CN110988172B - Hibiscus sabdariffa endogenous cytokinin extractant and application method thereof - Google Patents
Hibiscus sabdariffa endogenous cytokinin extractant and application method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of endogenous hormone extraction, and particularly relates to a cottonrose hibiscus endogenous cytokinin extracting agent and a use method thereof. The invention relates to a cottonrose hibiscus endogenous cytokinin extracting agent which is prepared by mixing an agent A and an agent B according to the mass ratio of 1-2:1; the agent A is at least 2 of glycine, phthalic acid, disodium hydrogen phosphate, citric acid, sodium citrate, sodium hydroxide, acetic acid, sodium acetate and hydrochloric acid, and is prepared into a mixed aqueous solution with the pH value of 2-3 after being dissolved by redistilled water; the agent B is methanol. The extraction method of the endogenous cytokinin of the cottonrose hibiscus is simple, has low cost, has the extraction rate of 86-95% on the endogenous cytokinin of the cottonrose hibiscus, meets the requirements of the detection method generally accepted by the industry, and has the technical scheme of simplicity and feasibility and is more suitable for popularization.
Description
Technical Field
The invention belongs to the technical field of endogenous hormone extraction, and particularly relates to a cottonrose hibiscus endogenous cytokinin extracting agent and a use method thereof.
Background
The plant hormone is a natural organic compound which is produced by metabolism or biosynthesized in the normal development process or under special environmental conditions, generates obvious physiological response in a trace concentration, plays an important role in regulating various physiological processes of tissue and organ differentiation, morphogenesis, growth and development, aging and the like of plants, and also influences important characters such as crop yield, quality, resistance and the like. Six major classes of plant endogenous hormones currently accepted are auxins (auxins), gibberellins (GA), cytokins (CK), ethylene (ethylene), abscisic acid (ABA), brassinosteroids (BRs).
Cottonrose hibiscus (Hibiscus mutabilis Linn.) is also known as cottonrose hibiscus flower, frost-resistant flower, lotus, ground cottonrose hibiscus, hua Mu, and native China. It is warm and moist, not cold-resistant, and is prohibited from drought and water-wet resistance. The requirement on soil is not high, and barren lands can also grow. Is Malvaceae, shrub of Hibiscus or small arbor. Flowers are grown singly in the axilla of the branch, terminal and leaf. Flowers and leaves can be used as medicines, and have the effects of clearing heat and detoxicating, detumescence and expelling pus, cooling blood and stopping bleeding. The cotton rose endogenous cytokinin is a series of trace small molecular compounds synthesized in plants, and can play various physiological roles at extremely low concentration. The content of the endogenous cytokinin of the cottonrose hibiscus in the plant body is extremely low, the nature is unstable, and the quantitative detection of the endogenous cytokinin of the cottonrose hibiscus is always a hot spot and a difficult point of current research.
The publication number is CN103884810A, the invention is named as a method for efficiently separating and measuring endogenous cytokinin of turf grass, which adopts 0.5g of fresh sample, grinding the fresh sample by liquid nitrogen, adding 5ml of extracting solution, and extracting overnight at minus 20 ℃ to obtain leaching solution; the extracting solution is methanol: formic acid: water = 15:1:4, further 40mg/ml of 2, 6-di-tert-butyl-4-methylphenol as antioxidant was added in v/v. The publication number is CN103048404A, the name of the invention is a quantitative detection method of endogenous cytokinin in a plant sample, and acetonitrile is adopted as an extractant. The publication number is CN109991339A, the name is a sample pretreatment method for measuring endogenous hormones in trace plant samples, and wall breaking enzyme is adopted as an extracting agent.
Therefore, for analysis of plant endogenous hormones, firstly, a proper extractant is selected to realize nondestructive complete extraction of target substances, so that a plurality of interfering substances are avoided, the endogenous hormones are prevented from being destroyed, and secondly, various extraction or chromatographic steps are adopted to partially purify the endogenous hormones, and then the content of the endogenous hormones is measured. However, in the prior art, when endogenous hormones are extracted, the problems of large use amount of organic solvents, complex extraction method, high cost, low extraction rate, difficulty in avoiding interference of non-target compounds such as phenols, pigments, proteins, lipids, nucleic acids and the like on experimental results and the like generally exist. In addition, no published literature reports such as papers, patents, scientific achievements and the like for directly extracting endogenous cytokinin of tissues/organs of cottonrose hibiscus are found at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the extraction agent for extracting the endogenous cytokinin of the cottonrose hibiscus, which is favorable for simplifying the purification step of the leaching solution and avoiding the interference of excessive pigments and impurities on the detection result, and the application method thereof according to the characteristics of the endogenous hormone of the cottonrose hibiscus.
The invention provides a cottonrose hibiscus endogenous cytokinin extracting agent which is prepared by mixing an agent A and an agent B according to the mass ratio of 1-2:1;
the agent A is at least 2 of glycine, phthalic acid, disodium hydrogen phosphate, citric acid, sodium citrate, sodium hydroxide, acetic acid, sodium acetate and hydrochloric acid, and is prepared into a mixed aqueous solution with the pH value of 2-3 after being dissolved by redistilled water; the component proportion relation of the raw materials of the agent A is properly adjusted according to the molar mass and the physical and chemical property difference, so that the agent A meets the condition that the pH value of a mixed reagent is between 2 and 3, and the extracting solution is kept in an acidic medium environment, so that the activities of enzymes such as phosphatase and the like in plant tissues can be passivated, the hydrolysis process of the phosphatase and the like on cytokinin nucleotide can be prevented, and the influence on the detection result caused by the increase of the corresponding nucleoside recovery rate due to enzymolysis is avoided;
the agent B is methanol.
The cotton rose endogenous cytokinin extracting agent provided by the invention is divided into A, B agents, so that the proportion of the agent A to the agent B can be conveniently adjusted according to the content of impurities in a detected object, and non-target impurities such as pigments, phenols, proteins and the like can be reduced as much as possible and are extracted along the way.
Further, the extract of the endogenous cytokinin of the cottonrose hibiscus, wherein the A agent can be a mixed aqueous solution with the pH of 2.0-2.1 prepared by mixing and dissolving glycine, hydrochloric acid and redistilled water according to the solid-to-liquid ratio of 0.7500-0.7510 g:50mL:150mL, and the concentration of hydrochloric acid is 0.2mol/L;
more preferably, the agent A is a mixed aqueous solution with pH of 2.0 prepared by mixing and dissolving glycine, hydrochloric acid and water according to a solid-to-liquid ratio of 0.7505g:50mL:150 mL.
Further, the A agent can be a mixed aqueous solution with pH of 2.1-2.3 prepared by mixing and dissolving phthalic acid, hydrochloric acid and redistilled water according to a solid-to-liquid ratio of 0.2050-0.2055 g to 4.05-4.10 mL to 10mL, and then fixing the volume to 20mL with redistilled water, wherein the concentration of the hydrochloric acid is 0.2mol/L;
more preferably, the agent A is a mixed aqueous solution with pH of 2.2 prepared by mixing and dissolving phthalic acid, hydrochloric acid and redistilled water according to a solid-to-liquid ratio of 0.2054g:4.07mL:10mL, and then fixing the volume to 20mL with redistilled water.
Further, the A agent is prepared into a mixed aqueous solution with pH of 2.1-2.3 by mixing and dissolving citric acid, sodium hydroxide, hydrochloric acid and redistilled water according to a solid-to-liquid ratio of 2.05-2.15 g:0.80-0.85 g:1.50-1.70 g:50mL, and then fixing the volume to 100mL by using the redistilled water, wherein the mass concentration of the hydrochloric acid is 40%;
more preferably, the agent A is a mixed aqueous solution prepared by mixing citric acid, sodium hydroxide, hydrochloric acid and redistilled water according to a mass ratio of 2.10g to 0.84g to 1.60g to 50mL, and fixing the volume to 100mL by the redistilled water, wherein the pH value of the mixed aqueous solution is 2.2.
Further, the A agent is prepared by mixing and dissolving disodium hydrogen phosphate, citric acid and redistilled water according to a solid-to-liquid ratio of 0.061900-0.061920 g to 0.37430-0.37450 g to 10mL, and then fixing the volume to 20mL by using the redistilled water to prepare a mixed aqueous solution with the pH value of 2.5-2.7;
more preferably, the agent A is disodium hydrogen phosphate, citric acid and redistilled water according to the solid-to-liquid ratio of 0.061912g to 0.37439g to 10mL, and then the mixed solution is prepared into a mixed aqueous solution with pH of 2.6 by constant volume to 20mL with redistilled water.
Further, the A agent is prepared by mixing and dissolving citric acid, sodium citrate and redistilled water according to a solid-to-liquid ratio of 0.3900-0.3910 g:0.0400-0.0420 g:10mL, and then fixing the volume to 20mL by using the redistilled water to prepare a mixed aqueous solution with the pH value of 2.0-3.0;
more preferably, the agent A is citric acid, sodium citrate and redistilled water according to a solid-to-liquid ratio of 0.3907g:0.0411g:10mL, and then the mixed aqueous solution with pH of 3.0 is prepared by constant volume to 20mL with redistilled water.
The preparation of the agent A of the present invention is not limited to the above-mentioned preferable reagent preparation, and the ratio of the raw materials of the agent A is suitably adjusted according to the difference of molar mass and physicochemical properties so long as the agent A satisfies the condition that the agent A is a mixed aqueous solution with pH of 2-3 prepared by dissolving at least 2 of glycine, phthalic acid, disodium hydrogen phosphate, citric acid, sodium citrate, sodium hydroxide, acetic acid, sodium acetate and hydrochloric acid with redistilled water, and the pH of the mixed agent is within the scope of the present invention.
The invention also provides a cottonrose hibiscus endogenous cytokinin extracting agent and a use method thereof, comprising the following steps:
a. the preparation of the extractant: preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: mixing the agent A and the agent B according to the mass ratio of 1-2:1 to prepare the cotton rose endogenous cytokinin extracting agent, and preserving at a low temperature of 10 ℃ for later use;
the agent A is at least 2 of glycine, phthalic acid, disodium hydrogen phosphate, citric acid, sodium citrate, sodium hydroxide, acetic acid, sodium acetate and hydrochloric acid, and is prepared into a mixed aqueous solution with the pH value of 2-3 after being dissolved by redistilled water;
the agent B is methanol;
b. extracting a target: taking a crushed sample of the cotton rose tissue, mixing the crushed sample with the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:5-10 mL, standing for 4-8 h at the temperature lower than 10 ℃, carrying out suction filtration, collecting filtrate I, leaching the obtained filter residue I for 1-2 h by using the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:2.5-5 mL, carrying out suction filtration, and collecting filtrate II and filter residue II; c, leaching the filter residue II by using the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:1-2.5 mL for 1h, filtering, and collecting filtrate III and filter residue III;
c. purifying a target: combining the filtrates I, II and III obtained in the step c, extracting by PVPP solid-liquid extraction small column, and collecting column-passing liquid to obtain the endogenous cytokinin test solution of the cotton rose tissue sample;
furthermore, the cotton rose endogenous cytokinin extracting agent and the using method provided by the invention, wherein in the step b, the cotton rose tissue sample is at least one of cotton rose leaves, petals, flower stalks, calyx, branches or roots and stem tissues or organs.
Further, according to the method for extracting the endogenous cytokinin of the cottonrose hibiscus, the content of the endogenous cytokinin of the cottonrose hibiscus tissue sample is detected by adopting HPLC or LC-MS.
The invention has the following beneficial effects:
1. according to the hibiscus endogenous cytokinin extracting agent and the extracting method thereof, under the condition of ensuring that target compounds are fully extracted, the using amount of an organic solvent is reduced, and the leaching of non-target compounds such as phenols, pigments, proteins, lipids, nucleic acids and the like is reduced as much as possible, so that the purifying step of a leaching solution is simplified, and the interference of excessive pigments and impurities on detection results is avoided;
2. according to the invention, the activity of enzymes such as phosphatase in plant tissues is passivated by keeping the acidic medium environment of the extracting solution, so that the cytokinin nucleotide hydrolysis process caused by the phosphatase and the like is prevented, and the influence on the detection result due to the increase of the corresponding nucleoside recovery rate caused by enzymolysis is avoided;
3. the invention utilizes the acidic environment, is favorable for preventing the interference substances such as phospholipid and the like in the sample from forming emulsion, and promotes the water phase distribution of the object to be detected;
4. the extraction method of the endogenous cytokinin of the cottonrose hibiscus is simple, has low cost, has the extraction rate of 86-95% on the endogenous cytokinin of the cottonrose hibiscus, meets the requirements of the detection method generally accepted by the industry, and has the technical scheme of simplicity and feasibility and is more suitable for popularization.
Drawings
FIG. 1 is an HPLC detection profile of a standard sample;
FIG. 2 is an HPLC detection chart of a Hibiscus detection sample obtained in example 1.
FIG. 3 is an HPLC detection spectrum of a Hibiscus sample obtained by a conventional method.
Detailed Description
The present invention will be described in further detail by the following detailed description, but it should not be construed that the scope of the invention is limited to the following examples. Various substitutions and alterations are also within the scope of this disclosure, as will be apparent to those of ordinary skill in the art and by routine experimentation, without departing from the spirit and scope of the invention as defined by the foregoing description.
Example 1
An extract of endogenous cytokinin of Hibiscus Mutabilis is prepared by mixing agent A and agent B according to the mass ratio of 1.5:1;
the agent A is prepared by uniformly mixing 0.7505g glycine, 50mL hydrochloric acid with the concentration of 0.2mol/L and 150mL water, detecting the pH value to be 2.0, and preserving at low temperature;
the agent B is methanol;
the application method of the cotton rose endogenous cytokinin extractant comprises the following steps:
a. preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: taking the newly prepared cottonrose hibiscus endogenous cytokinin extractant, and preserving the cottonrose hibiscus endogenous cytokinin extractant at a low temperature for later use;
b. respectively taking 5g of tissue broken samples of petals, leaves, current annual fresh roots and 1 annual branches of Hibiscus sabdariffa, adding 25mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent in the step a, mixing, standing for 4h at the temperature lower than 10 ℃, carrying out suction filtration to obtain a filtrate I and a filter residue I, carrying out ultrasonic mixing on the obtained filter residue I and 10mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent, standing for 2h, and carrying out suction filtration to obtain a filtrate II and a filter residue II; the obtained filter residue II is continuously mixed with 5mL of the cotton rose endogenous cytokinin extracting agent in an ultrasonic way, and is stood for 1h, and the filtrate III and the filter residue III are obtained through suction filtration;
c. and b, combining the filtrates I, II and III, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose hibiscus tissue sample.
Example 2
An extract of endogenous cytokinin of Hibiscus Mutabilis is prepared by mixing agent A and agent B according to the mass ratio of 1:1;
the agent A is prepared by uniformly mixing 0.2054g of phthalic acid, 4.07mL of hydrochloric acid, 0.2mol/L of hydrochloric acid and 10mL of redistilled water, fixing the volume to 20mL, detecting the pH value to be 2.2, and preserving at low temperature;
the agent B is methanol.
The application method of the cotton rose endogenous cytokinin extractant comprises the following steps:
a. preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: taking the newly prepared cottonrose hibiscus endogenous cytokinin extractant, and preserving the cottonrose hibiscus endogenous cytokinin extractant at a low temperature for later use;
b. respectively taking 5g of tissue broken samples of petals, leaves, current annual fresh roots and 1 annual branches of Hibiscus sabdariffa, adding 25mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent in the step a, mixing, standing for 8h at the temperature lower than 10 ℃, carrying out suction filtration to obtain a filtrate I and a filter residue I, carrying out ultrasonic mixing on the obtained filter residue I and 10mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent, standing for 2h, and carrying out suction filtration to obtain a filtrate II and a filter residue II; the obtained filter residue II is continuously mixed with 5mL of the cotton rose endogenous cytokinin extracting agent in an ultrasonic way, and is stood for 1h, and the filtrate III and the filter residue III are obtained through suction filtration;
c. and b, combining the filtrates I, II and III, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose hibiscus tissue sample.
Example 3
An extract of endogenous cytokinin of Hibiscus Mutabilis is prepared by mixing agent A and agent B according to the mass ratio of 2:1;
the agent A is prepared by dissolving 2.1g of citric acid, 0.84g of sodium hydroxide (97 wt%), 1.6g of hydrochloric acid (40 wt%), 50mL of redistilled water, and performing constant volume to 100mL, detecting the pH value to be 2.2, and preserving at low temperature;
the agent B is methanol.
The application method of the cotton rose endogenous cytokinin extractant comprises the following steps:
a. preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: taking the newly prepared cottonrose hibiscus endogenous cytokinin extractant, and preserving the cottonrose hibiscus endogenous cytokinin extractant at a low temperature for later use;
b. respectively taking 5g of tissue broken samples of petals, leaves, current annual fresh roots and 1 annual branches of Hibiscus sabdariffa, adding 50mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent in the step a, mixing, standing for 8h at the temperature lower than 10 ℃, carrying out suction filtration to obtain a filtrate I and a filter residue I, carrying out ultrasonic mixing on the obtained filter residue I and 25mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent, standing for 2h, and carrying out suction filtration to obtain a filtrate II and a filter residue II; the obtained filter residue II is continuously mixed with 10mL of the cotton rose endogenous cytokinin extracting agent by ultrasonic, and is stood for 1h, and the filtrate III and the filter residue III are obtained by suction filtration;
c. and b, combining the filtrates I, II and III, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose hibiscus tissue sample.
Example 4
An extract of endogenous cytokinin of Hibiscus Mutabilis is prepared by mixing agent A and agent B according to the mass ratio of 1.5:1;
the agent A is prepared by dissolving 0.061912g of disodium hydrogen phosphate, 0.37439g of citric acid and 10mL of redistilled water, fixing the volume to 20mL, detecting the pH value to be 2.6, and preserving at low temperature;
the agent B is methanol.
The application method of the cotton rose endogenous cytokinin extractant comprises the following steps:
a. preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: taking the newly prepared cottonrose hibiscus endogenous cytokinin extractant, and preserving the cottonrose hibiscus endogenous cytokinin extractant at a low temperature for later use;
b. respectively taking 5g of tissue broken samples of petals, leaves, current annual fresh roots and 1 annual branches of Hibiscus sabdariffa, adding 40mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent in the step a, mixing, standing for 6h at the temperature lower than 10 ℃, carrying out suction filtration to obtain a filtrate I and a filter residue I, carrying out ultrasonic mixing on the obtained filter residue I and 20mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent, standing for 2h, and carrying out suction filtration to obtain a filtrate II and a filter residue II; the obtained filter residue II is continuously mixed with 10mL of the cotton rose endogenous cytokinin extracting agent by ultrasonic, and is stood for 1h, and the filtrate III and the filter residue III are obtained by suction filtration;
c. and b, combining the filtrates I, II and III, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose hibiscus tissue sample.
Example 5
An extract of endogenous cytokinin of Hibiscus Mutabilis is prepared by mixing agent A and agent B according to the mass ratio of 1.5:1;
the agent A is prepared by dissolving 0.3907g of citric acid, 0.0411g of sodium citrate and 10mL of redistilled water, fixing the volume to 20mL, detecting the pH value to be 3.0, and preserving at low temperature;
the agent B is methanol.
The application method of the cotton rose endogenous cytokinin extractant comprises the following steps:
a. preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: taking the newly prepared cottonrose hibiscus endogenous cytokinin extractant, and preserving the cottonrose hibiscus endogenous cytokinin extractant at a low temperature for later use;
b. respectively taking 5g of tissue broken samples of petals, leaves, current annual fresh roots and 1 annual branches of Hibiscus sabdariffa, adding 30mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent in the step a, mixing, standing for 8h at the temperature lower than 10 ℃, carrying out suction filtration to obtain a filtrate I and a filter residue I, carrying out ultrasonic mixing on the obtained filter residue I and 10mL of the Hibiscus sabdariffa endogenous cytokinin extracting agent, standing for 1h, and carrying out suction filtration to obtain a filtrate II and a filter residue II; the obtained filter residue II is continuously mixed with 5mL of the cotton rose endogenous cytokinin extracting agent in an ultrasonic way, and is stood for 1h, and the filtrate III and the filter residue III are obtained through suction filtration;
c. and b, combining the filtrates I, II and III, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose hibiscus tissue sample.
Comparative example 1
Referring to the conventional technical scheme disclosed by the prior art for extracting endogenous hormones of herbs and woody plants such as rice, corn, cucumber and oleander, the endogenous cytokinin of the cottonrose hibiscus is extracted, and the general scheme is as follows:
respectively taking 5g of tissue disruption samples of petals, leaves, current annual new roots and 1 annual branches of Hibiscus sabdariffa, mixing with 25mL of 80wt% methanol, standing for 8h, extracting, concentrating at 40 ℃ under negative pressure, decolorizing with petroleum ether, dissolving with ethyl acetate, regulating with acid and alkali, repeatedly extracting with n-butanol for at least 3 times, and continuously purifying to obtain the endogenous cytokinin solution to be detected of Hibiscus sabdariffa tissues/organs.
Preparing a standard to-be-tested sample according to a conventional technical method, and respectively detecting the content of the cytokinin in the endogenous cells of the cotton rose hibiscus tissue sample obtained by the standard to-be-tested sample, the cotton rose hibiscus tissue sample obtained by the embodiments 1-5 and the comparative example 1 by adopting the Beijing island HPLC to obtain an HPLC detection map, wherein the HPLC detection map is shown in figures 1, 2, 3 and table 1. Wherein, the high performance liquid chromatography operation conditions are as follows:
high performance liquid chromatograph (shimadzu-LC-20 AD): an ultraviolet visible light detector having a photodiode array;
chromatographic column: kromasil 100-5c18 stainless steel column 150mm x phi 4.6 mm;
solvent filter: the pore diameter of the filter membrane is 0.45 mu m;
mobile phase: ψ (methanol: water) =30:70;
detection wavelength: 265nm;
flow rate: 0.6ml/min;
sample introduction amount: 20. Mu.L;
column temperature: 30 DEG C
Retention time: about 10min
TABLE 1 detection results of endogenous cytokinin of Hibiscus tissue/organ 3 times (ug/kg. fresh weight) in examples 1-5
Note that: table 1 shows that there are no peaks in the sample spectrum that correspond to the peak time of the standard substance, # indicating that overlapping peaks consistent with the peak time corresponding to the standard substance appear in the sample spectrogram.
From the data in table 1, it can be seen that: the method disclosed in the embodiments 1-5 of the invention has higher extraction rate of the mitogen in the tissue-crushed samples of the petals, leaves, current annual fresh roots and 1 annual branches of the cottonrose hibiscus, and the detection results after three times of repeated extraction have smaller differences, while the method of the comparative example 1 has much lower extraction rate of the tissue-crushed samples of the petals, leaves, current annual fresh roots and 1 annual branches of the cottonrose hibiscus, and the detection results after three times of repeated extraction have larger differences, even the detection results can not be detected.
According to the detection results of the examples, as well as the results of fig. 1 (standard sample map), fig. 2 (sample map) and fig. 3 (comparative example 1), the endogenous cytokinin of cottonrose hibiscus extracted by using the extractant of the invention can be seen, and the chromatographic analysis detection shows that the pattern is clear, the peak shape is independent and complete, and the peak shape can be better separated from impurity peaks, thereby effectively avoiding the interference of non-target compounds such as phenols, pigments, proteins, lipids, nucleic acids and the like. The HPLC detection spectrum (figure 3) obtained after the extraction of the cottonrose hibiscus endogenous cytokinin by the conventional extraction technology shows that the peak shape is disordered, and more impurity peaks appear in the spectrum, which indicates that the cottonrose hibiscus endogenous cytokinin extracted by the conventional extraction technology cannot avoid the interference of non-target compounds such as phenols, pigments, proteins, lipids, nucleic acid and the like, and has poor extraction effect.
TABLE 2 results of test for verification of endogenous mitogen recovery from Hibiscus Mutabilis in example 1 and 3
From table 2, it can be derived that: the recovery rate of the test method is verified to be 80-95%, and the extraction agent and the technical scheme of the invention prove that the extraction agent can effectively extract endogenous cytokinin active substances in the cotton rose tissues/organs while reducing the extraction of non-target impurities such as pigment, phenols, protein and the like, and meet the requirements of detection technical schemes generally accepted in industry.
Claims (3)
1. The application method of the cotton rose endogenous cytokinin extracting agent comprises the step of mixing an agent A and an agent B according to the mass ratio of 1-2:1; the method is characterized by comprising the following steps of:
a. preparation: preparing an extracting agent of the endogenous cytokinin of the cottonrose hibiscus: mixing the agent A and the agent B according to the mass ratio of 1-2:1 to prepare the cotton rose endogenous cytokinin extracting agent, and preserving at a low temperature of 10 ℃ for later use;
the agent A is at least 2 of glycine, phthalic acid, disodium hydrogen phosphate, citric acid, sodium citrate, sodium hydroxide, acetic acid, sodium acetate and hydrochloric acid, and is prepared into a mixed aqueous solution with the pH value of 2-3 after being dissolved by redistilled water;
the agent B is methanol;
b. extracting: taking a crushed sample of the cotton rose tissue, mixing the crushed sample with the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:5-10 mL, standing for 4-8 h at the temperature lower than 10 ℃, carrying out suction filtration, collecting filtrate I, leaching the obtained filter residue I for 1-2 h by using the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:2.5-5 mL, carrying out suction filtration, and collecting filtrate II and filter residue II; c, leaching the filter residue II by using the cotton rose endogenous cytokinin extracting agent obtained in the step a according to the solid-to-liquid ratio of 1 g:1-2.5 mL for 1h, filtering, and collecting filtrate III and filter residue III;
c. purifying: and c, combining the filtrates I, II and III obtained in the step c, extracting by PVPP solid-liquid extraction small columns, and collecting column-passing liquid to obtain the endogenous cytokinin to-be-detected liquid of the cotton rose tissue sample.
2. The method of claim 1, wherein the tissue sample of hibiscus in step b is at least one of a hibiscus leaf, petal, flower stalk, calyx, shoot or root, stem tissue or organ.
3. The use method according to claim 1, wherein the endogenous cytokinin test solution of the cotton rose tissue sample obtained in the step c is detected by adopting HPLC or LC-MS, so as to obtain the content of the endogenous cytokinin of the cotton rose tissue sample.
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