CN1176378C - Test-paper testing method for diosgenin content - Google Patents
Test-paper testing method for diosgenin content Download PDFInfo
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- CN1176378C CN1176378C CNB021478767A CN02147876A CN1176378C CN 1176378 C CN1176378 C CN 1176378C CN B021478767 A CNB021478767 A CN B021478767A CN 02147876 A CN02147876 A CN 02147876A CN 1176378 C CN1176378 C CN 1176378C
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- chinese yam
- saponin
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- yam saponin
- diosgenin
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Abstract
The present invention discloses a test paper detecting method for the content of diosgenin, particularly a method for detecting the content of diosgenin in plant materials or relevant samples. In the present invention, one kind of protein coupled diosgenin is used as an antigen to obtain the antibodies of anti-diosgenin and relevant saponin; the other kind of protein coupled diosgenin is used as an encrusting substance, and is combined with a nitrocellulose membrane to be manufactured into test paper used as a spot for detecting the content of diosgenin in plant materials or relevant samples by using an enzyme linked immunoassay. The present invention has the advantages of simple and inexpensive instrument, little investment, low detection cost, short detection time, simple detection process, little sample amount and high sensitivity, and can satisfy requirements for detecting the content of diosgenin in relevant plant materials in batch quantity in relevant research enterprises, trade enterprises, processing enterprises and quality detecting departments.
Description
Technical field
The present invention relates to a kind of method that detects Chinese yam saponin content in the plant, specifically, relate to a kind of method with Chinese yam saponin content in test paper detecting method (dot enzyme-linked immuno method) the detection by quantitative plant.
Background technology
Chinese yam saponin is the most important raw material of synthesizing steroid hormone medicine.Because it is consuming time that conventional method detects the interior Chinese yam saponin content complex steps of plant, thereby Chinese yam saponin Determination on content method becomes the problem that presses for solution in the improvement plant.
As far as is known, in detection plant (normally yam) body there be the conventional method of Chinese yam saponin content:
1. gravimetric method: take by weighing chopping and dry (or 80 ℃ of oven dry, other surveys moisture) two parts of testing samples, every part of 30g adds 2N hydrochloric acid 300ml respectively, water-bath heating hydrolysis 3.5 hours, suction strainer, be washed to neutrality, residue is 80 ℃ of oven dry, the paper web of packing into then, in fatty extractor, (last extract 6-8 is dripped fully with sherwood oil (60-90 ℃) extracting, when volatilizing no crystallization on surface plate, expression has been carried), extract inspissation is put cold, filter is assembled brilliant, wash twice with sherwood oil, crystallization is weighed after 80 ℃ of oven dry, and washing lotion and filtrate merge a little crystallization again of back inspissation, the same oven dry is weighed, and is converted into percent content.
2. colourimetry: precision takes by weighing two parts of the testing samples of chopping oven dry, every part of 0.1-0.3g is put in the screw, be respectively charged in the fatty extractor, use the benzene degreasing, after flinging to benzene, sample is moved into conical flask, add 2N sulfuric acid 20ml, 120 ℃ of left and right sides heating hydrolysis 3.5 hours, put cold, thin up filters, and is washed till alkalescence with 1% sodium carbonate liquid heat earlier, be washed till neutrality with distilled water again, after 80 ℃ of oven dry, the fatty extractor of packing into is complete with sherwood oil (60-90 ℃) extracting, and extract is changed in the volumetric flask (50ml), behind 30 ℃ of constant volumes, draw 0.5ml extract evaporate to dryness respectively, add 6ml perchloric acid (G.R.) colour developing, carry out colorimetric (spectrophotometer 480nm carries out colorimetric) after a quarter of an hour.According to typical curve, be converted into percent content.
3. high performance liquid chromatography: precision takes by weighing two parts of the testing samples of chopping oven dry, every part of 0.1-0.3g is put in the screw, be respectively charged in the fatty extractor, use the benzene degreasing, after flinging to benzene, sample is moved into conical flask, add 2N sulfuric acid 20ml, 120 ℃ of left and right sides heating hydrolysis 3.5 hours, put coldly, thin up filters, earlier be washed till alkalescence with 1% sodium carbonate liquid heat, be washed till neutrality with distilled water again, after 80 ℃ of oven dry, the fatty extractor of packing into is complete with sherwood oil (60-90 ℃) extracting, extract is changed in the volumetric flask (50ml), at 30 ℃ of constant volumes.Get an amount of usefulness 0.45 μ m organic membrane and filter, for sample introduction.Sample size 20 μ l.C
8Post (5 μ m, 4.6mm * 250mm) acetonitrile-water (80: 20) is flow rate of mobile phase 1ml/min, detects wavelength 205nm, Chinese yam saponin reaches with other compositions and separates in the sample.Calculate the peak area integrated value, be converted into percent content according to typical curve.
Wherein wt method and colourimetry equipment needed thereby are simple, and shortcoming is time-consuming, detect when being difficult to carry out a large amount of sample, and error are bigger; The high effective liquid chromatography for measuring result is accurate, but consuming time equally, and equipment needed thereby costliness (commercially available liquid chromatograph need hundreds of thousands is to millions of units), complicated operation, and the operating personnel of need special training, not general enterprise or individual can reach.
Summary of the invention
The objective of the invention is: overcome above-mentioned conventional method existing shortcoming and defect, a kind of test paper detecting method of Chinese yam saponin content is provided.Specifically to solve following technical matters:
1. the Chinese yam saponin content fast detecting problem of sample in processing enterprise's raw material purchase and the process;
2. the fast detecting problem of batch sample, particularly micro-example in the correlative study process;
3. have loaded down with trivial details, the high problem of specimen preparation program of method now to equipment requirements.
The object of the present invention is achieved like this: as antigen, obtain the antibody of anti-Chinese yam saponin and relevant saponin with a kind of Chinese yam saponin of albumen coupling; , combine with nitrocellulose membrane and to make test paper as encrusting substance with the Chinese yam saponin of another kind of albumen coupling, detect the content of Chinese yam saponin in plant or the associated sample with detection paper method (dot enzyme-linked immuno method).
Relational term and abbreviation:
The BSA-bovine serum albumin(BSA);
The OVA-ovalbumin;
DCC-N, the N-dicyclohexylcarbodiimide;
NHS-N-maloyl imines;
The DMF-dimethyl formamide;
Nitrocellulose membrane: commercially available homemade nitrocellulose membrane;
Albumen dry powder: commercially available skimmed milk power;
Phosphate buffer-0.01mol/L pH7.8 phosphate buffer contains 0.9% sodium chloride;
Substrate: DAB (3, the 3-diaminobenzidine).
The Chinese yam saponin enzyme-linked immune detection method sets up process and testing process is as follows:
1. Chinese yam saponin and succinic anhydride react in pyridine and make it carboxylated
Chinese yam saponin and succinic anhydride are dissolved in pyridine, and airtight room temperature reaction is more than 3 days, evaporated under reduced pressure, and the gained material is dissolved in chloroform, and washing volatilizes chloroform more than 3 times.
2. use N, N-dicyclohexylcarbodiimide method is 1. Chinese yam saponin, the DCC (N of the succinic acid anhydridization of gained of antigen with carboxylated Chinese yam saponin and bovine serum albumin(BSA) coupling, the N-dicyclohexylcarbodiimide), NHS (N-maloyl imines) is dissolved in the middle room temperature reaction of DMF (dimethyl formamide) 1 day by 1: 1: 3 mol ratio, filter, gained solution dropwise adds in BSA (bovine serum albumin(BSA)) solution (4mg/ML), room temperature magnetic agitation 5 hours, gained liquid dialysis (0.1% anhydrous sodium acetate) 3 days is as immunogene.
3. be that encrusting substance takes by weighing 20mgEDC (water-soluble carbodiimide) and is dissolved in the 4ml water with water-soluble carbodlimide method with carboxylated Chinese yam saponin and ovalbumin coupling, get OVA (ovalbumin) solution that 3ml dropwise adds 7ml 2mg/ml, magnetic agitation, dropwise add succinic acid anhydridization saponin/DMF solution 0.4ml then, magnetic agitation, add remaining 1mlEDC solution behind the 10min in the solution that is stirring, stirred overnight at room temperature.Dialysis (0.1% anhydrous sodium acetate) 3 days.
4. use antigen-immunized animal, obtain the antibody of anti-Chinese yam saponin and relevant saponin
With the conjugate of 2. gained as immunogene, immune male new zealand white rabbit, after one month booster immunization once, the back bloodletting of two weeks obtain the antiserum of anti-Chinese yam saponin and relevant saponin; The antibody purification of saltouing.
5. combine with nitrocellulose membrane with encrusting substance and make test paper
With the encrusting substance point sample of 3. gained on nitrocellulose membrane, drying, test paper is made in albumen dry powder solution sealing 30 minutes.
6. with the Chinese yam saponin content in euzymelinked immunosorbent assay (ELISA) detection plant or the associated sample
Specimen preparation: testing sample methyl alcohol lixiviate, activated charcoal discolors;
Antibody titer detects: 4. gained antibody is diluted to gradient concentration with phosphate buffer, is added drop-wise to 5. on the gained test paper, adds enzyme mark goat-anti rabbit and substrate colour developing thereof more successively.Scanner scanning is surveyed gray-scale value, selects suitable dilute concentration as antiserum titre;
The making of typical curve: drip gradient concentration Chinese yam saponin standard items and the 4. mixed liquor of gained antibody (hatching 1h for 37 ℃) on the test paper, phosphate buffer washing → enzyme mark goat-anti rabbit (hatching 1h for 37 ℃), the colour developing of phosphate buffer washing → substrate, washing → scanner scanning is surveyed gray-scale value and drawing standard curve;
Sample detection: drip testing sample and the 4. mixed liquor of gained antibody (hatching 1h for 37 ℃) on the test paper, phosphate buffer washing → enzyme mark goat-anti rabbit (hatching 1h for 37 ℃), the colour developing of phosphate buffer washing → substrate, washing → scanner scanning, survey gray-scale value, converse the saponin content of testing sample by typical curve.
The present invention has the following advantages and good effect:
1. required instrument and equipment simple cheap only needs the plain scan instrument just can realize continuous fast detecting to batch samples, so less investment, and it is low to detect cost.
2. lack detection time, when particularly detecting simultaneously in enormous quantities for a plurality of samples, the average required time of single sample is short, makes things convenient for the actual use of scientific research and production.
3. testing process is simplified, and required sample size is few, detection highly sensitive.
Can satisfy the needs that related research units, processing enterprise, quality testing department detect Chinese yam saponin content in the corresponding plants body in a large number.
Embodiment
Specimen preparation:
Take by weighing an amount of pulverizing testing sample (0.01g-1g decides on the sample situation), add the ultrasonic lixiviate of methyl alcohol (as no ultrasonic equipment, standing over night gets final product) 3 times, each 3-5ml merges leaching liquor, constant volume, activated carbon decolorizing.
Content detection:
Drip the mixed liquor (hatching 1h for 37 ℃) of gradient concentration Chinese yam saponin standard items and anti-Chinese yam saponin and relevant saponin antibody on the test paper, phosphate buffer washing → enzyme mark goat-anti rabbit (hatching 1h for 37 ℃), the colour developing of phosphate buffer washing → substrate, washing → scanner scanning is surveyed gray-scale value and drawing standard curve;
Sample detection: the mixed liquor (hatching 1h for 37 ℃) that drips testing sample and anti-Chinese yam saponin and relevant saponin antibody on the test paper, phosphate buffer washing → enzyme mark goat-anti rabbit (hatching 1h for 37 ℃), the colour developing of phosphate buffer washing → substrate, washing → scanner scanning, survey gray-scale value, converse the saponin content of testing sample by typical curve.
Measure the required test paper for preparing, various antibody, standard items, substrate, damping fluid reagent etc., can provide by patented claim person is complete, the user operates according to above-mentioned steps, can detect the Chinese yam saponin content of at least two ten samples in about 3 hours.If the testing sample number is more, it is shorter that single sample detects the required time.
Claims (2)
1, the test paper detecting method of Chinese yam saponin content is characterized in that: as antigen, obtain the antibody of anti-Chinese yam saponin and relevant saponin with a kind of Chinese yam saponin of albumen coupling; , combine with nitrocellulose membrane and to make test paper as encrusting substance with the Chinese yam saponin of another kind of albumen coupling, detect the content of Chinese yam saponin in plant or the associated sample with the dot enzyme-linked immuno method; Its step is as follows:
1. Chinese yam saponin and succinic anhydride react in pyridine and make it carboxylated;
2. use N, N-dicyclohexylcarbodiimide method is an antigen with carboxylated Chinese yam saponin and bovine serum albumin(BSA) coupling;
3. be encrusting substance with water-soluble carbodlimide method with carboxylated Chinese yam saponin and ovalbumin coupling;
4. use antigen-immunized animal, obtain the antibody of anti-Chinese yam saponin and relevant saponin;
5. combine with nitrocellulose membrane with encrusting substance and make test paper;
6. use the Chinese yam saponin content in detection paper plant or the associated sample.
2, by the test paper detecting method of the described Chinese yam saponin content of claim 1, it is characterized in that: immune animal is male new zealand white rabbit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021478767A CN1176378C (en) | 2002-12-19 | 2002-12-19 | Test-paper testing method for diosgenin content |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021478767A CN1176378C (en) | 2002-12-19 | 2002-12-19 | Test-paper testing method for diosgenin content |
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| Publication Number | Publication Date |
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| CN1419126A CN1419126A (en) | 2003-05-21 |
| CN1176378C true CN1176378C (en) | 2004-11-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB021478767A Expired - Fee Related CN1176378C (en) | 2002-12-19 | 2002-12-19 | Test-paper testing method for diosgenin content |
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| US20170362631A1 (en) * | 2014-12-16 | 2017-12-21 | bioMérieux | Method and device for determining the presence of a micro-organism in stools with activated carbon pretreatment |
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