CN105717209B - The quantitative detecting method of the extraction of carotenoid component and its content in Pepper Leaves - Google Patents
The quantitative detecting method of the extraction of carotenoid component and its content in Pepper Leaves Download PDFInfo
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- CN105717209B CN105717209B CN201610064004.5A CN201610064004A CN105717209B CN 105717209 B CN105717209 B CN 105717209B CN 201610064004 A CN201610064004 A CN 201610064004A CN 105717209 B CN105717209 B CN 105717209B
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Abstract
The present invention provides the extracting method of carotenoid component in Pepper Leaves, and step is as follows：（1）Liquid nitrogen is added in fresh chilli blade to be fully ground to powdered；（2）Saponification：The KOH methanol saponification liquors that mass concentration is 15 30% are added in Pepper Leaves, in 40 80 DEG C of 60min of saponification 20；（3）Extract：In step（2）The mixing Extraction solvent that acetone ethyl acetate is added in the Pepper Leaves of the saponification of preparation carries out ultrasonic extraction, obtains carotenoid crude extract；（4）By step（3）Obtained carotenoid crude extract is post-processed, and obtains the solution containing carotenoid.The present invention also provides the high-efficiency liquid chromatography method for detecting of carotenoids component, can separate 10 Carotenoids in Pepper Leaves well, and be capable of the content of the Carotenoids of Simultaneous Determination 10.
Description
Technical field
The present invention relates to the extraction of ten Carotenoids components in Pepper Leaves and its RPLC content
Quantitative detecting method.
Background technology
Capsicum (Capsicum annuum. L.), Solanaceae Capsicum annual herb plant, China before more than 400 years just
There is cultivation to record, up to tens kinds of current cultivar has plantation in China, is also that northern area winter-spring season is set
The staple vegetable kind of cultivation is applied, northern area winter-spring season facility cultivation is mainly influenceed by low temperature and poor light, and then influenceed
Yield of hot pepper and quality, filtering out weak light resistance and the strong capsicum variety of tolerance to low temperature and weak light can tackle the problem at its root.
Carotenoid is the class natural pigment being widely present in nature, and 26S Proteasome Structure and Function is varied, class Hu trailing plants
Structure special Bu Su imparts their very special properties, and these properties determine its complicated biological function.Class Hu trailing plants
Bu Su can press down cancer health care, and enhancing is immune；Research is it is also shown that carotenoid is except being that plant carries out photosynthetic important auxiliary
Outside pigment, Photosynthetic is also protected to exempt from photo damage and the function such as anti-oxidant.A word used in person's names is built the research such as bright and shown, total in Pepper Leaves
The content of carotenoid has good dependency relation with kind weak light resistance and tolerance to low temperature and weak light, but determines capsicum product
The major carotenoids component for planting patience is still not clear.
Phytoene (Phytoene), lycopene (Lycopene), alpha-carotene (α-carotene), leaf are yellow
Plain (Lutein), epoxy lutein (Lutein epoxide), beta carotene (β-carotene), luteole
(Zeaxanthin), single antheraxanthin (Antheraxanthin), neoxathin (Neoxanthin), violaxanthin
Etc. (Violaxanthin) it is photosynthetic pigments important in plant, verifies decision capsicum variety weak light resistance and tolerance to low temperature and weak light
The major carotenoids component of property, both can illustrate capsicum variety by molecular biology and genetic engineering from molecular level
Tolerance mechanism, can provide foundation for screening tolerance to low temperature and weak light variety and germplasm again, have to pepper breeding from now on great
Directive significance.
In the prior art, by Extraction solvent of petroleum ether, saponification temperature be 80 DEG C, saponification time be 40min, saponification liquor matter
Amount concentration is that 20%, saponification liquor volume is that 10mL, solid-liquid ratio are 1：5, can separate Pepper Leaves Lutein, luteole and
Beta carotene；Also set up in the prior art using 10mL80% acetone as Extraction solvent, it is molten with 6mL 200g/L KOH- methanol
Liquid is saponification liquor, and 50 DEG C of saponification 30min are the extraction process of Saponification Conditions；With C18 (250mm × 4.6 mm, 5 μm) chromatogram
Post, mobile phase is A acetonitriles, B ethyl acetate, C water, and wavelength 440nm, flow velocity 1mL/min, 35 DEG C of column temperature are chromatographic test strip part
High-efficiency liquid chromatography method for detecting；It is also unfavorable but these key technologies cannot be used for separating while a variety of carotenoids components
In the detection after separation.It can set up in the prior art while separating a variety of carotenoid components and its content assaying method
Report is less.The ultra performance liquid chromatography assay method of carotenoid content in wheat, this method energy are provided in the prior art
It is enough thoroughly to separate the lutein in wheat with zeaxanthin, but this method is only limitted to lutein, zeaxanthin, β-Hu Luo
Bu Su, alpha-carotene, the extraction of 4 Carotenoids contents and measure；A kind of strawberry carotenoid is also provided in the prior art
The content assaying method of composition, make use of ultra-performance liquid chromatography simultaneously to strawberry fruit Lutein, β-carrotene, β-
Kryptoxanthin and the Carotenoids assay of lycopene 4, but limitation is strong.Currently with RPLC
The research that method determines carotenoid is also seldom, and none of these methods is applied to all examination materials, and capsicum leaf in existing research
The separation of a variety of carotenoid components and its current document report of content assaying method in piece there is not yet, more ununified row
Industry bioassay standard.Therefore, setting up one kind can be while separates a variety of carotenoid in Pepper Leaves, and carotenoid is a variety of
Constituent content is measured, so that a kind of detection method is provided for the research of capsicum variety patience, it is significant.
The content of the invention
, can the invention provides the quantitative detecting method of the extraction of carotenoid component and its content in Pepper Leaves
Ten Carotenoids in Pepper Leaves are extracted simultaneously, can be by Pepper Leaves using reversed-phased high performace liquid chromatographic
Separation and content quantitative are determined ten Carotenoids components well.
The present invention provides the extracting method of carotenoid component in Pepper Leaves, and step is as follows：
（1）Liquid nitrogen grinding：Liquid nitrogen is added in fresh chilli blade to be fully ground to powdered；Preferably, in grinding
Before be additionally added antioxidant DBPC 2,6 ditertiary butyl p cresol；
（2）By Pepper Leaves saponification：In step（1）The KOH- methanol saponification liquors that mass concentration is 15-30% are added, in 40-
80 DEG C of saponification 20-60min；Preferably, the mass concentration of the KOH- methanol saponification liquor is 20%；
（3）Extract：In step（2）The mixing that acetone and ethyl acetate is added in the Pepper Leaves of the saponification of preparation extracts molten
Agent carries out ultrasonic extraction, obtains carotenoid crude extract；The Pepper Leaves are 1g with the ratio for mixing Extraction solvent:(2-8)
ml；The volume ratio of acetone and ethyl acetate is 1 in the mixing Extraction solvent:2；
（4）By step（3）Obtained carotenoid crude extract is post-processed, and obtains the solution containing carotenoid.
Preferably, step（2）Described in the consumption ratio of Pepper Leaves and KOH- methanol solutions be 1g：（2-6）ml.
As further preferred, the consumption ratio of the Pepper Leaves and KOH- methanol solutions is preferably 1g：4ml.
Preferably, step（2）Described in Saponification Conditions be：55 DEG C of saponification 30min.
Preferably, step（3）Described in Pepper Leaves with mix Extraction solvent ratio be 1g:8ml.
Preferably, step（3）Described in ultrasonic extraction time be 10-50min, ultrasonic temperature be 30 DEG C.
Preferably, the time of the ultrasonic extraction is 40min.
Preferably, step（4）Described in post processing be that will centrifuge 15min at 4 DEG C of carotenoid crude extract, take supernatant
Liquid, 45 DEG C of rotary evaporations of supernatant is concentrated near dry.
Above-mentioned each optimum condition is for obtaining the optimal conditions of Extraction of carotenoid pigment in Pepper Leaves, is beneficial into one
Step improves purity and recovery rate.
The present invention also provides the quantitative detecting method of carotenoid content in Pepper Leaves, and step is as follows：
（1）Carotenoid in Pepper Leaves is extracted using any described methods of claim 1-7；
（2）The obtained solution containing carotenoid is settled to 10mL with acetone, 0.22 μm of organic filter membrane is crossed, obtains
Pepper Leaves carotenoid testing sample；The acetone is chromatographically pure；
（3）High performance liquid chromatography test is carried out to testing sample according to following chromatographic conditions：Chromatographic column is Welch
Ultimate C30 performance liquid chromatographic columns, specification is 250mm × 4.6mm, a diameter of 5 μm of filler；The column flow rate of mobile phase
1.0mL/min, 30 DEG C of column temperature, Detection wavelength is 450nm；
Mobile phase composition is：A：Acetonitrile, B：Water, C：The mixed liquor of methyl tertiary butyl ether(MTBE) and methanol, wherein, methyl tertbutyl
Ether is 1 with methanol volume ratio:1, D：Ethyl acetate；
Eluent gradient elution program is：0min~6min：B increases linearly to 10%, C by 0 and keeps 0, D linearly increasing by 0
To 10%；6min~12min：B increases linearly to 15%, C by 10% and keeps 0, D to increase linearly to 20% by 10%；12min~
20min：B keeps 0, D by 20% linear decrease to 10% by 15% linear decrease to 0, C；20min~22min：B is linearly increasing by 0
40%, D is increased linearly to by 10% linear decrease to 5% by 0 to 5%, C；22min~30min：B by 5% linear decrease to 0, C by
40% increases linearly to 50%, D increases linearly to 20% by 5%；30min~35min：A increases linearly to 100%；
（4）According to the retention time of carotenoid component in each single standard solution, Pepper Leaves carotenoids are determined
The retention time of 10 Carotenoids in plain testing sample, further determines that the peak area of various carotenoid components, will be each
The peak area of Carotenoids component is substituted into corresponding equation of linear regression respectively, obtains respective components in Pepper Leaves
The content of carotenoid.
Preferably, the retention time of carotenoid component is respectively in the single standard solution：Octahydro tomato red
Plain retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min, leaf are yellow
Plain retention time 19.183min, epoxy lutein retention time 25.026min, beta carotene retention time 36.067min, jade
Cream-coloured element retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin retention time
10.473min when 9.189min, violaxanthin retain.
Preferably, the acquisition methods of the equation of linear regression of the various carotenoid components are：
（1）Prepare the carotenoid standard sample of series concentration；
（2）According to step in claim 8（3）Shown chromatographic condition carries out high performance liquid chromatography test；
（3）According to the retention time of carotenoid component in each single standard solution, series concentration carotenoids are determined
The retention time of carotenoid each component in plain standard sample, using each component various concentrations as abscissa, corresponding peak area is
Ordinate, draws the equation of linear regression of carotenoid each component respectively.
Beneficial effects of the present invention are：
（1）The extracting method of the present invention can separate 10 Carotenoids in Pepper Leaves well, using the present invention
Reversed-phase liquid chromatography detection method be capable of the contents of the Carotenoids of Simultaneous Determination 10.
（2）The methodological science of the present invention is feasible, phytoene, lycopene, alpha-carotene, lutein, epoxy leaf
Flavine, beta carotene, luteole, single antheraxanthin, neoxathin, the content and chromatogram of the Carotenoids of violaxanthin 10
Detect that response peak area linear relation is good, 10 Carotenoids are thoroughly separated, and compartment between every kind of material peak and peak
Every larger, the carotenoid component differentiated in sample is facilitated.
（3）The present invention determines the condition that ten Carotenoids are determined, phytoene by high performance liquid chromatograph
Retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min, lutein
Retention time 19.183min, epoxy lutein retention time 25.026min, beta carotene retention time 36.067min, corn
Flavine retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin retention time 9.189min,
10.473min when violaxanthin retains.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, the reality with the present invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the chromatogram of carotenoid each component in standard sample；Wherein, 1- neoxathins, 2- violaxanthins, 3- is monocyclic
Oxygen luteole, 4- lutein, 5- luteoles, 6- epoxy lutein, 7- lycopenes, 8- phytoenes, 9- α-Hu
Radish element, 10- beta carotenes；
Fig. 2 is the chromatogram of carotenoid each component in Pepper Leaves；Wherein, 1- neoxathins, 2- violaxanthins, 3- is monocyclic
Oxygen luteole, 4- lutein, 5- luteoles, 6- epoxy lutein, 7- lycopenes, 8- phytoenes, 9- α-Hu
Radish element, 10- beta carotenes；
Fig. 3 a are the standard curve of phytoene in standard sample, wherein, abscissa is quality（μg）, ordinate is
Peak area（×10-4）；
Fig. 3 b are the standard curve of lycopene in standard sample, wherein, abscissa is quality（μg）, ordinate is peak face
Product（×10-4）；
Fig. 3 c are the standard curve of alpha-carotene in standard sample, wherein, abscissa is quality（μg）, ordinate is peak
Area（×10-4）；
Fig. 3 d are the standard curve of standard sample Lutein, wherein, abscissa is quality（μg）, ordinate is peak area
（×10-4）；
Fig. 3 e are the standard curve of epoxy lutein in standard sample, wherein, abscissa is quality（μg）, ordinate is peak
Area（×10-4）；
Fig. 3 f are the standard curve of beta carotene in standard sample, wherein, abscissa is quality（μg）, ordinate is peak
Area（×10-4）；
Fig. 3 g are the standard curve of luteole in standard sample, wherein, abscissa is quality（μg）, ordinate is peak face
Product（×10-4）；
Fig. 3 h are the standard curve of single antheraxanthin in standard sample, wherein, abscissa is quality（μg）, ordinate
For peak area（×10-4）；
Fig. 3 i are the standard curve of neoxathin in standard sample, wherein, abscissa is quality（μg）, ordinate is peak area
（×10-4）；
Fig. 3 j are the standard curve of violaxanthin in standard sample, wherein, abscissa is quality（μg）, ordinate is peak area
（×10-4）.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Reagent and material used in following embodiments, unless otherwise specified,
Obtain from commercial channels.
Agents useful for same of the present invention：
Analysis is pure：Ethyl acetate, acetone, methanol, potassium hydroxide, DBPC 2,6 ditertiary butyl p cresol, methyl tertiary butyl ether(MTBE)；Color
Spectrum is pure：Methanol, acetonitrile, ethyl ester acetonitrile.
It is used in following embodiments：
Neoxathin Neoxanthin, violaxanthin Violaxanthin, luteole Zeaxanthin, lutein Lutein,
Beta carotene β-Carotene are purchased from ChromaDex companies of the U.S.；Phytoene Phytoene, lycopene
（Lycopene）, single antheraxanthin（Antheraxanthin）, epoxy lutein（Lutein epoxide）Purchased from Switzerland
CaroteNature；Alpha-carotene（α-Carotene）Purchased from Japanese Wako companies；Purity is more than 95%.
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 4-12mL mass concentrations are 15-30%, 40-80 DEG C of water bath with thermostatic control 20-60min is placed in after sealing, so
It is cooled to room temperature with cold water immediately afterwards；
3）Extract：By step 2）16ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:2-8）Acetone-acetic acid
Ethyl ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 10-50min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 45 DEG C of ± 2 DEG C of rotary evaporations, i.e.,
The solution containing carotenoid can be obtained.
Through experiment, it can be good at separating the carotenoid in Pepper Leaves, 10 Carotenoids using the above method
Thoroughly separated, separation spacing is larger between every kind of material peak and peak.
Embodiment 1
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 8mL mass concentrations are 20%, 55 DEG C of water bath with thermostatic control 30min are placed in after sealing, cold water is used immediately after
It is cooled to room temperature；
3）Extract：By step 2）16ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:8）Acetone-acetic acid second
Ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 40min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 45 DEG C of rotary evaporations, you can
To the solution containing carotenoid.
Embodiment 2
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 12mL mass concentrations are 15%, 80 DEG C of water bath with thermostatic control 20min are placed in after sealing, immediately after with cold
It is water-cooled to room temperature；
3）Extract：By step 2）4ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:2）Acetone-acetic acid second
Ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 10min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 43 DEG C of rotary evaporations, you can
To the solution containing carotenoid.
Embodiment 3
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 4mL mass concentrations are 30%, 40 DEG C of water bath with thermostatic control 60min are placed in after sealing, cold water is used immediately after
It is cooled to room temperature；
3）Extract：By step 2）12ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:6）Acetone-acetic acid second
Ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 50min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 47 DEG C of rotary evaporations, you can
To the solution containing carotenoid.
Embodiment 4
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 6mL mass concentrations are 25%, 50 DEG C of water bath with thermostatic control 50min are placed in after sealing, cold water is used immediately after
It is cooled to room temperature；
3）Extract：By step 2）8ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:4）Acetone-acetic acid second
Ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 20min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 44 DEG C of rotary evaporations, you can
To the solution containing carotenoid.
Embodiment 5
The extracting method of carotenoid is specific as follows in Pepper Leaves：
1）Ice bath is ground, and weighs the Pepper Leaves sample that 2.000g has been dispensed, and adds 0.010g quartz sands and 0.1000g is anti-
Liquid nitrogen is added in oxidant BHT (BHT), mortar quickly to grind, and is fully ground and is obtained Pepper Leaves powder
End；
2）Saponification：By step 1）Pepper Leaves powder after ice bath grinding is positioned in 50mL lucifuge brown reagent bottles, plus
Enter the KOH- methanol saponification liquors that 10mL mass concentrations are 18%, 70 DEG C of water bath with thermostatic control 30min are placed in after sealing, immediately after with cold
It is water-cooled to room temperature；
3）Extract：By step 2）10ml is added in Pepper Leaves after gained saponification（Solid-liquid ratio is 1:5）Acetone-acetic acid second
Ester（V：V=1：2）Mixing Extraction solvent, ultrasonic extraction 35min, ultrasonic temperature is 30 DEG C；
4）Centrifugation：By step 3）Gained crude extract is 4 DEG C on low-temperature and high-speed centrifuge, 8000r/min centrifugations 15min；
5）Rotary evaporation：By step 4）Gained supernatant on a rotary evaporator near do by 46 DEG C of rotary evaporations, you can
To the solution containing carotenoid.
The drafting of the Carotenoids standard curve of embodiment 6 10
A) preparation of single standard solution：
Phytoene, lycopene, alpha-carotene, lutein, epoxy lutein, β-carrot are accurately weighed respectively
Element, luteole, single antheraxanthin, neoxathin, violaxanthin standard items 1mg with the dissolving of chromatographically pure organic solvent and constant volume in
In 25mL brown volumetric flasks, preserved as single standard items storing solution, concentration is 40 μ g/mL.
And take 0.2 μ L to mix respectively, filtered, as hybrid standard product storing liquid, protected with 0.22 μm of micropore organic phase filter membrane
It is stored in -20 DEG C of refrigerators stand-by.
B) preparation of series concentration carotenoid standard sample
By step b）Prepare 10 Carotenoids standard solution, in chromatographically pure organic solvent, in be hybridly prepared into
Illumination and high temperature should be avoided in a series of carotenoid standard sample of concentration, process for preparation, is then entered by following chromatographic condition
Row high performance liquid chromatography is tested.
C) according to following chromatographic condition to step a）Single standard solution and step b）Series concentration carotenoid
Standard sample carries out high performance liquid chromatography (HPLC) test：The INSTRUMENT MODEL of high performance liquid chromatography is Waters 2695, detection
Device is Waters2487 UV-detectors, and Detection wavelength is 450nm；Chromatographic column is Welch Ultimate C30 high-efficient liquid phase colors
Post is composed, specification is 250mm × 4.6mm, a diameter of 5 μm of filler, the column flow rate 1.0mL/min of mobile phase, performance liquid chromatographic column
30 DEG C of column temperature；25 DEG C of sample room temperature, the μ L of sample size 20.
Mobile phase：Mobile phase composition A is acetonitrile, and B is water, and C is the mixed liquor of methyl tertiary butyl ether(MTBE) and methanol, wherein, first
Base tertbutyl ether：Methanol (V:V=1:1), D is ethyl acetate.
Eluent gradient elution program is as shown in table 1.
The eluent gradient of table 1 is eluted
In table 1：0min~6min：B increases linearly to 10%, C by 0 and keeps 0, D to increase linearly to 10% by 0；6min~
12min：B increases linearly to 15%, C by 10% and keeps 0, D to increase linearly to 20% by 10%；12min~20min：B is linear by 15%
It is decremented to 0, C and keeps 0, D by 20% linear decrease to 10%；20min~22min：It is linearly increasing by 0 that B increases linearly to 5%, C by 0
To 40%, D by 10% linear decrease to 5%；22min~30min：B increases linearly to 50%, D by 5% linear decrease to 0, C by 40%
20% is increased linearly to by 5%；30min~35min：A increases linearly to 100%.
e）The retention time of carotenoid component in each single standard solution is recorded respectively（As shown in figure 1, octahydro kind
Lycopene retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min,
Lutein retention time 19.183min, epoxy lutein retention time 25.026min, beta carotene retention time
36.067min, luteole retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin are protected
10.473min when staying time 9.189min, violaxanthin reservation）, and series concentration carotenoid mark is determined according to the retention time
Corresponding each component in quasi- sample, then determines phytoene under each concentration, lycopene, alpha-carotene, leaf yellow
Element, epoxy lutein, beta carotene, luteole, single antheraxanthin, neoxathin, the corresponding peak area of violaxanthin；And
Using each component various concentrations as abscissa, corresponding peak area is ordinate, and the linear of carotenoid each component is drawn respectively and is returned
Return equation.As a result referring to Fig. 3 and table 2.
Regression equation, the range of linearity and the coefficient correlation of the carotenoid each component of table 2
X- mass (x, μ g)；Y- peak areas (× 10-4)。
The assay method of carotenoid content in the capsicum of embodiment 7
Those skilled in the art know that the assay method of carotenoid content and the kind of capsicum are unrelated in capsicum, in order to
The method of the present invention is discussed in detail, the embodiment is illustrated by taking No. 5 capsicums of Gansu Province green pepper as an example.
First, the collection of Pepper Leaves：Test and carried out in November, 2013 in College of Horticulture of Gansu Agriculture University proving ground,
It is heliogreenhouse tolerance to low temperature and weak light kind for examination capsicum：Gansu Province green pepper No. 5, seedling medium is provided by College of Horticulture of Gansu Agriculture University,
The volume proportion of matrix is：Cow dung:Maize straw:Turf:Vermiculite=5:1:2:2, select color, grain type, uniform in size consistent
Pepper seed, 55 DEG C of 30min that hot water treatment of seeds stir to room temperature, are placed on vernalization in growth cabinet, and sowing is in 50 cave caves when showing money or valuables one carries unintentionally
In disk, Routine Management when pepper seedling length to 7-8 piece true leaves, takes 3-4 piece functional leafs, every part of fresh leaf 2.000g uses liquid
Nitrogen rapid freeze-drying, be stored in -80 DEG C it is stand-by；
2nd, in Pepper Leaves carotenoid extraction：It can be extracted well using the method for the present invention, herein,
Extracted using the method in embodiment 1；
3rd, the preparation of Pepper Leaves carotenoid testing sample：Contain carotenoid by what is obtained with chromatogram pure acetone
Solution be settled to 10mL, cross 0.22 μm of organic filter membrane, be contained in 2ml browns chromatography column feed materials bottle, be stored in -20 DEG C it is stand-by,
Produce Pepper Leaves carotenoid testing sample.
4th, with the step c of embodiment 6）Method to Pepper Leaves carotenoid testing sample carry out high efficiency chromatography test.
5th, according to the retention time of carotenoid component in each single standard solution, Pepper Leaves carotenoids are determined
The retention time of 10 Carotenoids in plain testing sample（As shown in Figure 2）, further determine that the peak of various carotenoid
Area, the peak area of various carotenoid is substituted into corresponding equation of linear regression respectively, obtains respective sets in Pepper Leaves
The content of classification carrotene.
With the method for the present invention, the actual content of 10 Carotenoids in Pepper Leaves is measured, takes 5 parts to weigh up
Sample, determine 5 parts of samples in 10 Carotenoids average contents, i.e., the actual content of 10 Carotenoids in sample.Knot
Fruit is as shown in table 3.
The present embodiment of table 3 surveys ten Carotenoids content in Pepper Leaves
The precision test of the inventive method of embodiment 8
Sample is measured with the RPLC detection method of the present invention, to carotenoids under the same terms
Plain extract solution carries out 5 replications, result of the test such as table 4, as shown in Table 4, neoxathin, violaxanthin, list antheraxanthin,
Luteole, epoxy lutein, lutein, lycopene, phytoene, alpha-carotene, the species of beta carotene 10 are recklessly
The response time of radish element is fluctuated within 0.1min, the standard deviation of response time is respectively 0.074,0.074,0.080,
0.050th, 0.087,0.035,0.034,0.055,0.030,0.007, show that the precision of the inventive method is very good, can be used for
The detection of 10 Carotenoids in Pepper Leaves.
The precision test result of carotenoid in the HPLC of table 4 detection Pepper Leaves
The determination of recovery rates of the inventive method of embodiment 9
Take 4 parts of samples, 1 part is control, is added without standard items, other 3 parts be separately added into basic, normal, high concentration standard items,
Chromatogram detection is carried out with the method for the present invention, and every part of sample introduction 5 times, the rate of recovery is that the content of mark-on sample subtracts and is not added with standard specimen product
Content is again divided by the amount of standard sample that adds is obtained, result of the test as shown in table 5, be separately added into low content, middle content,
The standard items of high content, calculate obtained sample recovery rate more than 80%, and 10 Carotenoids repeat to survey in sample
The standard deviation for determining result is respectively：Phytoene 1.21%, lycopene 0.97%, alpha-carotene 0.58%, lutein
1.20%, epoxy lutein 1.87%, beta carotene 1.72%, luteole 1.35%, single antheraxanthin 1.99%, neoxathin
1.02%, violaxanthin 1.81%.
The Recovery test result of carotenoid in the application HPLC detection method Pepper Leaves of table 5
Testing result reappearance is examined：5 parts of samples weighed up are taken, the RPLC set up according to the present invention is examined
Survey method is measured to carotenoid in Pepper Leaves, and the reappearance to detection method is tested, result of the test
Show, neoxathin, violaxanthin, luteole, lutein, beta carotene, epoxy lutein, monocyclic oxygen corn in Pepper Leaves
Flavine, lycopene, phytoene, the Carotenoids measurement result standard deviation of alpha-carotene 10 are in 2% scope
It is interior, meet the requirement of chromatographic detection method.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (12)
1. the quantitative detecting method of carotenoid content in Pepper Leaves, it is characterised in that：Step is as follows：
（1）Extract carotenoid in Pepper Leaves；The extracting method is：
A, liquid nitrogen grinding：Liquid nitrogen is added in fresh chilli blade to be fully ground to powdered；
B, saponification：The KOH- methanol saponification liquors that mass concentration is 15-30% are added in step, in 40-80 DEG C of saponification 20-
60min；
C, extraction：The mixing Extraction solvent that acetone and ethyl acetate is added in the Pepper Leaves of the step B saponification prepared is surpassed
Sound is extracted, and obtains carotenoid crude extract；The Pepper Leaves are 1g with the ratio for mixing Extraction solvent:(2-8)ml；It is described
The volume ratio for mixing acetone and ethyl acetate in Extraction solvent is 1:2；
D, the carotenoid crude extract that step C is obtained post-processed, obtain the solution containing carotenoid；
（2）The obtained solution containing carotenoid is settled to 10mL with acetone, 0.22 μm of organic filter membrane is crossed, obtains capsicum
Carotenoid in Leaves testing sample；The acetone is chromatographically pure；
（3）High performance liquid chromatography test is carried out to testing sample according to following chromatographic conditions：Chromatographic column is Welch Ultimate
C30 performance liquid chromatographic columns, specification is 250mm × 4.6mm, a diameter of 5 μm of filler；The column flow rate 1.0mL/min of mobile phase, post
30 DEG C of temperature, Detection wavelength is 450nm；
Mobile phase composition is：A：Acetonitrile, B：Water, C：The mixed liquor of methyl tertiary butyl ether(MTBE) and methanol, wherein, methyl tertiary butyl ether(MTBE) with
Methanol volume ratio is 1:1, D：Ethyl acetate；
Eluent gradient elution program is：0min~6min：B increases linearly to 10%, C by 0 and keeps 0, D to be increased linearly to by 0
10%；6min~12min：B increases linearly to 15%, C by 10% and keeps 0, D to increase linearly to 20% by 10%；12min~20min：B
0, D is kept by 20% linear decrease to 10% by 15% linear decrease to 0, C；20min~22min：B by 0 increase linearly to 5%, C by
0 increases linearly to 40%, D by 10% linear decrease to 5%；22min~30min：B is by 5% linear decrease to 0, C by 40% linear increasing
It is added to 50%, D and increases linearly to 20% by 5%；30min~35min：A increases linearly to 100%；
（4）According to the retention time of carotenoid component in each single standard solution, determine that Pepper Leaves carotenoid is treated
The retention time of 10 Carotenoids in test sample product, further determines that the peak area of various carotenoid components, will be various types of
The peak area of carrotene component is substituted into corresponding equation of linear regression respectively, obtains the class of respective components in Pepper Leaves recklessly
The content of radish element.
2. according to the method described in claim 1, it is characterised in that：The step of the method for carotenoid in Pepper Leaves are extracted
In rapid A, antioxidant BHT is additionally added before the milling.
3. according to the method described in claim 1, it is characterised in that：The step of the method for carotenoid in Pepper Leaves are extracted
In rapid B, the mass concentration of the KOH- methanol saponification liquor is 20%.
4. according to the method described in claim 1, it is characterised in that：Step（2）In, the Pepper Leaves and KOH- methanol solutions
Consumption ratio be 1g：（2-6）ml.
5. method according to claim 4, it is characterised in that：Step（2）In, the Pepper Leaves and KOH- methanol solutions
Consumption ratio be 1g：4ml.
6. according to the method described in claim 1, it is characterised in that：Step（2）Described in Saponification Conditions be：55 DEG C of saponification
30min。
7. according to the method described in claim 1, it is characterised in that：Step（3）Described in Pepper Leaves with mixing Extraction solvent
Ratio be 1g:8ml.
8. according to the method described in claim 1, it is characterised in that：Step（3）Described in ultrasonic extraction time be 10-
50min, ultrasonic temperature is 30 DEG C.
9. method according to claim 8, it is characterised in that：The time of the ultrasonic extraction is 40min.
10. according to the method described in claim 1, it is characterised in that：Step（4）Described in post processing be by carotenoid it is thick
15min is centrifuged at 4 DEG C of extract, supernatant is taken, 45 DEG C of rotary evaporations of supernatant are concentrated near dry.
11. the method according to claim any one of 1-10, it is characterised in that：Class Hu trailing plants in the single standard solution
The retention time of Bu Su components is respectively：Phytoene retention time 33.168min, lycopene retention time
When 29.231min, alpha-carotene retention time 34.971min, lutein retention time 19.183min, epoxy lutein retain
Between 25.026min, beta carotene retention time 36.067min, luteole retention time 20.398min, monocyclic oxygen maize
10.473min when plain retention time 17.950min, neoxathin retention time 9.189min, violaxanthin retain.
12. the method according to claim any one of 1-10, it is characterised in that：The line of the various carotenoid components
The acquisition methods of property regression equation are：
（1）Prepare the carotenoid standard sample of series concentration；
（2）According to step in claim 1（3）Shown chromatographic condition carries out high performance liquid chromatography test；
（3）According to the retention time of carotenoid component in each single standard solution, series concentration carotenoid mark is determined
The retention time of carotenoid each component in quasi- sample, using each component various concentrations as abscissa, corresponding peak area is vertical seat
Mark, draws the equation of linear regression of carotenoid each component respectively.
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| CN106631953B (en) * | 2016-12-29 | 2019-01-22 | 河南中大恒源生物科技股份有限公司 | The preparation method of beta carotene in a kind of capsicum |
| CN107144651B (en) * | 2017-05-05 | 2019-06-25 | 华中农业大学 | The Structural Identification method of Arctic Sea fuchsin coccus B7740 production isoprenoid |
| CN108008036A (en) * | 2017-11-23 | 2018-05-08 | 广州市辉乐医药科技有限公司 | A kind of method for measuring Flos Tagetis Erectae extract Lutein content |
| CN108680683B (en) * | 2018-04-17 | 2020-12-15 | 中国农业科学院郑州果树研究所 | Mobile phase formula for detecting carotenoid in fruits by liquid chromatography and HPLC (high performance liquid chromatography) method for detecting carotenoid in fruits |
| CN111141840B (en) | 2019-12-18 | 2021-06-29 | 西安交通大学 | Method for simultaneously measuring fat-soluble vitamins and carotenoids in serum |
| CN112557544B (en) * | 2020-12-09 | 2023-06-30 | 宁夏农林科学院枸杞科学研究所 | Detection method of carotenoid metabolites of wolfberry |
| CN112964797A (en) * | 2021-02-05 | 2021-06-15 | 石河子大学 | Method for extracting carotenoid components in pepper fruits and measuring content of carotenoid components |
| CN113588577A (en) * | 2021-07-30 | 2021-11-02 | 河北东之星生物科技股份有限公司 | Method for detecting content of lutein in chrysanthemum granules |
| CN115078567B (en) * | 2022-05-19 | 2023-10-24 | 江苏艾兰得营养品有限公司 | Method for determining lutein ester and zeaxanthin ester |
| CN115078590B (en) * | 2022-07-16 | 2023-08-04 | 福建省产品质量检验研究院(福建省缺陷产品召回技术中心) | Detection method for measuring 8 carotenoid types by two-dimensional liquid chromatography |
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