CN104792890B - The method of sesamin content in mensuration sesame seed - Google Patents
The method of sesamin content in mensuration sesame seed Download PDFInfo
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- CN104792890B CN104792890B CN201510132312.2A CN201510132312A CN104792890B CN 104792890 B CN104792890 B CN 104792890B CN 201510132312 A CN201510132312 A CN 201510132312A CN 104792890 B CN104792890 B CN 104792890B
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Abstract
The invention discloses a kind of can quickly, the method for sesamin content in Accurate Determining sesame seed, comprise the following steps: that (1) prepares sesamin standard solution；(2) sesamin standard curve is drawn；(3) preparation testing sample solution；(4) supersound process and filtration；(5) detection.Testing result of the present invention more accurately, stable, it is possible to for quickly detection Semen Sesami quality, it is simple to be that Semen Sesami carries out grade classification according to sesamin content；Also being able to provide theoretical reference into the sesamin in extraction sesame seed quick, efficient, extensive, for improving the extraction ratio of sesamin in sesamin production field, shortening extraction time has great significance simultaneously.
Description
Technical field
The present invention relates to sesamin assay technical field, be specifically related to a kind of measure the method for sesamin content in sesame seed.
Background technology
Semen Sesami (Sesame) is one of important oil crop of China, and the Semen Sesami per mu yield of current China reaches 92kg.Sesamin (Sesamin) belongs to wood lipid, and the content in Oleum sesami is 0.4%～0.8%；There is removing toxic substances, resisting hypertension, reduce cholesterol absorption, the liver protecting, regulating lipid metabolism, the multiple physiologically active such as anticancer and regulation immunity.In the planted area of China extensively, the Semen Sesami quality that different regions produce is uneven, and its sesamin content has the biggest difference for Semen Sesami.The different indexs of national standard foundation are by Semen Sesami divided rank, but do not specify the content of sesamin.Different grades of Semen Sesami price variance is big, and in view of the high added value of sesamin, etalon sesamin content being listed in division Semen Sesami grade is of practical significance.It is thus desirable to a kind of method setting up simple, fast mensuration sesamin content.
At present, the mensuration of sesamin content is mainly used chromatography, such as thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC) etc., but the detection object of these methods is all Oleum sesami, and before detection, need Oleum sesami is carried out the pretreatment of complexity, complex steps during detection, required time are long, and Oleum sesami sample and solvent-oil ratio are big, and testing result error is bigger.A kind of new technique for extracting sesamin is disclosed in Chinese patent literature CN101092421B, it it is extracting directly sesamin from sesame seed, eliminate oil press process, but the method is primarily directed to sesamin production field, it still has process complexity, the shortcoming of required time length, is not particularly suited for quick, accurate, quantitative analysis detection.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of quickly, the method for sesamin content in Accurate Determining sesame seed.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that
Design and a kind of measure the method for sesamin content in sesame seed, comprise the following steps:
(1) preparation sesamin standard solution: weigh sesamin standard substance and add absolute methanol dissolving, be configured to 0.2mg/
The sesamin standard solution of mL；
(2) sesamin standard curve is drawn: take the sesamin standard solution of step (1) gained, the absolute methanol being separately added into corresponding amount is configured to the sesamin standard substance methanol solution of gradient concentration, chromatograph of liquid is used respectively above-mentioned solution to be carried out chromatography, draw out sesamin standard working curve according to measurement result, or matching draws the linear relationship equation of sesamin content and peak area；
(3) preparation testing sample solution: sesame seed to be measured is pulverized, and cross the sieve of 0.35 μm, the black sesame powder sample then taking gained adds, by the solid-to-liquid ratio of 0.1～0.4g:5～10mL, methanol or the ethanol that volumetric concentration is 80%～100%；
(4) supersound process and filtration: the mixed solution of step (3) gained is carried out ultrasonic Treatment 20～50min under conditions of 30～60 DEG C, then filters with the filter membrane of 0.45 μm；
(5) detection: take the filtrate 9 μ l of step (4) gained, use chromatograph of liquid that this filtrate is carried out sample detection, search according to sesamin standard working curve and draw the content of sesamin in sesame seed to be measured, or draw the content of sesamin in sesame seed to be measured according to gained linear relationship Equation for Calculating.
Preferably, the gradient concentration of sesamin standard substance methanol solution described in step (2) be respectively 200,100,50,40,20,8ug/mL.
Linear relationship equation described in step (2) is preferably:
A=587.525675x-4.531582, wherein A is peak area, and x is sesamin content.
Chromatograph of liquid testing conditions in step (2) and step (5) is preferably:
Chromatographic column: Agilent L1260, Eclipse
XDB-C18-USP L1 analytical column, specification (mm): 4.6*250, particle diameter: 5um；Flowing phase: methanol: water=80:20(V/V)；Flow velocity: 1mL/min；Detection wavelength: 287nm；Sampling volume: 9ul；Retention time: 15min.
In step (4), the condition of ultrasonic Treatment is preferably: using supersonic frequency is the ultrasonic cleaner of 40KHZ, carries out supersound process under the ultrasonic power of 70～100W.
Preferably, using oscillator that this mixed solution is carried out concussion process in step (4) while carrying out supersound process, the oscillator power of use is 70～100W.
Preferably, before filtering in step (4), first it is centrifuged solution processing: be centrifuged processing 2min under conditions of 5000rpm.
The beneficial effects of the present invention is:
1. can directly survey, measure the sesamin content in sesame seed rapidly, it is not necessary to first sesame seed be squeezed into Oleum sesami and detect again, eliminate the operation of oil expression；The preprocessing process complicated also without carrying out step before detection, greatly simplify step and operation that sesamin measures, detection required time is greatly shortened.
2. the various process parameters of optimized choice sample treatment and the conditional parameter of detection so that testing result more accurately, stable, improve sensitivity, it is possible to for quickly detection Semen Sesami quality, it is simple to be Semen Sesami carries out grade classification according to sesamin content；Also being able to provide reference into the sesamin in extraction sesame seed quick, efficient, extensive, for improving the extraction ratio of sesamin in sesamin production field, shortening extraction time has great significance simultaneously.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of sesamin standard solution；
Fig. 2 is the various extracting conditions influence curve figure to example sesame element extracted amount；
Fig. 3 is the ultrasonic position influence curve figure to example sesame element extracted amount；
Fig. 4 is the sonication treatment time influence curve figure to example sesame element extracted amount；
Fig. 5 is the supersound process temperature influence curve figure to example sesame element extracted amount；
Fig. 6 is the centrifuge speed influence curve figure to example sesame element extracted amount；
Fig. 7 is the solid-liquid ratio influence curve figure to example sesame element extracted amount.
Detailed description of the invention
The detailed description of the invention of the present invention is described with embodiment below in conjunction with the accompanying drawings, but following example are used only to describe the present invention in detail, and limit the scope of the present invention never in any form.
Embodiment 1: a kind of measure the method for sesamin content in sesame seed, comprises the following steps:
(1) preparation sesamin standard solution: weigh sesamin standard substance 10mg, adds absolute methanol and dissolves and be diluted to 50mL, then filters with the filter membrane of 0.45 μm, obtains the sesamin standard solution of 0.2mg/ mL；
(2) sesamin standard curve is drawn: take the sesamin standard solution 20 of step (1) gained, 10, 5, 4, 2, 0.8mL, it is separately added into absolute methanol 0, 10, 15, 16, 18, 19.2mL, shake up and be configured to gradient concentration and be respectively 200, 100, 50, 40, 20, the sesamin standard substance methanol solution of 8ug/mL, chromatograph of liquid is used respectively above-mentioned solution to be carried out chromatography, sesamin standard curve (liquid chromatogram of sesamin standard solution sees Fig. 1) is drawn according to measurement result, and obtain the linear relationship equation of sesamin content and peak area: A=587.525675x-4.531582, wherein A is peak area, x is sesamin content；
(3) preparation testing sample solution: using Henan No. 11 sesame seeds of sesame is testing sample, and sesame seed Universalpulverizer to be measured is pulverized 30s, then by the sieve of 0.35 μm, takes the black sesame powder sample 0.2g of gained, adds the ethanol 5mL that concentration is 100%；
(4) supersound process and filtration: the mixed solution of step (3) gained is carried out under conditions of 30～60 DEG C ultrasonic Treatment 20～50min, then with the filter membrane of 0.45 μm, the solution after supersound process is filtered；
The condition of ultrasonic Treatment is: use supersonic frequency is 40KHZ, model is the numerical control ultrasonic cleaner of KQ3200DE, carries out supersound process under the ultrasonic power of 70～100W.
(5) detection: take the filtrate 9 μ l of step (4) gained, uses chromatograph of liquid that this filtrate is carried out sample detection, draws the content of sesamin in sesame seed to be measured according to sesamin standard curve, gained linear relationship Equation for Calculating.
Wherein, the chromatograph of liquid testing conditions in step (2) and step (5) is:
Chromatographic column: Agilent L1260, Eclipse
XDB-C18-USP L1 analytical column, specification (mm): 4.6*250, particle diameter: 5um；Flowing phase: methanol: water=80:20(V/V)；Flow velocity: 1mL/min；Detection wavelength: 287nm；Sampling volume: 9ul；Retention time: 15min.
Embodiment 2: be with the difference of embodiment 1, in step (4), uses oscillator that this mixed solution is carried out concussion process while carrying out supersound process, the oscillator power of use is 70～100W；Before filtration, first it is centrifuged solution processing: be centrifuged processing 2min under conditions of 5000rpm.
Embodiment 3: be with the difference of embodiment 1, in step (3), the black sesame powder sample taken is 0.1g, join in black sesame powder sample be 10ml concentration be the methanol of 90%.
Instrument and equipment involved in the embodiment above if no special instructions, is routine instrument device；The involved raw material of industry if no special instructions, is commercially available regular industrial raw material.
The comprehensive test study of testing conditions: use the technical thought of the present invention, carries out substantial amounts of experimental analysis research at different conditions, analyzes the situation of change of sesamin extracted amount, and experimentation and analysis result are as follows:
1. concussion and the ultrasound condition impact on example sesame element extracted amount
In the case of other condition determinations are identical, in the step (4) of technical solution of the present invention, screening uses the concussion of ultrasonic Treatment, oscillator, ultrasonic Treatment+three kinds of modes of oscillator concussion to process mixed solution respectively.Result shows, after three kinds of modes process mixed solution 30min, ultrasonic Treatment, ultrasound wave+concussion processes the sesamin content that both modes are extracted, and is all remarkably higher than the sample that only concussion is the most ultrasonic, sees Fig. 2.Ultrasound wave is more conducive to extract example sesame cellulose content, and find that this processing mode does not destroy sesamin sample after detection, find that ultrasound wave is highly advantageous to the pretreatment of fluid sample, and can promote and accelerate the extraction process of sample, and it is avoided that the high temperature destruction to target component, and method is simple to operate, side-product is few, object is easily separated, can reach more preferably to extract result.
2. ultrasonic position, temperature and time and the ultrasonic power impact on example sesame element extracted amount
In the case of other experiment conditions are identical, measure the content of sesamin in sample by temperature, sonication treatment time and ultrasonic power when the most different ultrasonic position, supersound process, contrast different ultrasonic position, different temperatures, different time and the impacts on example sesame element extracted amount of the different ultrasonic power conditions.By the extracted amount of sesamin after comparing sample and carrying out supersound process on the upper left of ultrasound wave, lower-left, centre, upper right, position, 5, bottom right, see Fig. 3, although result shows that sesamin extracted amount is had a certain impact by ultrasonic position, but all it is not up to significant difference, in practical operation, can be suitable adjust sample position on ultrasound wave, with reach eliminate test error purpose.By comparing the impact on sesamin extracted amount of 5min, 10min, 20min, 30min, 40min, 50min ultrasonic time, see Fig. 4, result shows, in 5～30min ultrasonic range, sesamin extracted amount presents the trend being stepped up, in 30～50min ultrasonic range, sesamin extracted amount is relatively stable, not up to significant difference.By comparing 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, the impact on extracting sesamin content of 60 DEG C of ultrasonic temperature conditions, seeing Fig. 5, result shows, under 20 DEG C of ultrasound conditions, sesamin content is minimum；Under 30～60 DEG C of ultrasound conditions, example sesame cellulose content is without significant difference, but sample is under 60 DEG C of ultrasound conditions, and example sesame element extracted amount is the highest and the most stable.Relatively under the conditions of ultrasonic power is 50W, 70W, 80W, 100W, example sesame element extracted amount, in the range of result proves 70～100W, sesamin extracted amount is relatively stable, without significant difference.
3. the sample extracting solution type impact on example sesame element extracted amount
In the case of other experiment conditions are identical, the ethanol of Select to use variable concentrations and methanol configuration sample solution.The impact on example sesame element extracted amount of the methanol of relatively 100%, 90%, 80%, 70% concentration, result shows, compared with the methanol that concentration is 100%, 80%, 70%, concentration be 90% methanol extraction sesamin content the highest.The relatively ethanol of 70%, 80%, 85%, 90%, 95%, the 100% concentration impact on example sesame element extracted amount, result shows, relatively low to example sesame element extracted amount less than the ethanol of 80% concentration, the ethanol of 80% and 100% concentration is the highest to example sesame element extracted amount.
4. sample centrifugal speed and the time impact on sesamin extracted amount
In the case of other experiment conditions are identical, observe different centrifugal speed, the centrifugation time impact on example sesame element extracted amount.It is centrifuged sample solution respectively processing 2min under conditions of 5000 turns, 8000 turns, 10000 turns and 12000 turns, result shows, under different centrifugal speeds, sesamin extracted amount is without significant difference, and under conditions of 5000 turns, example sesame element extracted amount is of a relatively high, sees Fig. 6.Under conditions of 5000rpm, carrying out 2min, 4min, 6min, 8min centrifugal treating, result shows that under different centrifugal condition, sesamin extracted amount is without significant difference.
5. the different feed liquid ratio impact on sesamin extracted amount
By to sesamin extracted amount under the conditions of different feed liquid ratio, observing the impact of different feed liquid comparison sesamin extracted amount.Taking 0.1g black sesame powder sample, the methanol using 3ml, 5ml, 7ml and 10ml concentration to be 100% respectively is as extracting solution, and result shows that 10ml extracting solution is the most abundant to 0.1g sample extraction, sees Fig. 7.
Consider above each factor, and combine substantial amounts of verification experimental verification, obtain the preferably embodiment of technical solution of the present invention and correspondence.
Above in conjunction with drawings and Examples, the present invention is described in detail, but, person of ordinary skill in the field it will be appreciated that, on the premise of without departing from present inventive concept, each design parameter in above-described embodiment can also be changed, form multiple specific embodiment, be the common excursion of the present invention, describe in detail the most one by one at this.
Claims (4)
1. one kind measures the method for sesamin content in sesame seed, it is characterised in that comprise the following steps:
(1) preparation sesamin standard solution: weigh sesamin standard substance and add absolute methanol dissolving, be configured to the sesamin standard solution of 0.2mg/ mL；
(2) sesamin standard curve is drawn: take the sesamin standard solution of step (1) gained, the absolute methanol being separately added into corresponding amount is configured to the sesamin standard substance methanol solution of gradient concentration, chromatograph of liquid is used respectively above-mentioned solution to be carried out chromatography, draw out sesamin standard working curve according to measurement result, or matching draws the linear relationship equation of sesamin content and peak area；
Wherein, the gradient concentration of described sesamin standard substance methanol solution be respectively 200,100,50,40,20,8ug/mL；
Described linear relationship equation is: A=587.525675x-4.531582, and wherein A is peak area, and x is sesamin content；
(3) preparation testing sample solution: sesame seed to be measured is pulverized, and cross the sieve of 0.35 μm, the black sesame powder sample then taking gained adds, by the solid-to-liquid ratio of 0.1～0.4g:5～10mL, methanol or the ethanol that volumetric concentration is 80%～100%；
(4) supersound process and filtration: the mixed solution of step (3) gained is carried out ultrasonic Treatment 20～50min under conditions of 30～60 DEG C, then filters with the filter membrane of 0.45 μm；
(5) detection: take the filtrate 9 μ l of step (4) gained, use chromatograph of liquid that this filtrate is carried out sample detection, search according to sesamin standard working curve and draw the content of sesamin in sesame seed to be measured, or draw the content of sesamin in sesame seed to be measured according to gained linear relationship Equation for Calculating；
Chromatograph of liquid testing conditions in step (2) and step (5) is:
Chromatographic column: Agilent L1260, Eclipse XDB-C18-USP L1 analytical column, specification (mm): 4.6*250, particle diameter: 5um；Flowing phase: methanol: water=80:20(V/V)；Flow velocity: 1mL/min；Detection wavelength: 287nm；Sampling volume: 9ul；Retention time: 15min.
The method of sesamin content in mensuration sesame seed the most according to claim 1, it is characterized in that, in step (4), the condition of ultrasonic Treatment is: using supersonic frequency is the ultrasonic cleaner of 40KHZ, carries out supersound process under the ultrasonic power of 70～100W.
The method of sesamin content in mensuration sesame seed the most according to claim 1, it is characterised in that using oscillator that this mixed solution is carried out concussion process while carrying out supersound process in step (4), the oscillator power of use is 70～100W.
The method of sesamin content in mensuration sesame seed the most according to claim 1, it is characterised in that before filtering in step (4), is first centrifuged solution processing: be centrifuged processing 2min under conditions of 5000rpm.
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| CN1895396A (en) * | 2006-06-27 | 2007-01-17 | 天津中新药业集团股份有限公司达仁堂制药厂 | Chinese-medicinal preparation with Yin-nourishing, intestin-moistening and bowls-relaxing functions |
| CN103389342A (en) * | 2012-05-08 | 2013-11-13 | 上海良友（集团）有限公司 | Method for detecting where leached sesame oil is doped in pressed sesame oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1895396A (en) * | 2006-06-27 | 2007-01-17 | 天津中新药业集团股份有限公司达仁堂制药厂 | Chinese-medicinal preparation with Yin-nourishing, intestin-moistening and bowls-relaxing functions |
| CN103389342A (en) * | 2012-05-08 | 2013-11-13 | 上海良友（集团）有限公司 | Method for detecting where leached sesame oil is doped in pressed sesame oil |
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