wavelength place; The said test solution that adds refers to 2mL DPPH
*+ 2mL need testing solution, so Ai is 2mLDPPH
*+ 2mL need testing solution is being measured the absorbance at wavelength place; Said blank solution refers to 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 wavelength place.
The adjustment of E, application of sample amount
For the convenience of tiring and calculating, need be according to the estimation of tiring of preliminary experiment and test sample, application of sample amount to test sample is carried out necessary adjustment, guarantee response intensity around clearance rate (P) (± 50%) 50% Shang Xia (refer to data processing and tire calculatings), and guarantee the amount effect relation curve parallel (referring to interpretation of result) that test sample reacts with the reference substance of working.
F, data processing and the calculating of tiring
1) according to following formula, calculate and measure liquid to DPPH
*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
For convenience of calculating, set (the P that tires of work reference substance
s) be 1.0Umg
-1(crude drug), according to parallel line assay principle adopt quantitative response parallel line assay method (22) method (according to version " Chinese Pharmacopoeia " in January, 2010. two appendix XI V Bioassay-statistical methods) 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:
According to the basic demand pair of medicine biological standardization: the 1. precision of detection method is 3. stability of repeatability 2., verifies the science of method for building up and rationality to determine.
As the basis of prepared rhizome of rehmannia method for evaluating quality (anti-oxidant titration method) research, first carried out prepared rhizome of rehmannia and removed DPPH
*the linearity of active detection and dose-effect relationship is as shown in Fig. 1 Fig. 2.
As can be seen from Figure 1 between reaction rate and log10 dose, be good the symmertrical "S" curve, meet conventional pharmacological reaction rule.
From Fig. 2 to 0.125gmL
-1～0.500gmL
-1in dosage range, sample is removed DPPH
*dose-effect relationship carry out linear analysis, can clearly find out at 0.125gmL
-1～0.500gmL
-1in dosage range, prepared rhizome of rehmannia is removed DPPH
*dose-effect relationship present good linearity (r>=99%).Testing sample and work reference substance response curve in the range of linearity are good parallel relation (meet 2.4.2 described in Bioassay-statistical method basic demand).
According to the requirement of medicine biological standardization, in above-mentioned research, (prepared rhizome of rehmannia is removed DPPH
*active detection research) on basis, by representative sample and 20 batch samples are repeatedly detected repeatedly, carry out methodology checking, consequently,
1) precision
According to the inventive method, get the test liquid (0.250gmL with a prepared rhizome of rehmannia sample
-1), carry out continuously 5 absorbance measurements and calculate DPPH
*clearance rate, be respectively: 70.1%, 71.2%, 68.6%, 67.7%, 65.4%, the coefficient of variation RSD of result 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, by upper method, contrasts calibrating respectively with work reference substance solution, and calculating tires is respectively: 0.87Umg
-1, 0.83Umg
-1, 0.94Umg
-1, 0.86Umg
-1, 0.79Umg
-1.The RSD that tires measuring for 5 times is 6.46%, shows that the method has good repeatability.
3) stability
Respectively at 0,1,2,3 and 4h the same need testing solution that is stored in 4 ℃ is carried out to bioactivity, result is respectively: 0.86Umg
-1, 0.84Umg
-1, 0.92Umg
-1, 0.85Umg
-1, 0.80Umg
-1.Its RSD is 5.08%, shows that it is stable that need testing solution is preserved 4h under 4 ℃ of conditions.
According to said method, 20 batches of cultivated land samples and work reference substance contrasts to detection, according to above-mentioned data processing and the computing method of tiring (" Chinese Pharmacopoeia " in January, 2010 version. two appendix XI V Bioassay-statistical methods) calculating of tiring.Result is as following table:
Above-mentioned different batches sample demonstrates different valence values.If remove DPPH with cultivated land
*active is the index calculating of tiring, and in order to characterize cultivated land quality, can carry out quantitative evaluation to different places of production different batches cultivated land sample.The applicability of illustration method is better, method is feasible.Can be effective to the quality evaluation of Chinese medicine cultivated land, to determine the quality of prepared rhizome of rehmannia quality, select and excellently wash in a pan badly, guarantee prepared rhizome of rehmannia quality and drug effect.The present invention compared with prior art, has significant feature, first by DPPH
*the determination method of removing ability, for the antioxidation activity in vitro of cultivated land is measured, proposes and works 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 free radical ability.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 detecting based on biologically active, is characterized in that, by following steps, is realized:
The preparation of A, test sample:
1) prepared rhizome of rehmannia is placed in to comminutor, under-40 ℃ of states, makes 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 brown bottle;
3) take 10 times of ethanol that powder 2g adds powder weight in conical flask, the ultrasonic extraction of normal temperature 30 minutes, gained extract filters with filter paper, discards residue, puts in surface plate, and ethanol is flung in water-bath, and dry thing is preserved in sealing;
4) will be dried thing and be dissolved in ethanol, making respectively concentration is that every mL is 1.0000g, 0.5000g, 0.2500g, 0.1250g, 0.0625g containing crude drug, becomes test sample;
The preparation of B, work reference substance:
By 20 batches of prepared rhizome of rehmannia uniform samplings, pulverize respectively, the powder of choosing equivalent fully mixes, as work object of reference, by the step 3 of the steps A)～4) be prepared into solution, as work reference substance;
The preparation of C, DPPH* solution, wherein, DPPH* solution is 1,1-diphenyl-2-trinitrobenzen hydrazine solution:
Take 40mg DPPH*, be first dissolved in 480mL absolute ethyl alcohol, then add absolute ethyl alcohol to 500mL, put in refrigerator and keep in Dark Place;
D, application of sample and detection:
By 2mL DPPH* solution+2mL absolute ethyl alcohol, by 2mL DPPH*+2mL need testing solution, by 2mL absolute ethyl alcohol+2mL need testing solution, be placed in respectively test tube, fully mix, the standing 30min of room temperature, in 517nm wavelength place, measure absorbance, with absolute ethyl alcohol, do blank;
The adjustment of E, application of sample amount:
For the convenience of tiring and calculating, need be according to the estimation of tiring of preliminary experiment and test sample, application of sample amount to test sample is carried out necessary adjustment, guarantee response intensity around the clearance rate P of DPPH* 50% upper and lower, and guarantee that test sample is parallel with the amount effect relation curve that work reference substance reacts;
F, data processing and the calculating of tiring:
1) according to following formula, calculate and measure the clearance rate P of liquid to DPPH*:
P=[1-(Ai-Aj)/Ac] * 100%, wherein Ac is that the solution of 2mL DPPH* solution+2mL absolute ethyl alcohol is in the absorbance at mensuration wavelength place; Ai is that 2mL DPPH*+2mL need testing solution is in the absorbance of measuring wavelength place; Aj is that 2mL absolute ethyl alcohol+2mL need testing solution is in the absorbance of measuring 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 principle, according to version " Chinese Pharmacopoeia " in January, 2010. and 22 methods in the parallel line analysis determination method of stipulating under two appendix XIV Bioassay-statistical method items, carry out test sample and examine and determine with the contrast that work reference substance is tired.
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| CN102914506B (en) * | 2012-10-09 | 2015-05-20 | 福建省农业科学院食用菌研究所 | Novel method for evaluating lucid ganoderma extract quality |
| 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 |
| 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 |
| 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 |
| CN110702809B (en) * | 2019-10-12 | 2023-01-31 | 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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| 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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| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 朱德新.《中草药对DPPH·自由基的清除作用的研究》.《天津化工》.2008,第22卷(第2期),全文. * |
| 王宏洁等.《鲜、生、熟地黄药材中3种活性成分含量的比较》.《中国中药杂志》.2008,第33卷(第15期),全文. * |
| 郭威等.《熟地黄中活性成分甘露三糖提取工艺研究》.《上海中医药大学学报》.2007,第21卷(第6期),全文. * |
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