CN1546992A - Method for rapidly and quantitatively determining the triterpenoid content in ganoderma lucidum - Google Patents
Method for rapidly and quantitatively determining the triterpenoid content in ganoderma lucidum Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 240000008397 Ganoderma lucidum Species 0.000 title claims abstract description 10
- 235000001637 Ganoderma lucidum Nutrition 0.000 title claims abstract description 10
- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 title 1
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Abstract
The invention is a method for measuring the content of triterpenes compound in Ganoderma Lucidum, especially a method for using ultraviolet splitting luminosity to measure, the invention refers to biology technology field. The technology project is: the Ganodeerma Lucidum is put into a cleaned tube, adds chloroform to immerse for 0.5-1 hours, and acquires the supernate after being centrifugated, uses the saturate NaHCO3 solvent to extract for 2-3 times, collects the NaHCO3 layer, acquires the liquid for test, measures the volume accurately, uses ultraviolet splitting luminosity method to measure the light absorbing valve under 250-260nm wavelength, works out the content of triterpenes in the sample according to the standard curve. The method saves the acidification, chloroform extraction, reduced pressure distillation and drying steps, the period of measurement is shortened greatly.
Description
One. technical field
The present invention is the method for triterpene compound content in a kind of quantitative determination glossy ganoderma, is meant especially with ultraviolet spectrophotometry to come method quick, quantitative measurement, relates to biological technical field.
Two. background technology
The separation and purification of relevant glossy ganoderma [red sesame Ganoderma lucidum (Fr.) Karst] polysaccharide composition, the research of constituent structure, pharmacology, immune antitumor activity and clinical practice extensively are seen in document, separation and purification, structure evaluation and biologically active for triterpene compound also have some reports, and be an important directions of glossy ganoderma research in the world, its biologically active comprises and suppresses tumor cell proliferation, antiviral, antibacterial, analgesia, liver protecting etc. always.The extraction process of triterpene compound is roughly in the glossy ganoderma: the smart powder of ganoderma lucidum fruitbody is through alcohol extract, and chloroform dissolves, saturated NaHCO
3Extraction, the HCl acidifying, chloroform extraction, steps such as vacuum distillation drying can obtain the faint yellow solid crystallization.(referring to: Wang Bangwu etc.The extraction separation of glossy ganoderma acidic components and bacteriostatic activity research.Beijing Institute of Technology's journal, 2002,22 (1): 125-128)
At present ganoderma lucidum product is of a great variety on the home market, a lot of products with triterpenes as the effect component.Because this type of active constituent is not also had simple and practical detection method, cause and to detect fast and effectively and quantitative measurement its quality, so often occur the propaganda that falls short of the reality on the market.What the present invention described is a kind of method of coming triterpenes content in the fast measuring glossy ganoderma test sample with ultraviolet spectrophotometry, has the shortening minute, simplifies characterization processes, good reproducibility, and cost is lower, implements advantages such as easy.
Three. summary of the invention
The objective of the invention is to set up a kind of method with triterpene compound content in the ultraviolet spectrophotometry quantitative determination glossy ganoderma test sample.
1. the used instrument of the present invention is: UV, visible light double beam spectrophotometer, high pressure liquid chromatograph, chromatographic column, ultraviolet detection analyser.
2. material required for the present invention is: glossy ganoderma (Ganoderma lucidum (Fr.) Karst) fructification; Methyl alcohol and acetic acid are chromatographically pure reagent; Methenyl choloride, sodium bicarbonate etc. are analytical reagent; 0.45 μ m miillpore filter.
3. indication measuring object of the present invention is the triterpene compound in glossy ganoderma (Ganoderma lucidum (Fr.) Karst) fructification, has following features:
A. its outward appearance is the faint yellow solid crystallization.
B. chromatogram feature: thin-layer chromatography condition: GF-254 silica gel plate, developping agent V (n-C
6H
14): V (CH
2Cl
2): V (CHCl
3): V (MeOH)=4: 3: 2.5: 0.5.Under wavelength 254nm detects, Rf value 0.35～0.90.High pressure liquid chromatography condition: detect wavelength 250～260nm, spectral bandwidth 16, reference wavelength 360nm, spectral bandwidth 100, dissolve with methanol sample, concentration 1mg/mL, sample size 10 μ L, flow velocity 0.8mL/min, 40 ℃ of column temperatures; Moving phase: 5% acetic acid-methyl alcohol 60: 40.Hewlett-Packard-1100 type high pressure liquid chromatograph analysis shows 8 principal character peaks, and relative retention time α is respectively 0.78,0.88,0.92,1.00,1.12,1.27,1.48,1.61,8 main peak area percent more than 70%.
C. spectral signature: its methyl alcohol, chloroform, saturated NaHCO
3In the uv-vis spectra of solution, a maximum absorption band is arranged all at 250～260nm place; In its infrared spectrum, at 3400cm
-1, 1025cm
-1The hydroxyl absorption peak is arranged, 1710cm
-1Hexa-atomic cyclic ketones occurs, CH appears in 2955cm-1
3-C-group, 2922cm
-1And 2851cm
-1Appearance-C-CH
2The stretching vibration peak that-C-is strong.
D. chemical constitution: 5 kinds of different mother nucleus structures are arranged, see accompanying drawing 1.Fig. 1 has shown five kinds of structure parent nucleus that utilize triterpene compound in the detected glossy ganoderma of ultraviolet spectrophotometry, belongs to highly oxidized lanostane analog derivative, can be divided into C by contained carbon atom number in the molecule
30, C
27And C
24Three major types.In these five kinds of basic skeleton structures, the two keys on the ring are positioned at Δ mostly
8 (9)The position, most of at C
11Position and C
23Carbonyl is arranged, and at C
3, C
7, C
15The position is also many to be replaced by carbonyl or hydroxyl.And triterpene alcohol, aldehyde and superoxide structure in, mostly exist two unsaturated double-bonds, its position at Δ on the ring
7 (8), Δ
9 (11)The position, C
11Position and C
23There is not carbonyl in the position yet, and the substituting group on the ring obviously reduces.
4. the preparation of standard items and typical curve
A. the preparation of standard items and testing result.Preparation process is roughly: extract total triterpene compound according to literature method from the smart powder of glossy ganoderma earlier, utilize repeatedly silica gel column chromatogram method to separate again, with chloroform: methyl alcohol=98: 2,95: 5,50: 50 gradient elutions are collected the cut that 98: 2 wash-outs go out and are detected.Thin-layer chromatography detects selects developping agent V (n-C
6H
14): V (CH
2Cl
2): V (CHCl
3): V (MeOH)=4: 3: 2.5: 0.5, ultraviolet detection analyser (detecting wavelength 254nm) and silica gel plate GF-254.The cut of selecting testing result to present single spot carries out high performance liquid chromatography and prepares a kind of unimodal material.Detect sample concentration 1mgmL with the analytic type high performance liquid chromatography
-1, applied sample amount 10 μ L, moving phase is 5% acetic acid-methyl alcohol (60: 40), flow velocity 1mLmin
-1, detect wavelength 250～260nm, reference wavelength 360nm.
Testing result is: the thin-layer chromatography result is shown as single spot, and the Rf value is 0.43.Further use high pressure liquid chromatographic analysis, display standard product purity reaches 100% as a result, and its retention time is 22.26min.In its infrared spectrum, at 3400cm
-1, 1025cm
-1The hydroxyl absorption peak is arranged, 1710cm
-1Hexa-atomic cyclic ketones appears, 2955cm
-1CH appears
3-C-group, 2922cm
-1And 2851cm
-1Appearance-C-CH
2The stretching vibration peak that-C-is strong.
B. the preparation method of typical curve and drafting: accurately take by weighing the triterpene compound standard items, be made into initial concentration 200 μ gmL with saturated sodium bicarbonate solution
-1Fully after the dissolving, 2 times are diluted to series concentration 200,100,50,25,12.5 μ gmL
-1With the saturated sodium bicarbonate solution is that blank is carried out baseline zeroing, spectral scan scope 200～400nm, sampling interval 0.5nm, spectral bandwidth 2nm.After determining maximum absorption wavelength, under this wavelength, carry out the quantitative measurement of standard specimen, calculate equation of linear regression and carry out significance test.
Typical curve is drawn: standard items series concentration all has 1 maximum absorption band in 200～400nm scanning wavelength scope, its maximum absorption wavelength scope is 250～260nm, equation of linear regression is y=-0.00574+0.01048x, correlation coefficient r is between 0.9990～0.9999, SD=0.01355, P＜0.0001 (n=6).
5. test sample preparation method: with reference to the accompanying drawings 2, accurately take by weighing 0.2～2.0g glossy ganoderma test sample and place clean test tube, add chloroform, vibration shakes up, and soaks behind 0.5h～1h in 2000rm
-1Centrifugal, sampler is accurately drawn supernatant and is got extract I, and surplus liquid is put into separating funnel, uses saturated NaHCO
3Extract 2～3 times, collect chloroform layer and NaHCO
3Layer gets extract II and III.
Emphasis of the present invention:
The standard program of triterpene compound content fast detecting is in the glossy ganoderma test sample: accurately take by weighing 0.2～2g ganoderma lucidum fruitbody and place clean tube, add chloroform lixiviate 0.5～1h, chloroform volume ml number is 10: 1～20: 1 with the ratio of example weight g number.The centrifugal supernatant that gets is with the saturated NaHCO of equivalent
3Solution extraction 2～3 times is collected extract III, i.e. the NaHCO that carries of alkali
3Layer be a test solution, accurately measure its volume after, dilution uses ultraviolet spectrophotometry to measure the absorbance value of test solution under maximum absorption wavelength 250～260nm, can calculate the content of triterpene compound in the sample according to typical curve.The present invention utilizes ultraviolet spectrophotometry in the interstage of extracting triterpene compound technology, i.e. the NaHCO that carries of alkali
3Layer detects, and has saved the steps such as acidifying, chloroform extraction, vacuum distillation drying of back, makes minute shorten greatly.
Wherein utilize extract III, i.e. the NaHCO that carries of alkali
3Layer carry out quantitative measurement former because: with reference to the extract preparation flow of accompanying drawing 2, respectively contained triterpene compound among 3 kinds of extract I, II of test sample, the III has been carried out the uv-spectrophotometric quantitative measurement, repeatedly experimental result shows: the maximum absorption wavelength of extract I and III is 257nm, and II then has two peak value 247nm and 283nm.Though component II does not have absorption maximum at 257nm, absorption value shows that also up to 1.856 non-triterpenes component also has stronger absorption at the 257nm place among the extract I, therefore can not be used for detecting.Therefore the extract III that contains the triterpenes component is the same with standard items only to have absorption maximum at the 257nm place, can measure among the III triterpenes content with the content of triterpenes in the representative sample.
Major advantage of the present invention is as follows:
1. easy quick and good reproducibility.In the interstage of extracting triterpene compound technology, i.e. the NaHCO that carries of alkali
3Layer detects, and has saved the steps such as acidifying, chloroform extraction, vacuum distillation drying of back, has the shortening minute, simplifies characterization processes, advantage such as is easy to repeat, and can be applied to the quality evaluation of glossy ganoderma and Related product thereof.
2. cost is low: it is less to analyze required sample size, only is 0.2～2g;
3. operating process is simple, and used medicine and instrument are common being easy to get, and can implement in common lab or pharmaceutical factory;
Four. Figure of description
Fig. 1 has shown five kinds of structure parent nucleus that utilize triterpene compound in the detected glossy ganoderma of ultraviolet spectrophotometry.
Fig. 2 has shown triterpene compound content quick determination process flow diagram in the glossy ganoderma.
Five. embodiment
Embodiment 1
1 instrument and material
1.1 instrument TU-1901 type twin-beam ultraviolet-visible pectrophotometer; High pressure liquid chromatograph (1100series of Hewlett-Packard); ODS RP-C
18Chromatographic column: 5 μ m fillers, 250mm * 4.6mm.Portable ultraviolet detection analyser UV-III type (detecting wavelength 254nm).
1.2 material glossy ganoderma (Ganoderma lucid um (Fr.) Karst) fructification (Tai'an, Shandong, the place of production).Methyl alcohol and acetic acid are chromatographically pure reagent, and methenyl choloride, sodium bicarbonate etc. is an analytical reagent.0.45 μ m miillpore filter (homemade).
2 experimental techniques
2.1 standard items preparation and detection
Earlier extract total triterpene compound according to literature method from the smart powder of glossy ganoderma, utilize repeatedly silica gel column chromatogram method to separate again, with chloroform: methyl alcohol=98: 2,95: 5,50: 50 gradient elutions were collected the cut that 98: 2 wash-outs go out and are detected.(Thin Layer Chromatography TLC) detects selection developping agent V (n-C to thin-layer chromatography
6H
14): V (CH
2Cl
2): V (CHCl
3): V (MeOH)=4: 3: 2.5: 0.5, ultraviolet detection analyser (detecting wavelength 254nm), silica gel plate GF-254.The cut of selecting testing result to present single spot carries out high performance liquid chromatography and prepares a kind of unimodal material.Detect sample concentration 1mgmL with the analytic type high performance liquid chromatography
-1, applied sample amount 10 μ L, moving phase is 5% acetic acid-methyl alcohol (60: 40), flow velocity 1mLmin
-1, detect wavelength 257nm, reference wavelength 360nm.
2.2 typical curve preparation
Accurately take by weighing the triterpene compound standard items, be made into initial concentration 200 μ gmL with saturated sodium bicarbonate solution
-1Fully after the dissolving, 2 times are diluted to series concentration 200,100,50,25,12.5 μ gmL
-1With the saturated sodium bicarbonate solution is that blank is carried out baseline zeroing, spectral scan scope 200～400nm, sampling interval 0.5nm, spectral bandwidth 2nm.After determining maximum absorption wavelength, under this wavelength, carry out the quantitative measurement of standard specimen, calculate equation of linear regression and carry out significance test.
2.3 test sample preparation
According to Fig. 2, accurately take by weighing 1g glossy ganoderma test sample and place clean test tube, add chloroform 15mL, vibration shakes up, and soaks behind the 0.5h in 2000rm
-1Centrifugal 10min, sampler is accurately drawn supernatant and is got extract I (volume V
1Be 9.7mL).Get and carry out spectral scan and quantitative measurement after 300 μ L extract I dilute 20 times, surplus liquid is put into the 50mL separating funnel, uses saturated NaHCO
3Extraction, each 10mL totally 3 times, collects chloroform layer and NaHCO
3Layer gets extract II and III (III volume V
2Be 30.0mL), after diluting 3 times and 4 times respectively, II and III carry out spectral scan and quantitative measurement.
3 results
3.1 standard items testing result
TLC result detects with the 254nm uviol lamp, and standard items show single spot, and the Rf value is 0.43.Further use high pressure liquid chromatographic analysis, display standard product purity reaches 100% as a result, and its retention time is 22.26min.In its infrared spectrum, at 3400cm
-1, 1025cm
-1The hydroxyl absorption peak is arranged, 1710cm
-1Hexa-atomic cyclic ketones appears, 2955cm
-1CH appears
3-C-group, 2922cm
-1And 2851cm
-1Appearance-C-CH
2The stretching vibration peak that-C-is strong.
3.2 typical curve is drawn
Standard items series concentration all has 1 maximum absorption band in 200～400nm scanning wavelength scope, its maximum absorption wavelength is 257nm, and the reappearance of each peak maximum absorption wavelength is better, and accuracy of instrument is higher.
0～200.0 μ g/mL triterpene compound standard items are dissolved in saturated NaHCO
3In, in maximum absorption band 257nm place's photometry absorption value, through Origin 6.1 software processes analyses, its equation of linear regression is y=-0.00574+0.01048x, coefficient R=0.99988, SD=0.01355, P＜0.0001 (n=6).The concentration and the absorbance that show standard solution are the conspicuousness linear dependence, and this method measurement result in this concentration range is more accurate.
3.3 determining of test sample preparation condition
Whether can be directly used in the content of measuring the triterpene compound in the glossy ganoderma test sample in order to inquire into extract I, in this experiment 3 kinds of extract I, II, III carried out UV spectrophotometry.The result shows: the maximum absorption wavelength of extract I and III is 257nm, and II then has two peak value 247nm and 283nm.Though component II does not have absorption maximum at 257nm, absorption value shows that also up to 1.856 non-triterpenes component also has stronger absorption at the 257nm place among the extract I, therefore can not be used for detecting.Therefore the extract III that contains the triterpenes component is the same with standard items only to have absorption maximum at the 257nm place, can measure among the III triterpenes content with the content of triterpenes in the representative sample.Triterpene compound content can calculate according to following formula:
After the smart powder of conclusion 1g glossy ganoderma soaked 0.5h with the 15mL chloroform, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 3 times, the content of triterpenes among the 257nm Detection and Extraction liquid III.Through 3 mensuration, the triterpenes average content is 1.48%.
Embodiment 2
Repeat embodiment 1 by described same steps as, but glossy ganoderma test sample and chloroform rate of charge 1 (g): 20 (ml), after chloroform soaked 0.5h, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 3 times is collected extract III, i.e. NaHCO
3Layer, the content of triterpenes during 257nm detects.Through three mensuration, the triterpenes average content is 1.51%.
Embodiment 3
Repeat embodiment 1 by described same steps as, but glossy ganoderma test sample and chloroform rate of charge 1 (g): 10 (ml), after chloroform soaked 0.5h, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 3 times is collected NaHCO
3Layer, the content of triterpenes among the 257nm Detection and Extraction liquid III.Through three mensuration, the triterpenes average content is 1.49%.
Embodiment 4
Repeat embodiment 1 by described same steps as, but glossy ganoderma test sample and chloroform rate of charge 1 (g): 15 (ml), after chloroform soaked 1h, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 2 times is collected NaHCO
3Layer, the content of triterpenes among the 257nm Detection and Extraction liquid III.Through three mensuration, the triterpenes average content is 1.50%.
Embodiment 5
Repeat embodiment 1 by described same steps as, but glossy ganoderma test sample and chloroform rate of charge 1 (g): 20 (ml), after chloroform soaked 1h, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 2 times is collected NaHCO
3Layer, the content of triterpenes among the 257nm Detection and Extraction liquid III.Through three mensuration, the triterpenes average content is 1.52%.
Embodiment 6
Repeat embodiment 1 by described same steps as, but glossy ganoderma test sample and chloroform rate of charge 1 (g): 10 (ml), after chloroform soaked 1h, the centrifugal supernatant that gets was with the saturated NaHCO of equivalent
3Solution extraction 2 times is collected NaHCO
3Layer, the content of triterpenes among the 257nm Detection and Extraction liquid III.Through three mensuration, the triterpenes average content is 1.50%.
Claims (6)
1. the method for triterpene compound content in the quantitative determination glossy ganoderma is characterized in that: with ultraviolet spectrophotometry ganoderma lucidum triterpene compounds is carried out quick, quantitative mensuration.
2. the method for triterpene compound content in a kind of quantitative determination glossy ganoderma according to claim 1, it is characterized in that: the maximum absorption wavelength scope of ultraviolet detection by quantitative is at 250nm～260nm.
3. the method for triterpene compound content in a kind of quantitative determination glossy ganoderma according to claim 1 is characterized in that: utilize ultraviolet spectrophotometry in the interstage of extracting triterpene compound technology, i.e. the NaHCO that carries of alkali
3Layer detects, and has saved the steps such as acidifying, chloroform extraction, vacuum distillation drying of back, makes minute shorten greatly.
4. the method for triterpene compound content in a kind of quantitative determination glossy ganoderma according to claim 1 is characterized in that: standard items adopt the saturated sodium bicarbonate solution dissolving; And standard items are at 0～400.0 μ gmL
-1In the concentration range, correlation coefficient r is between 0.9990～0.9999.
5. the standard items in a kind of quantitative determination glossy ganoderma according to claim 4 in the method for triterpene compound content, its preparation method is characterised in that: the smart powder of ganoderma lucidum fruitbody is through alcohol extract, chloroform dissolving, saturated NaHCO
3Extraction and HCl acidifying obtain total triterpene, utilize repeatedly silica gel column chromatogram method separation again, with chloroform: methyl alcohol=98: 2,95: 5,50: 50 gradient elutions are collected cut and are carried out the thin-layer chromatography detection, select to prepare monomeric substance than pure fraction by high pressure liquid chromatography.
6. the method for triterpene compound content in a kind of quantitative determination glossy ganoderma according to claim 1, it is characterized in that: glossy ganoderma test sample preparation method is, accurately take by weighing the glossy ganoderma test sample and place clean test tube, add chloroform lixiviate 0.5～1h, chloroform volume ml number is 10: 1～20: 1 with the ratio of example weight g number, the centrifugal supernatant that gets is with the saturated NaHCO of equivalent
3Extract 2～3 times, collect NaHCO
3Layer is also accurately measured its volume, is test solution.
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| CN 200310117111 CN1261749C (en) | 2003-12-03 | 2003-12-03 | Method for rapidly and quantitatively determining the triterpenoid content in ganoderma lucidum |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100491999C (en) * | 2006-09-30 | 2009-05-27 | 吴敏 | Method for detecting glossy ganoderma spore oil content in glossy ganoderma oil preparation |
| CN103163128A (en) * | 2013-03-13 | 2013-06-19 | 重庆大学 | Method for determining content of amylopectin and amylose in broomcorn |
| CN108426974A (en) * | 2018-05-21 | 2018-08-21 | 浙江省中医药研究院 | A kind of TLC Identification of ganoderma lucidum and lucidum spore powder |
| CN109503696A (en) * | 2019-01-04 | 2019-03-22 | 云南中烟工业有限责任公司 | A kind of triterpene compound with antibacterial functions and preparation method thereof and the application in electronic cigarette |
| CN109730359A (en) * | 2019-01-04 | 2019-05-10 | 云南中烟工业有限责任公司 | Triterpene compound in a kind of adiantum cpilus-veneris and preparation method thereof and the application in electronic cigarette |
| CN110411917A (en) * | 2019-08-01 | 2019-11-05 | 河南师范大学 | The method for being attached to human hands dust quality is assessed by standard curve |
-
2003
- 2003-12-03 CN CN 200310117111 patent/CN1261749C/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100491999C (en) * | 2006-09-30 | 2009-05-27 | 吴敏 | Method for detecting glossy ganoderma spore oil content in glossy ganoderma oil preparation |
| CN103163128A (en) * | 2013-03-13 | 2013-06-19 | 重庆大学 | Method for determining content of amylopectin and amylose in broomcorn |
| CN103163128B (en) * | 2013-03-13 | 2015-07-22 | 重庆大学 | Method for determining content of amylopectin and amylose in broomcorn |
| CN108426974A (en) * | 2018-05-21 | 2018-08-21 | 浙江省中医药研究院 | A kind of TLC Identification of ganoderma lucidum and lucidum spore powder |
| CN109503696A (en) * | 2019-01-04 | 2019-03-22 | 云南中烟工业有限责任公司 | A kind of triterpene compound with antibacterial functions and preparation method thereof and the application in electronic cigarette |
| CN109730359A (en) * | 2019-01-04 | 2019-05-10 | 云南中烟工业有限责任公司 | Triterpene compound in a kind of adiantum cpilus-veneris and preparation method thereof and the application in electronic cigarette |
| CN110411917A (en) * | 2019-08-01 | 2019-11-05 | 河南师范大学 | The method for being attached to human hands dust quality is assessed by standard curve |
| CN110411917B (en) * | 2019-08-01 | 2022-03-25 | 河南师范大学 | Method for evaluating quality of dust attached to human hand through standard curve |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1261749C (en) | 2006-06-28 |
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