CN105717209A - Method for extracting carotenoid components in pepper leaves and quantitatively detecting content of carotenoid components - Google Patents
Method for extracting carotenoid components in pepper leaves and quantitatively detecting content of carotenoid components Download PDFInfo
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Abstract
The invention provides a method for extracting carotenoid components in pepper leaves. The method includes the steps that 1, liquid nitrogen is added into the fresh pepper leaves, and the fresh pepper leaves are fully ground into powder; 2, a KOH-methanol saponifying solution with the mass concentration of 15-30% is added into the pepper leaves to carry out saponification for 20-60 min at the temperature of 40-80 DEG C; 3, an acetone-ethyl acetate mixed extracting solvent is added into the saponified pepper leaves prepared in the step 2 to conduct supersonic extraction to obtain a carotenoid crude extract; 4, the carotenoid crude extract obtained in the step 3 is subjected to aftertreatment to obtain a solution containing carotenoid. The invention further provides a high performance liquid chromatography detection method for the carotenoid components, 10 kinds of carotenoid in the pepper leaves can be separated well, and the content of the 10 kinds of carotenoid can be quantitatively measured at the same time.
Description
Technical field
The present invention relates to extraction and the quantitative detecting method of reversed-phase high-performance liquid chromatography content thereof of ten Carotenoids components in Pepper Leaves.
Background technology
Fructus Capsici (Capsicum annuum. L.), Solanaceae Capsicum annual herb plant, China just had cultivation record before more than 400 years, cultivar up to tens kinds at present, the most all having plantation, be also the vegetables kind of northern area winter-spring season facility cultivation, northern area winter-spring season facility cultivation is mainly affected by low temperature and poor light, and then affect yield of hot pepper and quality, filter 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, and the special structure of carotenoid imparts the character that they are the most special, and these character determine the biological function of its complexity.Carotenoid can press down cancer health care, strengthens immunity；Research is it is also shown that carotenoid is except being that in addition to plant carries out photosynthetic important accessory pigments, the most protected Photosynthetic exempts from the function such as photo damage and antioxidation.A word used in person's names is built research such as bright grade and is shown, in Pepper Leaves, the content of total carotinoid and kind weak light resistance and tolerance to low temperature and weak light have good dependency relation, but determine that the major carotenoids component of capsicum variety patience is still not clear.
nullPhytoene (Phytoene)、Lycopene (Lycopene)、Alpha-carotene (α-carotene)、Phylloxanthin (Lutein)、Epoxy phylloxanthin (Lutein epoxide)、Beta-carotene (β-carotene)、Zeaxanthin (Zeaxanthin)、Single antheraxanthin (Antheraxanthin)、Neoxathin (Neoxanthin)、Violaxanthin (Violaxanthin) etc. is photosynthetic pigments important in plant，Verify decision capsicum variety weak light resistance and the major carotenoids component of tolerance to low temperature and weak light，Both molecular biology and genetic engineering can have been passed through，The Tolerance mechanism of capsicum variety is illustrated from molecular level，Can be again that screening tolerance to low temperature and weak light variety and germplasm provides foundation，Pepper breeding from now on had great directive significance.
In prior art, with petroleum ether as Extraction solvent, saponification temperature be 80 DEG C, saponification time as 40min, saponification liquor mass concentration be 20%, saponification liquor volume as 10mL, solid-liquid ratio is as 1:5, it is possible to separate Pepper Leaves Lutein, zeaxanthin and beta-carotene；Having also set up with 10mL80% acetone as Extraction solvent in prior art, with the KOH-methanol solution of 6mL 200g/L as saponification liquor, 50 DEG C of saponification 30min are the extraction process of Saponification Conditions；With C18 (250mm × 4.6 mm, 5 μm)
Chromatographic column, flowing is A acetonitrile mutually, and B ethyl acetate, C water, wavelength 440nm, flow velocity 1mL/min, column temperature 35 DEG C is the high-efficiency liquid chromatography method for detecting of chromatographic test strip part；But these key technologies separate while cannot be used for multiple types Radix Dauci Sativae component, are also unfavorable for the detection after separating.Prior art can set up to concurrently separate the report of multiple types carotene component and content assaying method thereof less.Prior art provides the Ultra Performance Liquid Chromatography assay method of carotenoid content in Semen Tritici aestivi, phylloxanthin in Semen Tritici aestivi can thoroughly be separated by the method with cryptoxanthin, but the method is only limitted to phylloxanthin, cryptoxanthin, beta-carotene, alpha-carotene, the extraction of 4 Carotenoids content and mensuration；Prior art also provides for the content assaying method of a kind of Fructus Fragariae Ananssae carotenoid composition, make use of ultra-performance liquid chromatography simultaneously to strawberry fruit Lutein, β-carotene, beta-cryptoxanthin and lycopene 4 Carotenoids assay, but limitation is strong.Research currently with rp-hplc determination carotenoid is the most little, existing research does not have a kind of method be applicable to all of examination material, and multiple types carotene Component seperation and content assaying method current document report thereof there is not yet in Pepper Leaves, the most unified industry bioassay standard.Therefore, set up one and can concurrently separate multiple types carotene in Pepper Leaves, and carotenoid various ingredients content is measured, thus the research for capsicum variety patience provides a kind of detection method, significant.
Summary of the invention
The invention provides the extraction of carotenoid component in Pepper Leaves and the quantitative detecting method of content thereof, can extract ten Carotenoids in Pepper Leaves, ten Carotenoids components in Pepper Leaves can well be separated by application reversed phase high-performance liquid chromatography and content quantitative measures simultaneously.
The present invention provides the extracting method of carotenoid component in Pepper Leaves, and step is as follows:
(1) liquid nitrogen grinding: add liquid nitrogen in fresh chilli blade and be fully ground to powder；As preferably, it is additionally added antioxidant 2,6 ditertiary butyl p cresol before the milling；
(2) by Pepper Leaves saponification: add, in step (1), the KOH-methanol saponification liquor that mass concentration is 15-30%, in 40-80 DEG C of saponification 20-60min；As preferably, the mass concentration of described KOH-methanol saponification liquor is 20%；
(3) extract: the mixed extraction solvent adding acetone and ethyl acetate in the Pepper Leaves of the saponification prepared in step (2) carries out supersound extraction, obtains carotenoid crude extract；Described Pepper Leaves is 1g:(2-8 with the ratio of mixed extraction solvent) ml；In described mixed extraction solvent, acetone is 1:2 with the volume ratio of ethyl acetate；
(4) the carotenoid crude extract that step (3) obtains is carried out post processing, obtain the solution containing carotenoid.
As preferably, Pepper Leaves described in step (2) is 1g:(2-6 with the consumption ratio of KOH-methanol solution) ml.
As further preferably, described Pepper Leaves is preferably 1g:4ml with the consumption ratio of KOH-methanol solution.
As preferably, described in step (2), Saponification Conditions is: 55 DEG C of saponification 30min.
As preferably, Pepper Leaves described in step (3) is 1g:8ml with the ratio of mixed extraction solvent.
As preferably, the time of supersound extraction described in step (3) is 10-50min, and ultrasonic temperature is 30 DEG C.
As preferably, the time of described supersound extraction is 40min.
As preferably, post processing described in step (4) is by 15min centrifugal at carotenoid crude extract 4 DEG C, takes supernatant, is concentrated into by 45 DEG C of rotary evaporations of supernatant near dry.
Above-mentioned each optimum condition is for obtaining the optimal conditions of Extraction of carotenoid pigment in Pepper Leaves, is beneficial to improve further purity and extraction ratio.
The present invention also provides for the quantitative detecting method of carotenoid content in Pepper Leaves, and step is as follows:
(1) the arbitrary described method of application claim 1-7 extracts carotenoid in Pepper Leaves；
(2) with acetone, the solution containing carotenoid obtained is settled to 10mL, crosses the 0.22 organic filter membrane of μm, obtain Pepper Leaves carotenoid testing sample；Described acetone is chromatographically pure；
(3) according to following chromatographic condition, testing sample carried out high performance liquid chromatography test: chromatographic column is Welch Ultimate C30 performance liquid chromatographic column, and specification is 250mm × 4.6mm, a diameter of 5 μm of filler；The column flow rate 1.0mL/min of flowing phase, column temperature 30 DEG C, detection wavelength is 450nm；
Mobile phase composition is: A: acetonitrile, B: water, C: methyl tertiary butyl ether(MTBE) and the mixed liquor of methanol, and wherein, methyl tertiary butyl ether(MTBE) and methanol volume ratio are 1:1, D: ethyl acetate；
Eluent gradient elution program is: 0min～6min:B is increased linearly to 10% by 0, and C keeps 0, and D is increased linearly to 10% by 0；6min～12min:B is increased linearly to 15% by 10%, and C keeps 0, and D is increased linearly to 20% by 10%；12min～20min:B is kept 0 by 15% linear decrease to 0, C, and D is by 20% linear decrease to 10%；20min～22min:B is increased linearly to 5% by 0, and C is increased linearly to 40% by 0, and D is by 10% linear decrease to 5%；22min～30min:B is increased linearly to 50% by 5% linear decrease to 0, C by 40%, and D is increased 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 the retention time of 10 Carotenoids in Pepper Leaves carotenoid testing sample, further determine that the peak area of various types of carotene component, the peak area of various types of carotene component is substituted in corresponding equation of linear regression respectively, obtains the content of the carotenoid of respective components in Pepper Leaves.
As preferably, in described single standard solution the retention time of carotenoid component be respectively as follows: phytoene retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min, phylloxanthin retention time 19.183min, epoxy phylloxanthin retention time 25.026min, beta-carotene retention time 36.067min, zeaxanthin retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin retention time 9.189min, violaxanthin 10.473min when retaining.
As preferably, the acquisition methods of the equation of linear regression of described various types of carotene component is:
(1) the carotenoid standard sample of series concentration is prepared；
(2) high performance liquid chromatography test is carried out according to chromatographic condition shown in step in claim 8 (3)；
(3) according to the retention time of carotenoid component in each single standard solution, determine the retention time of each component of carotenoid in series concentration carotenoid standard sample, with each component variable concentrations as abscissa, corresponding peak area is vertical coordinate, draws the equation of linear regression of each component of carotenoid respectively.
The invention have the benefit that
(1) 10 Carotenoids in Pepper Leaves can well be separated by the extracting method of the present invention, and the reversed-phase liquid chromatography detection method of the application present invention can the content of Simultaneous Determination 10 Carotenoids.
(2) methodological science of the present invention is feasible, phytoene, lycopene, alpha-carotene, phylloxanthin, epoxy phylloxanthin, beta-carotene, zeaxanthin, list antheraxanthin, neoxathin, the content of violaxanthin 10 Carotenoids and chromatograph detection response peak area linear relation are good, 10 Carotenoids are thoroughly separated, and between every kind of material peak and peak, separation spacing is relatively big, facilitates the carotenoid component differentiated in sample.
(3) present invention determines the condition that ten Carotenoids measure, 10.473min when phytoene retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min, phylloxanthin retention time 19.183min, epoxy phylloxanthin retention time 25.026min, beta-carotene retention time 36.067min, zeaxanthin retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin retention time 9.189min, violaxanthin retain by high performance liquid chromatograph.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is the chromatogram of each component of carotenoid in standard sample；Wherein, 1-neoxathin, 2-violaxanthin, the mono-antheraxanthin of 3-, 4-phylloxanthin, 5-zeaxanthin, 6-epoxy phylloxanthin, 7-lycopene, 8-phytoene, 9-alpha-carotene, 10-beta-carotene；
Fig. 2 is the chromatogram of each component of carotenoid in Pepper Leaves；Wherein, 1-neoxathin, 2-violaxanthin, the mono-antheraxanthin of 3-, 4-phylloxanthin, 5-zeaxanthin, 6-epoxy phylloxanthin, 7-lycopene, 8-phytoene, 9-alpha-carotene, 10-beta-carotene；
Fig. 3 a is the standard curve of phytoene in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 b is the standard curve of lycopene in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 c is the standard curve of alpha-carotene in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 d is the standard curve of standard sample Lutein, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 e is the standard curve of epoxy phylloxanthin in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 f is the standard curve of beta-carotene in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 g is the standard curve of zeaxanthin in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 h is the standard curve of single antheraxanthin in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 i is the standard curve of neoxathin in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4)；
Fig. 3 j is the standard curve of violaxanthin in standard sample, and wherein, abscissa is quality (μ g), and vertical coordinate is peak area (× 10-4).
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Reagent used in following embodiment and material, if no special instructions, the most commercially obtain.
Agents useful for same of the present invention:
Analytical pure: ethyl acetate, acetone, methanol, potassium hydroxide, DBPC 2,6 ditertiary butyl p cresol, methyl tertiary butyl ether(MTBE)；Chromatographically pure: methanol, acetonitrile, ethyl ester acetonitrile.
Used by following embodiment:
Neoxathin Neoxanthin, violaxanthin Violaxanthin, zeaxanthin Zeaxanthin, phylloxanthin Lutein, beta-carotene β-Carotene are purchased from ChromaDex company of the U.S.；Phytoene Phytoene, lycopene (Lycopene), single antheraxanthin (Antheraxanthin), epoxy phylloxanthin (Lutein epoxide) are purchased from Switzerland CaroteNature；Alpha-carotene (α-Carotene) is purchased from Wako company of Japan；Purity is all more than 95%.
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, add the KOH-methanol saponification liquor that 4-12mL mass concentration is 15-30%, sealing is placed on 40-80 DEG C of water bath with thermostatic control 20-60min, is cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 16ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:2-8) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 10-50min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: by 45 DEG C on a rotary evaporator ± 2 DEG C rotary evaporations of step 4) gained supernatant near dry, i.e. can obtain the solution containing carotenoid.
Through test, should can be good in aforementioned manners separating the carotenoid in Pepper Leaves, 10 Carotenoids are thoroughly separated, and between every kind of material peak and peak, separation spacing is the biggest.
Embodiment 1
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, adds the KOH-methanol saponification liquor that 8mL mass concentration is 20%, seals and be placed on 55 DEG C of water bath with thermostatic control 30min, be cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 16ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:8) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 40min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: step 4) gained supernatant 45 DEG C of rotary evaporations on a rotary evaporator, near dry, i.e. can be obtained the solution containing carotenoid.
Embodiment 2
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, adds the KOH-methanol saponification liquor that 12mL mass concentration is 15%, seals and be placed on 80 DEG C of water bath with thermostatic control 20min, be cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 4ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:2) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 10min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: step 4) gained supernatant 43 DEG C of rotary evaporations on a rotary evaporator, near dry, i.e. can be obtained the solution containing carotenoid.
Embodiment 3
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, adds the KOH-methanol saponification liquor that 4mL mass concentration is 30%, seals and be placed on 40 DEG C of water bath with thermostatic control 60min, be cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 12ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:6) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 50min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: step 4) gained supernatant 47 DEG C of rotary evaporations on a rotary evaporator, near dry, i.e. can be obtained the solution containing carotenoid.
Embodiment 4
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, adds the KOH-methanol saponification liquor that 6mL mass concentration is 25%, seals and be placed on 50 DEG C of water bath with thermostatic control 50min, be cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 8ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:4) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 20min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: step 4) gained supernatant 44 DEG C of rotary evaporations on a rotary evaporator, near dry, i.e. can be obtained the solution containing carotenoid.
Embodiment 5
In Pepper Leaves, the extracting method of carotenoid is specific as follows:
1) ice bath grinds, and weighs the Pepper Leaves sample that 2.000g subpackage is good, adds 0.010g quartz sand and 0.1000g antioxidant 2,6 ditertiary butyl p cresol (BHT), adds liquid nitrogen and quickly grinds, be fully ground and obtain Pepper Leaves powder in mortar；
2) saponification: the Pepper Leaves powder after being ground by step 1) ice bath is positioned in 50mL lucifuge brown reagent bottle, adds the KOH-methanol saponification liquor that 10mL mass concentration is 18%, seals and be placed on 70 DEG C of water bath with thermostatic control 30min, be cooled to room temperature with cold water immediately after；
3) extract: by step 2) to add 10ml(solid-liquid ratio in Pepper Leaves after gained saponification be 1:5) the mixed extraction solvent of acetone and ethyl acetate (V:V=1:2), supersound extraction 35min, ultrasonic temperature is 30 DEG C；
4) centrifugal: by step 3) gained crude extract on low-temperature and high-speed centrifuge 4 DEG C, 8000r/min is centrifuged 15min；
5) rotary evaporation: step 4) gained supernatant 46 DEG C of rotary evaporations on a rotary evaporator, near dry, i.e. can be obtained the solution containing carotenoid.
The drafting of embodiment 6 10 Carotenoids standard curve
A) preparation of single standard solution:
The most accurately weigh phytoene, lycopene, alpha-carotene, phylloxanthin, epoxy phylloxanthin, beta-carotene, zeaxanthin, single antheraxanthin, neoxathin, violaxanthin standard substance 1mg chromatographically pure organic solvent dissolves and constant volume is in 25mL brown volumetric flask, preserving as single standard substance storing solution, concentration is 40 μ g/mL.
And take 0.2 μ L mixing respectively, with 0.22 μm micropore organic facies membrane filtration, store liquid as hybrid standard product, be stored in-20 DEG C of refrigerators stand-by.
B) preparation of series concentration carotenoid standard sample
The standard solution of 10 Carotenoids prepared by step b), at chromatographically pure organic solvent, in be hybridly prepared into the carotenoid standard sample of a series of concentration, process for preparation should be avoided illumination and high temperature, then carry out high performance liquid chromatography test by following chromatographic condition.
C) according to following chromatographic condition, the single standard solution of step a) and the series concentration carotenoid standard sample of step b) are carried out high performance liquid chromatography (HPLC) to test: the INSTRUMENT MODEL of high performance liquid chromatography is Waters 2695, detector is Waters2487 UV-detector, and detection wavelength is 450nm；Chromatographic column is Welch Ultimate C30 performance liquid chromatographic column, and specification is 250mm × 4.6mm, a diameter of 5 μm of filler, the column flow rate 1.0mL/min of flowing phase, the column temperature of performance liquid chromatographic column 30 DEG C；Sample room temperature 25 DEG C, sample size 20 μ L.
Flowing 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, methyl tertiary butyl ether(MTBE): methanol (V:V=1:1), and D is ethyl acetate.
Eluent gradient elution program is as shown in table 1.
Table 1 eluent gradient eluting
In table 1: 0min～6min:B is increased linearly to 10% by 0, C keeps 0, and D is increased linearly to 10% by 0；6min～12min:B is increased linearly to 15% by 10%, and C keeps 0, and D is increased linearly to 20% by 10%；12min～20min:B is kept 0 by 15% linear decrease to 0, C, and D is by 20% linear decrease to 10%；20min～22min:B is increased linearly to 5% by 0, and C is increased linearly to 40% by 0, and D is by 10% linear decrease to 5%；22min～30min:B is increased linearly to 50% by 5% linear decrease to 0, C by 40%, and D is increased linearly to 20% by 5%；30min～35min:A increases linearly to 100%.
nullE) retention time of carotenoid component is recorded in each single standard solution respectively (as shown in Figure 1，Phytoene retention time 33.168min、Lycopene retention time 29.231min、Alpha-carotene retention time 34.971min、Phylloxanthin retention time 19.183min、Epoxy phylloxanthin retention time 25.026min、Beta-carotene retention time 36.067min、Zeaxanthin retention time 20.398min、Single antheraxanthin retention time 17.950min、Neoxathin retention time 9.189min、10.473min when violaxanthin retains)，And determine corresponding each component in series concentration carotenoid standard sample according to this retention time，Then phytoene under each concentration is measured、Lycopene、Alpha-carotene、Phylloxanthin、Epoxy phylloxanthin、Beta-carotene、Zeaxanthin、Single antheraxanthin、Neoxathin、The peak area that violaxanthin is corresponding；And with each component variable concentrations as abscissa, corresponding peak area is vertical coordinate, draw the equation of linear regression of each component of carotenoid respectively.Result sees Fig. 3 and Biao 2.
Regression equation, the range of linearity and the correlation coefficient of each component of table 2 carotenoid
X-mass (x, g)；Y-peak area (× 10-4)。
The assay method of carotenoid content in embodiment 7 Fructus Capsici
Those skilled in the art know, and in Fructus Capsici, the assay method of carotenoid content and the kind of Fructus Capsici are unrelated, and for the method that the present invention is discussed in detail, this embodiment illustrates as a example by No. 5 Fructus Capsicis of Gansu Province green pepper.
One, the collection of Pepper Leaves: test and carry out in College of Horticulture of Gansu Agriculture University proving ground in November, 2013, it it is heliogreenhouse tolerance to low temperature and weak light kind for examination Fructus Capsici: Gansu Province green pepper No. 5, seedling medium is provided by College of Horticulture of Gansu Agriculture University, the volume proportion of substrate is: cattle manure: corn straw: the peat composed of rotten mosses: Vermiculitum=5:1:2:2, select color, grain type, the pepper seed of size uniformity, 55 DEG C of 30min that hot water treatment of seeds, stirring is to room temperature, it is placed on accelerating germination in growth cabinet, when showing money or valuables one carries unintentionally, sowing is in 50 cave hole tray, Routine Management, when pepper seedling length to 7-8 sheet true leaf, take 3-4 sheet functional leaf, every part of fresh leaf 2.000g, use liquid nitrogen rapid freeze-drying, be stored in-80 DEG C stand-by；
Two, the extraction of carotenoid in Pepper Leaves: the method for the application present invention all can well be extracted, herein, uses the method in embodiment 1 to extract；
Three, the preparation of Pepper Leaves carotenoid testing sample: the solution containing carotenoid obtained is settled to 10mL with chromatographically pure acetone, cross the 0.22 organic filter membrane of μm, be contained in 2ml brown chromatography column feed materials bottle, be saved in-20 DEG C stand-by, obtain Pepper Leaves carotenoid testing sample.
Four, with the method for embodiment 6 step c), Pepper Leaves carotenoid testing sample is carried out high efficiency chromatography test.
Five, according to the retention time of carotenoid component in each single standard solution, determine the retention time (as shown in Figure 2) of 10 Carotenoids in Pepper Leaves carotenoid testing sample, further determine that the peak area of various types of carotene, the peak area of various types of carotene is substituted in corresponding equation of linear regression respectively, obtains the content of respective components carotenoid in Pepper Leaves.
By the method for the present invention, the actual content of 10 Carotenoids in Pepper Leaves is measured, takes 5 parts of samples weighed up, measure 10 Carotenoids average contents in 5 parts of samples, the i.e. actual content of 10 Carotenoids in sample.Result is as shown in table 3.
Ten Carotenoids content in the surveyed Pepper Leaves of table 3 the present embodiment
The precision test of embodiment 8 the inventive method
By the reversed-phase high-performance liquid chromatography detection method of the present invention, sample is measured, under the same terms, Extraction of carotenoid pigment liquid is carried out 5 replications, result of the test such as table 4, as shown in Table 4, neoxathin, violaxanthin, single antheraxanthin, zeaxanthin, epoxy phylloxanthin, phylloxanthin, lycopene, phytoene, alpha-carotene, the response time of beta-carotene 10 Carotenoids fluctuates within 0.1min, the standard deviation of response time is respectively 0.074, 0.074, 0.080, 0.050, 0.087, 0.035, 0.034, 0.055, 0.030, 0.007, show that the precision of the inventive method is the best, can be used for the detection of 10 Carotenoids in Pepper Leaves.
The precision test result of carotenoid in table 4 HPLC detection Pepper Leaves
The determination of recovery rates of embodiment 9 the inventive method
nullTake 4 parts of samples，1 part is comparison，It is added without standard substance，Other 3 parts are separately added into low、In、The standard substance of high concentration，Chromatograph detection is carried out by the method for the present invention，Every part of sample introduction 5 times，The response rate is that the content of mark-on sample deducts and is not added with the content of the standard specimen product amount again divided by the standard sample added and obtains，Result of the test is as shown in table 5，It is separately added into low content、Middle content、The standard substance of high-load，Calculated sample recovery rate is all more than 80%，And the standard deviation of 10 Carotenoids replication results is respectively as follows: phytoene 1.21% in sample，Lycopene 0.97%，Alpha-carotene 0.58%，Phylloxanthin 1.20%，Epoxy phylloxanthin 1.87%，Beta-carotene 1.72%，Zeaxanthin 1.35%，Single antheraxanthin 1.99%，Neoxathin 1.02%，Violaxanthin 1.81%.
The Recovery test result of carotenoid in HPLC detection method Pepper Leaves applied by table 5
Testing result repeatability is checked: take 5 parts of samples weighed up, carotenoid in Pepper Leaves is measured by the reversed-phase high-performance liquid chromatography detection method set up according to the present invention, the repeatability of detection method is tested, result of the test shows, in Pepper Leaves, neoxathin, violaxanthin, zeaxanthin, phylloxanthin, beta-carotene, epoxy phylloxanthin, single antheraxanthin, lycopene, phytoene, alpha-carotene 10 Carotenoids measurement result standard deviation are all in the range of 2%, meet the requirement of chromatographic detection method.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature is carried out equivalent.All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (10)
1. the extracting method of carotenoid component in Pepper Leaves, it is characterised in that: step is as follows:
(1) liquid nitrogen grinding: add liquid nitrogen in fresh chilli blade and be fully ground to powder；As preferably, it is additionally added antioxidant 2,6 ditertiary butyl p cresol before the milling；
(2) saponification: add the KOH-methanol saponification liquor that mass concentration is 15-30% in (1), in 40-80 DEG C of saponification 20-60min；As preferably, the mass concentration of described KOH-methanol saponification liquor is 20%；
(3) extract: the mixed extraction solvent adding acetone and ethyl acetate in the Pepper Leaves of the saponification prepared in step (2) carries out supersound extraction, obtains carotenoid crude extract；Described Pepper Leaves is 1g:(2-8 with the ratio of mixed extraction solvent) ml；In described mixed extraction solvent, acetone is 1:2 with the volume ratio of ethyl acetate；
(4) the carotenoid crude extract that step (3) obtains is carried out post processing, obtain the solution containing carotenoid.
Method the most according to claim 1, it is characterised in that: Pepper Leaves described in step (2) is 1g:(2-6 with the consumption ratio of KOH-methanol solution) ml；
As preferably, described Pepper Leaves is 1g:4ml with the consumption ratio of KOH-methanol solution.
Method the most according to claim 1, it is characterised in that: described in step (2), Saponification Conditions is: 55 DEG C of saponification 30min.
Method the most according to claim 1, it is characterised in that: Pepper Leaves described in step (3) is 1g:8ml with the ratio of mixed extraction solvent.
Method the most according to claim 1, it is characterised in that: the time of supersound extraction described in step (3) is 10-50min, and ultrasonic temperature is 30 DEG C.
Method the most according to claim 5, it is characterised in that: the time of described supersound extraction is 40min.
Method the most according to claim 1, it is characterised in that: post processing described in step (4) is by 15min centrifugal at carotenoid crude extract 4 DEG C, takes supernatant, is concentrated into by 45 DEG C of rotary evaporations of supernatant near dry.
8. the quantitative detecting method of carotenoid content in Pepper Leaves, it is characterised in that: step is as follows:
(1) the arbitrary described method of application claim 1-7 extracts carotenoid in Pepper Leaves；
(2) with acetone, the solution containing carotenoid obtained is settled to 10mL, crosses the 0.22 organic filter membrane of μm, obtain Pepper Leaves carotenoid testing sample；Described acetone is chromatographically pure；
(3) according to following chromatographic condition, testing sample carried out high performance liquid chromatography test: chromatographic column is Welch Ultimate C30 performance liquid chromatographic column, and specification is 250mm × 4.6mm, a diameter of 5 μm of filler；The column flow rate 1.0mL/min of flowing phase, column temperature 30 DEG C, detection wavelength is 450nm；
Mobile phase composition is: A: acetonitrile, B: water, C: methyl tertiary butyl ether(MTBE) and the mixed liquor of methanol, and wherein, methyl tertiary butyl ether(MTBE) and methanol volume ratio are 1:1, D: ethyl acetate；
Eluent gradient elution program is: 0min～6min:B is increased linearly to 10% by 0, and C keeps 0, and D is increased linearly to 10% by 0；6min～12min:B is increased linearly to 15% by 10%, and C keeps 0, and D is increased linearly to 20% by 10%；12min～20min:B is kept 0 by 15% linear decrease to 0, C, and D is by 20% linear decrease to 10%；20min～22min:B is increased linearly to 5% by 0, and C is increased linearly to 40% by 0, and D is by 10% linear decrease to 5%；22min～30min:B is increased linearly to 50% by 5% linear decrease to 0, C by 40%, and D is increased 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 the retention time of 10 Carotenoids in Pepper Leaves carotenoid testing sample, further determine that the peak area of various types of carotene component, the peak area of various types of carotene component is substituted in corresponding equation of linear regression respectively, obtains the content of the carotenoid of respective components in Pepper Leaves.
Method the most according to claim 8, it is characterized in that: in described single standard solution, the retention time of carotenoid component is respectively as follows: phytoene retention time 33.168min, lycopene retention time 29.231min, alpha-carotene retention time 34.971min, phylloxanthin retention time 19.183min, epoxy phylloxanthin retention time 25.026min, beta-carotene retention time 36.067min, zeaxanthin retention time 20.398min, single antheraxanthin retention time 17.950min, neoxathin retention time 9.189min, 10.473min when violaxanthin retains.
Method the most according to claim 8, it is characterised in that: the acquisition methods of the equation of linear regression of described various types of carotene component is:
(1) the carotenoid standard sample of series concentration is prepared；
(2) high performance liquid chromatography test is carried out according to chromatographic condition shown in step in claim 8 (3)；
(3) according to the retention time of carotenoid component in each single standard solution, determine the retention time of each component of carotenoid in series concentration carotenoid standard sample, with each component variable concentrations as abscissa, corresponding peak area is vertical coordinate, draws the equation of linear regression of each component of carotenoid respectively.
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