CN107064391B - Method for determining zeatin in magnolia subgenus plant - Google Patents
Method for determining zeatin in magnolia subgenus plant Download PDFInfo
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Abstract
The invention discloses a method for measuring zeatin in magnolia subgenus plants, which comprises a zeatin extraction step, a standard solution preparation step, a high performance liquid chromatography analysis step and a standard solution measurement step, wherein in the zeatin extraction step, the zeatin is effectively extracted by using modes of low-temperature grinding, low-temperature light-resistant extraction, PVPP extraction and the like. The method has the advantages of better peak shape, high separation degree, no trailing phenomenon, good repeatability, high recovery rate, accuracy and reliability.
Description
Technical Field
The invention belongs to the field of biochemistry, relates to a qualitative and quantitative analysis technology of phytohormone, and particularly relates to a determination method of endogenous hormone in magnolia subordinates, in particular to a determination method of Zeatin (ZT) in magnolia rubra and magnolia alba in magnolia subordinates.
Background
The Magnolia grandiflora (Magnolia wufengensis L.Y.Ma et L.R.Wang) is a new species of Magnolia subgenus of Magnolia genus of Magnoliaceae discovered in the 3 rd month in Hubei of 2004 in the fifth peak of Hubei, the number of the cover pieces of the Magnolia grandiflora is 9 (-11), the characteristics of red inside and outside and uniform flower color are rare. Then, in the investigation of the area, the wild group with 12, 15, 18, 24 and the like of the number of the perianth of Magnolia denudata, even as many as 46 pieces, is found, and the group with the similar color to the perianth of Magnolia denudata and the increased number of the perianth is formally named as the Magnolia denudata (Magnolia wufengensis var. muttepala l.y.ma.et l.r.wang). With the continuous promotion of breeding and reproduction of new variety of magnolia denudata, industrialization and commercialization of magnolia denudata seedlings have become a necessary trend. Plant endogenous hormones such as gibberellin (GA3), auxin (IAA), abscisic acid (ABA) and Zeatin (ZT) have important regulating effects on the growth and development of plants. Understanding the change law of magnolia sieboldii phytohormone level is the theoretical basis for improving ornamental performance by applying plant growth regulators in production.
The phytohormone is contained in a plant in a very small amount, and the composition of the phytohormone in the plant is complicated, and the phytohormone is sensitive to conditions such as temperature because many interfering components coexist with each other, so that the measurement of the phytohormone is difficult. High Performance Liquid Chromatography (HPLC) is a relatively ideal method for analyzing endogenous hormones in plants, the HPLC determination of the phytohormones is applied to fruits such as apricot blossom buds, sweet cherries and pears for kernel use and seeds such as rapes and lettuce, and the HPLC determination of the phytohormones mainly focuses on the determination of the hormone content of seeds such as gingkoes, hawthorns, sweet cherries, schisandra chinensis, early honey pears and golden pears for plant seeds. At present, few reports are reported on the research of the plant hormone of the magnolia subgenus, and a universal plant hormone determination method is not established yet.
In the process of implementing the invention, the inventor finds that at least the following problems exist in the prior art:
1. in the high performance liquid chromatography determination method of the zeatin in different plants, the differences of the pretreatment method, the elution system and the detection system are very obvious, and the content of the zeatin, particularly the zeatin of the magnolia denudata and the zeatin of the magnolia denudata can not be determined by directly using the high performance liquid chromatography determination method of the zeatin of other plants.
2. Tender plant tissues of magnolia liliiflora, especially flower buds, are easy to brown in the grinding process, and the accuracy of the inspection result is affected.
3. During the pretreatment, the extraction solvent is difficult to select directly. In the process of completing the invention, the problems of non-ideal pigment removing effect, the problem that the extract liquid is emulsified and can not be layered, the problems of the ionization of the zeatin to be detected and the like, complicated extraction process and low extraction efficiency can occur when the conventional solvent in the prior art is used.
4. The components of a sample to be detected are complex, the polarity is strong, and chromatographic peaks are overlapped seriously and have a tailing phenomenon by using a conventional column chromatography technology.
In the process of completing the invention, the inventor also finds that the technical requirements of a pretreatment method and a column chromatography method of three plant endogenous hormones, namely Zeatin (ZT) and gibberellin (GA3), auxin (IAA) and abscisic acid (ABA), in a magnolia subgenus plant, particularly magnolia sieboldii and magnolia alba have great difference. The invention mainly aims to detect the Zeatin (ZT) content in magnolia, particularly in magnolia grandiflora and magnolia denudata.
Disclosure of Invention
The invention aims to solve the first technical problem of overcoming the defect that the prior art lacks a method for determining phytohormone in magnolia subgenus plants, in particular a method for determining Zeatin (ZT) in magnolia zenii and a method for determining Zeatin (ZT) in magnolia zenii.
The second technical problem to be solved by the invention is to overcome at least one of the problems of unsatisfactory pigment removal effect, incapability of layering due to emulsification of extract and ionization of zeatin to be detected in a pretreatment method of magnolia subgenus plants, particularly magnolia grandiflora or magnolia alba.
The third technical problem to be solved by the present invention is to overcome the problem of serious overlapping of chromatographic peaks in liquid chromatography.
The inventor provides a method for measuring zeatin in magnolia subgenus plants by long-term exploration and trial, multiple experiments and efforts and continuous innovation, and in order to solve the technical problems, the technical scheme provided by the invention comprises a zeatin extraction step, a standard solution preparation step, a high performance liquid chromatography analysis step and a standard solution measurement step;
the extraction steps of the zeatin are as follows:
A) grinding the plant tissue sample at low temperature to powder, adding a low-temperature methanol solution, and continuously grinding to homogenate;
B) transferring the homogenate into a first centrifuge tube, and leaching at a first low temperature in a dark place;
C) first centrifugation, sucking the first supernatant, adding a methanol solution into the precipitate, and second low-temperature light-proof leaching;
D) second centrifuging, sucking the second supernatant, and combining the first supernatant and the second supernatant to a first heart-shaped bottle;
E) ammonia water is dripped into the first heart-shaped bottle, and the mixture is decompressed, rotated and evaporated to a water phase at the temperature of 35-40 ℃;
F) completely transferring the water phase to a second centrifuge tube, adding PVPP into the second centrifuge tube, and performing third centrifugation after shaking by a shaking table; absorbing the third supernatant, adjusting the pH to 7.5-8.0 by using PBS phosphate buffer, then adding n-butanol saturated by the same volume of PBS for extraction, and repeating the step for 1-3 times;
G) mixing the extractive solutions, and concentrating under reduced pressure to dry;
H) dissolving with initial mobile phase, filtering with microporous membrane to obtain sample solution, and storing at low temperature.
According to a further embodiment of the method for determining zeatin in magnolia subgenus according to the invention, the hplc analysis step comprises:
the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-C18; mobile phase A: methanol, mobile phase B: 0.1M aqueous acetic acid; sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; detection wavelength: 270 nm;
quantitative determination was performed by external standard method.
According to a further embodiment of the method for determining zeatin in magnolia, the hplc step employs a gradient elution under the following conditions: 0-3 min, 20% -20% A; 3-8 min, 20% -30% A; 8-11 min, 30-30% A; 11-16 min, 30-50% A; 16-25 min, 50-50% A; 25-30 min, 50% -20% A, flow rate: 1 mL/min.
According to a preferred embodiment of the method for determining zeatin in a magnolia subgenus plant according to the invention, the magnolia subgenus plant is magnolia denudata or magnolia alba; the plant tissue is flower bud, flower quilt sheet or leaf of Magnolia liliiflora, or flower quilt sheet or leaf of Magnolia liliiflora.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plants, the standard solution is prepared by accurately weighing 0.0025g of zeatin standard substance, diluting to 10mL with methanol to obtain a standard stock solution with a concentration of 250mg/L, sealing, and storing at-20 ℃ in a dark place; sucking a certain amount of standard stock solution by a pipette, diluting into a mixed standard solution with the original concentration of 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 and 1/1024, and storing at low temperature in a dark place for later use.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant of the present invention, the standard solution determination step is specifically, injecting a standard stock solution of zeatin under determined chromatographic conditions, respectively, and recording retention time, thereby performing qualitative determination; repeatedly injecting the mixed standard solution for 3 times under the determined chromatographic condition, recording retention time and peak area, and calculating relative standard deviation; and (4) drawing a standard working curve by taking each sample injection concentration as a horizontal coordinate x and a peak area as a vertical coordinate y, and calculating a regression equation and a correlation coefficient so as to quantify.
According to a preferred embodiment of the method for determining zeatin in magnolia subgenus plants according to the invention, the cryogrinding is performed using a pre-cooled mortar, preferably a liquid nitrogen pre-cooled mortar; the low-temperature methanol solution is a precooled methanol solution, preferably a 4 ℃ precooled methanol solution, and more preferably a 4 ℃ precooled methanol solution with a volume fraction of 80%; the methanol solution is 80% by volume.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant, the first low-temperature dark leaching is performed at 1-4 ℃ for 15-21 h.
Preferably, the first low-temperature light-proof leaching is leaching at 4 ℃ for 15-21 h.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant according to the invention, the second low-temperature light-proof leaching is performed at 1-4 ℃ for 1-3 hours.
Preferably, the second low-temperature light-proof leaching is leaching at 4 ℃ for 2 h.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant of the present invention, the first centrifugation is performed at 1-4 ℃ and 10000-14000 r/min for 8-12 min.
Preferably, the first centrifugation is a centrifugation at 12000r/min at 4 ℃ for 10 min.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant of the present invention, the second centrifugation is performed at 10000-14000 r/min for 8-12 min and at 1-4 ℃.
Preferably, the second centrifugation is a centrifugation at 12000r/min at 4 ℃ for 10 min.
According to a further embodiment of the method for determining zeatin in magnolia subgenus plant according to the invention, the third centrifugation is 10000-14000 r/min centrifugation for 8-12 min.
Preferably, the third centrifugation is 12000r/min for 10 min.
According to a further embodiment of the method for determining zeatin in magnolia plants, the shaking table is shaking for 15-25 min at normal temperature.
Preferably, the shaking table oscillation is normal temperature shaking table oscillation for 20 min.
According to a preferred embodiment of the method for determining zeatin in magnolia subgenus according to the invention, the PBS phosphate buffer is a pH 8.0PBS saturated n-butanol solution; the initial mobile phase was 20% methanol, 80% 0.1M aqueous acetic acid; the microporous filter membrane is a 0.45 mu m microporous filter membrane; the low-temperature storage is storage at 4 ℃.
According to a preferred embodiment of the method for determining zeatin in magnolia, the steps for extracting zeatin are as follows:
accurately weighing 0.5000g of a magnolia sieboldii plant tissue sample, putting the magnolia sieboldii plant tissue sample into a precooled mortar, adding liquid nitrogen, grinding the mixture into powder, adding 8mL of precooled 80% methanol, grinding the mixture into homogenate, transferring the homogenate into a 10mL first centrifuge tube, and placing the homogenate into a refrigerator at 4 ℃ for first low-temperature dark extraction for 15-21 h; carrying out first centrifugation at 12000r/min at 4 ℃ for 10min, sucking the first supernatant, adding 4mL of precooled 80% methanol into the precipitate, and carrying out second low-temperature light-proof leaching for 2 h; centrifuging at 12000r/min for 10min at 4 deg.C, sucking second supernatant, and mixing the first supernatant and the second supernatant into 100mL first heart-shaped bottle; adding 1 drop of ammonia water, performing reduced pressure rotary evaporation at 35-40 ℃ to obtain a water phase, transferring the water phase to a 10mL centrifuge tube, adding 2mL of ultrapure water into the first heart-shaped bottle, cleaning, and combining the water phases; weighing 0.1000g PVPP in a centrifuge tube, and oscillating for 20min in a normal temperature shaking table; centrifuging at 12000r/min for 10min, sucking third supernatant, and adjusting pH to 7.5-8.0 with PBS phosphate buffer solution with pH of 8.0; adding n-butanol saturated with PBS (pH 8.0) with the same volume, extracting, and repeating for 3 times; mixing n-butanol phases, pouring into a second heart-shaped flask, and concentrating under reduced pressure at 60 deg.C to dry; dissolving with 0.5mL of initial mobile phase, filtering with 0.45 μm microporous membrane to obtain sample solution to be tested, and storing in 4 deg.C refrigerator for testing zeatin; the initial mobile phase was 20% methanol, 80% 0.1M aqueous acetic acid.
Compared with the prior art, one of the technical solutions has the following advantages:
a) the inventor finds that tender plant tissues, especially flower buds, of the magnolia sieboldii are extremely easy to brown in the grinding process in the process of completing the invention. And a low-temperature mortar is used before grinding, and if the mortar is precooled by liquid nitrogen, the grinding process ensures that the low-temperature environment can prevent browning to a certain extent.
b) The invention uses PVPP to remove pigments and phenolic substances, on one hand, the negative influence of emulsification on ethyl acetate extraction is not caused, on the other hand, the impurity removal requirement can be met, and the extraction process is simplified.
c) The invention adopts a gradient elution method, and 20 percent of methanol is selected: 80% 0.1M acetic acid (0.575% acetic acid solution) is used as the initial mobile phase, the peak shape is better, the separation degree is high, and no tailing phenomenon exists.
d) The gradient design of the elution system, the base line is stably recovered after the peak emergence in the whole program operation process, and the continuity of automatic sample introduction and equal precision measurement of samples are ensured.
e) The column temperature is selected to ensure that the separation degree of the zeatin is optimal.
f) Too fast a flow rate will result in overlapping chromatographic peaks, too slow a flow rate will result in "flat peaks", with the peak shape of zeatin being best when the flow rate is 1.0 mL/min.
g) The distribution coefficient of the zeatin in the n-butyl alcohol is highest, so that the efficiency of extracting the zeatin by using the n-butyl alcohol is highest. The pH value of the water-saturated n-butanol is 5.6, and if the water-saturated n-butanol is directly used for extraction, the pH value of an extracting solution adjusted to pH 7.5-8.0 is reduced to acidity, so that alkaline zeatin is not easily subjected to molecularization, and the extraction efficiency is reduced.
h) The invention has good repeatability, high recovery rate, accuracy and reliability.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a liquid chromatogram of a standard zeatin product of example 1.
FIG. 2 is a liquid chromatogram of a sample in example 1 of the present invention.
FIG. 3 is a liquid chromatogram of the sample plus standard in example 1 of the present invention.
Fig. 4 shows the mobile phase as 40% methanol: sample chromatogram of 60% water (0.75% acetic acid in it).
In the figure, 1 is ZT.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
Example 1
In this example, the following instruments and reagents were used.
The instrument comprises the following steps: an American Agilent 1260 high performance liquid chromatograph comprises a quaternary pump, an automatic sample injector, a column incubator and an ultraviolet detector; 3-30K high speed refrigerated centrifuge (SIGMA, Germany); Hei-VAP rotary evaporator (Heidolph, Germany); PB-21 acidimeters (Sartorius, Germany); quintix 224-1CN electronic balance (Sartorius, Germany); pipettors (Eppendorf, germany); SHB-III circulating water type multipurpose vacuum pump (Zhengzhou great wall science and trade Co., Ltd.).
Reagent: polyvinylpolypyrrolidone (PVPP) (Sigma in the usa); methanol (chromatographically pure, fischer scientific, usa); glacial acetic acid, ethyl acetate, n-butanol, ammonia, sodium dihydrogen phosphate, disodium hydrogen phosphate (analytical grade, Beijing chemical plant); ultrapure water; zeatin standard (HPLC grade, purity ≥ 98%, Sigma in USA).
The method for measuring the zeatin in the magnolia subgenus plant can be used for measuring the zeatin content in the flower buds, the flower quilt sheets or the leaves of the magnolia denudata, and can also be used for measuring the zeatin content in the flower quilt sheets or the leaves of the magnolia denudata. In this example, magnolia zenii bud was used as an extraction target. The magnolia wufengensis plant tissue samples mentioned below refer to magnolia wufengensis buds unless otherwise specified.
The method for determining zeatin in magnolia subgenus plants comprises a zeatin extraction step, a standard solution preparation step, a high performance liquid chromatography analysis step and a standard solution determination step.
1. Extraction of zeatin
Accurately weighing 0.5000g of a magnolia sieboldii plant tissue sample, putting the magnolia sieboldii plant tissue sample into a precooled mortar, adding liquid nitrogen, grinding the mixture into powder, adding 8mL of precooled 80% methanol, grinding the mixture into homogenate, transferring the homogenate into a 10mL first centrifuge tube, and placing the homogenate into a refrigerator at 4 ℃ for first low-temperature dark extraction for 15-21 h. The first centrifugation is carried out for 10min at 12000r/min at 4 ℃, the first supernatant is absorbed, and then precooled 4mL 80% methanol is added into the sediment for second low-temperature light-proof leaching for 2 h. Centrifuging at 12000r/min for 10min at 4 deg.C, sucking the second supernatant, and combining the first supernatant and the second supernatant into a 100mL first heart-shaped bottle. Adding 1 drop of ammonia water, performing rotary evaporation at the temperature of 35-40 ℃ under reduced pressure until the water phase is reduced by about 2/3, transferring the water phase into a 10mL second centrifuge tube, adding 2mL of ultrapure water into the first heart-shaped bottle, cleaning, and combining the water phases. 0.1000g PVPP was weighed into a second centrifuge tube and shaken on a shaker at room temperature for 20 min. And (3) centrifuging at 12000r/min for 10min, sucking a third supernatant, and adjusting the pH to 7.5-8.0 by using PBS phosphate buffer solution with the pH being 8.0. An equal volume of n-butanol saturated with PBS at pH 8.0 was added for extraction and repeated 3 times. The n-butanol phases were combined and poured into a second heart-shaped flask and concentrated to dryness at 60 ℃ under reduced pressure. After dissolving with 0.5mL of initial mobile phase (20% methanol, 80% 0.1M acetic acid), the solution was filtered through a 0.45 μ M microporous membrane to obtain a sample solution to be tested, which was stored in a refrigerator at 4 ℃ for the determination of zeatin.
The first, second, and third components only have the function of distinguishing objects, and do not have actual meanings. In the embodiment, the extraction method of zeatin is based on the prior art, and a great deal of exploration and research are carried out, so that an improved optimization scheme is finally obtained. The inventor finds that tender plant tissues, especially flower buds, of the magnolia denudata are extremely easy to brown in the grinding process in the extraction process. And the mortar is precooled by liquid nitrogen before grinding, and the low-temperature environment can be ensured to prevent browning to a certain extent in the grinding process. The known literature indicates that the interference of pigment can be mostly removed by adding petroleum ether after the crude extraction of 80% methanol, but the inventor finds that the use of petroleum ether in the process of the invention can influence the extraction of ethyl acetate in the extraction process, so that the extraction solution is emulsified and cannot be separated. In order to solve the emulsification phenomenon, researchers add an equal volume of 5g/L NaOH before the pretreatment stage of extracting the phytohormone sample. However, the inventors have found that a strong alkaline substance not only destroys the phytohormone but also increases the pH of the extract solution to ionize the acidic phytohormone, thereby significantly reducing the extraction efficiency. The inventor shows through further research that the emulsification phenomenon of the extracting solution can be eliminated by adding a certain saturated NaCl solution, and the influence on the hormone is small. However, tests show that the decolorizing effect of petroleum ether is very limited, and the extraction process is more complicated. Finally, the inventor screens and uses PVPP to remove pigments and phenolic substances, and after the PVPP is added, the influence on butanol extraction is avoided, the impurity removal requirement can be met, and the extraction process is simplified.
It should be noted that none of the above mentioned prior art methods are directed to the extraction of zeatin from magnolia, particularly magnolia zenii. But also the extraction method of the zeatin in other plants.
2. Preparation procedure of Standard solution
Accurately weighing 0.0025g of zeatin standard substance, diluting to 10mL with methanol to obtain standard stock solutions with concentration of 250mg/L, sealing, and storing at-20 deg.C in dark. Sucking a certain amount of standard stock solution of zeatin by a pipette, diluting into a mixed standard solution with the original concentration of 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 and 1/1024, and storing at low temperature in a dark place for later use.
3. High performance liquid chromatography analysis procedure
Chromatographic conditions
A chromatographic column: agilent ZORBAX SB-C18 (4.6X 150mm, 5 μm); mobile phase A: methanol, mobile phase B: 0.1M aqueous acetic acid.
0-3 min, 20% -20% A; 3-8 min, 20% -30% A; 8-11 min, 30-30% A; 11-16 min, 30-50% A; 16-25 min, 50-50% A; 25-30 min, 50% -20% A; run time 35 min. Flow rate: 1 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; detection wavelength: 270 nm. Quantitative determination was performed by external standard method.
The gradient elution schedule is shown in the following table:
the diisobutyl bonding phase Agilent ZORBAX SB-C18 column is adopted in the embodiment, so that the acidic, alkaline and neutral components can be analyzed under the conditions of low pH and high temperature, and the advantages of excellent peak shape and long column life are achieved.
In the prior art, the mobile phase composition of methanol-water is the combination commonly used at present. Since zeatin is an alkaline hormone, the inventors tested 40% methanol: the isocratic elution effect of 60% water shows that the sample components of the magnolia sieboldii have strong polarity, the chromatographic peaks are seriously overlapped, and the phenomenon of tailing exists. It is well documented that a suitable amount of acetic acid improves tailing and improves the degree of separation. In the test of improving the composition of a mobile phase, the inventor adopts 40 percent methanol: the chromatographic peak of hormone can not be separated from the impurity peak even in 60% water (containing 0.75% of acetic acid), and the chromatogram is shown in figure 4, and the result shows that the magnolia grandiflora sample has complex components and strong polarity, and the isocratic elution HPLC method is not suitable for detecting the zeatin.
Finally, the inventors selected a gradient elution method, and finally 20% methanol by comparative experiments: 80% 0.1M aqueous acetic acid (0.575% aqueous acetic acid) was used as the initial mobile phase. Decreasing the proportion of the organic phase increases the elution strength and increasing the proportion of the organic phase at the end of the gradient elution causes the polar components remaining in the sample to be eluted. Selecting 20% methanol: when 80% water is used as the mobile phase, the chromatographic peak appearance time of the zeatin is seriously overlapped with the impurity peak with strong polarity. The reaction was changed to 20% methanol: 80% 0.1M acetic acid (0.575% acetic acid solution) works best as the initial mobile phase. The proportion of methanol is 20% in 0-3 min; the methanol proportion is increased from 20% to 30% in 3-8 min, and the zeatin peak appearance is finished; the proportion of methanol is kept at 30% in 8-11 min; the proportion of methanol is changed from 30% to 50% in 11-16 min, and the proportion of 50% is kept in 16-25 min, so that most of impurities are eluted in the process; the proportion of the methanol is reduced to 20 percent in 25-30 min. The whole program was run for 35min, and the baseline eventually returned to a steady state.
In liquid chromatography, the column temperature is increased to shorten the peak-producing time and improve the sensitivity. In the test, chromatograms at 25 ℃, 30 ℃, 35 ℃ and 40 ℃ are respectively compared, and the result shows that the separation degree of the zeatin is optimal under the column temperature condition of 40 ℃.
Increasing the mobile phase velocity can shorten the retention time and narrow the peak shape. The experiment compares 4 flow rates of 0.6mL/min, 0.8mL/min, 1.0mL/min and 1.2mL/min, and the result shows that the chromatographic peaks are overlapped when the flow rate is too high, the 'flat head peak' appears when the flow rate is too low, and the peak shape of the zeatin is the best when the flow rate is 1.0 mL/min.
4. Standard solution determination procedure
Respectively injecting standard stock solutions of the zeatin under a determined chromatographic condition, and recording the retention time of the zeatin, thereby performing qualitative analysis. The mixed standard solution was injected repeatedly 3 times under defined chromatographic conditions, the retention time and peak area were recorded, and the relative standard deviation was calculated. And (4) drawing a standard working curve by taking each sample injection concentration as a horizontal coordinate x and a peak area as a vertical coordinate y, and calculating a regression equation and a correlation coefficient so as to quantify. The calculated regression equation and the correlation coefficient are shown in table 1. The lowest concentration of the zeatin detected when the signal-to-noise ratio S/N is 3 is taken as the detection limit of the method, and the result is shown in table 1, wherein the zeatin concentration and the peak area have a good linear relationship, and the correlation coefficient is greater than 0.9990.
TABLE 1 Standard working curves and detection limits for zeatin
Sample determination and recovery from spiking
Weighing 0.5000g of magnolia liliiflora flower bud, extracting and detecting according to the steps, adding a certain amount of standard solution with known concentration into a detected sample, repeatedly injecting samples for 5 times, and calculating the recovery rate of added standard. As shown in Table 2, the standard deviation of the retention time of zeatin was 0.006, the recovery of zeatin was 98.11%, and the relative standard deviation RSD was 0.31%. The method has the advantages of good repeatability, high recovery rate, accuracy and reliability. As shown in fig. 1, the chromatogram of the zeatin standard has a sharp peak shape and is not interfered by a solvent peak; as shown in fig. 2, zeatin does not overlap with impurity peaks and has a good degree of separation; as shown in fig. 3, the peak was superimposed with the hormone in the sample at the same retention time after the sample was added to the standard.
TABLE 2 recovery of zeatin by addition of standard
*Mean±SD。
Example 2
Replacing the plant tissue of the magnolia wufengensis in the example 1, namely the bud of the magnolia wufengensis with the perianth wufengensis slice, the magnolia wufengensis leaf slice, the magnolia wufengensis slice and the magnolia wufengensis leaf slice respectively, and performing zeatin determination and sample injection for 3 times repeatedly, wherein other steps are the same. The zeatin content of each plant tissue is shown in table 3, the zeatin content of the magnolia sieboldii leaves is the highest, and is 3.77 +/-0.25 mu g/g.FW; the magnolia denudata flower bud sample is the flower bud in the dormancy stage, and the zeatin content is the lowest and is 1.20 +/-0.01 mu g/g.
TABLE 3 zeatin content of different plant tissues
*Mean±SD。
The result shows that the method is simultaneously suitable for the zeatin determination of the magnolia wufengensis and the magnolia bailii, the standard deviation is small, and the accuracy is high. The method has wide applicability and strong practicability, and provides technical support for the research work of the growth and development rule of the magnolia sieboldii.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (13)
1. A method for determining zeatin in a magnolia subgenus plant is characterized by comprising a zeatin extraction step, a standard solution preparation step, a high performance liquid chromatography analysis step and a standard solution determination step;
the extraction steps of the zeatin are as follows:
A) grinding the plant tissue sample to powder at low temperature by adopting a mortar precooled by liquid nitrogen, then adding a methanol solution precooled at 4 ℃, and continuously grinding to homogenate;
B) transferring the homogenate into a first centrifuge tube, performing first low-temperature light-proof leaching, and performing light-proof leaching at 1-4 ℃ for 15-21 h;
C) performing first centrifugation, namely centrifuging at 10000-14000 r/min for 8-12 min at 1-4 ℃, absorbing a first supernatant, adding a low-temperature methanol solution into a precipitate, performing second low-temperature light-proof extraction, and performing light-proof extraction at 1-4 ℃ for 1-3 h;
D) centrifuging for 8-12 min at 10000-14000 r/min at 1-4 ℃, sucking a second supernatant, and combining the first supernatant and the second supernatant to a first heart-shaped bottle;
E) ammonia water is dripped into the first heart-shaped bottle, and the mixture is decompressed, rotated and evaporated to a water phase at the temperature of 35-40 ℃;
F) completely transferring the water phase to a second centrifuge tube, adding PVPP into the second centrifuge tube, shaking for 15-25 min by a normal-temperature shaking table, centrifuging for the third time, and centrifuging for 8-12 min at a speed of 10000-14000 r/min; absorbing the third supernatant, adjusting the pH to 7.5-8.0 by using PBS phosphate buffer, then adding n-butanol saturated by the same volume of PBS for extraction, and repeating the step for 1-3 times;
G) mixing the extractive solutions, and concentrating under reduced pressure to dry;
H) dissolving with initial mobile phase, filtering with microporous membrane to obtain sample solution, and storing at low temperature; the initial mobile phase was 20% methanol, 80% 0.1M aqueous acetic acid;
in the step of high performance liquid chromatography, gradient elution is adopted, and the ratio of a mobile phase A: methanol, mobile phase B: 0.1M aqueous acetic acid, elution conditions: 0-3 min, 20% -20% A; 3-8 min, 20% -30% A; 8-11 min, 30-30% A; 11-16 min, 30-50% A; 16-25 min, 50-50% A; 25-30 min, 50% -20% A, flow rate: 1 mL/min; the high performance liquid chromatography analysis step comprises:
the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-C18; sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; detection wavelength: 270 nm;
quantitative determination is carried out by an external standard method;
the said sub-genus plant of Yulan magnolia is flos Carthami Yulan or white Yulan magnolia; the plant tissue is flower bud, flower quilt sheet or leaf of Magnolia liliiflora, or flower quilt sheet or leaf of Magnolia liliiflora.
2. The method for determining zeatin in magnolia subgenus plant according to claim 1, wherein the standard solution is prepared by accurately weighing 0.0025g ZT standard, diluting to 10mL with methanol, preparing into standard stock solution with concentration of 250mg/L, sealing, and storing at-20 deg.c in dark; sucking a certain amount of standard stock solution by a pipette, diluting into a mixed standard solution with the original concentration of 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 and 1/1024, and storing at low temperature in a dark place for later use.
3. The method for determining zeatin in magnolia subgenus plant according to claim 1, wherein the standard solution determination step is characterized in that a standard stock solution of ZT is injected under certain chromatographic conditions, and retention time is recorded for qualitative determination; repeatedly injecting the mixed standard solution for 3 times under the determined chromatographic condition, recording retention time and peak area, and calculating relative standard deviation; and (4) drawing a standard working curve by taking each sample injection concentration as a horizontal coordinate x and a peak area as a vertical coordinate y, and calculating a regression equation and a correlation coefficient so as to quantify.
4. The method of claim 1, wherein the low temperature methanol solution is a pre-cooled methanol solution; the methanol solution is 80% by volume.
5. The method of claim 1, wherein said low temperature methanolic solution is a 4 ℃ pre-cooled 80% volume fraction methanol solution.
6. The method for determining zeatin in magnolia subgenus plant according to claim 1, wherein the first low temperature dark leaching is dark leaching at 4 ℃ for 15-21 h.
7. The method of claim 1, wherein the second low temperature light-protected extraction is a 4 ℃ light-protected extraction for 2 hours.
8. The method of claim 1, wherein the first centrifugation is at 12000r/min at 4 ℃ for 10 min.
9. The method of claim 1, wherein the second centrifugation is at 12000r/min at 4 ℃ for 10 min.
10. The method of claim 1, wherein the third centrifugation is 12000r/min for 10 min.
11. The method of claim 1, wherein the table shaking is performed for 20min at room temperature.
12. The method for assaying zeatin in magnolia subgenus according to claim 1, wherein the PBS phosphate buffer is a pH 8.0PBS saturated n-butanol solution; the microporous filter membrane is a 0.45 mu m microporous filter membrane; the low-temperature storage is storage at 4 ℃.
13. The method for detecting zeatin in magnolia subgenus as claimed in claim 1, wherein the steps of extracting zeatin are as follows:
accurately weighing 0.5000g of a magnolia sieboldii plant tissue sample, putting the magnolia sieboldii plant tissue sample into a precooled mortar, adding liquid nitrogen, grinding the mixture into powder, adding 8mL of precooled 80% methanol, grinding the mixture into homogenate, transferring the homogenate into a 10mL first centrifuge tube, and placing the homogenate into a refrigerator at 4 ℃ for first low-temperature dark extraction for 15-21 h; centrifuging for 10min at 12000r/min at 4 ℃, sucking the first supernatant, adding 4mL of 80% methanol into the precipitate, and leaching for 2h in a low-temperature and dark manner; centrifuging at 12000r/min for 10min at 4 deg.C, sucking second supernatant, and mixing the first supernatant and the second supernatant into 100mL first heart-shaped bottle; adding 1 drop of ammonia water, performing reduced pressure rotary evaporation at 35-40 ℃ to obtain a water phase, transferring the water phase to a 10mL centrifuge tube, adding 2mL of ultrapure water into the first heart-shaped bottle, cleaning, and combining the water phases; weighing 0.1000g PVPP in a centrifuge tube, and oscillating for 20min in a normal temperature shaking table; centrifuging at 12000r/min for 10min, sucking third supernatant, and adjusting pH to 7.5-8.0 with PBS phosphate buffer solution with pH of 8.0; adding n-butanol saturated with PBS (pH 8.0) with the same volume, extracting, and repeating for 3 times; mixing n-butanol phases, pouring into a second heart-shaped flask, and concentrating under reduced pressure at 60 deg.C to dry; dissolving with 0.5mL of initial mobile phase, filtering with 0.45 μm microporous membrane to obtain sample solution to be tested, storing in 4 deg.C refrigerator for determining ZT; the initial mobile phase was 20% methanol, 80% 0.1M aqueous acetic acid.
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