Detection Method of Phytosterols in Peony Pollen and Stamens

Abstract

This invention discloses a method for detecting phytosterol content in peony pollen and stamens. The method for detecting phytosterols in peony pollen and stamens provided by this invention includes the following steps: (1) hydrolysis; (2) extraction; (3) washing with water; (4) derivatization; (5) standard preparation; and (6) GC-MS / MS analysis. To ensure accurate detection of phytosterol components in peony pollen, this invention employs gas chromatography-mass spectrometry (GC-MS), and simultaneously investigates the effects of alkaline hydrolysis and acid-base hydrolysis on the detection of sterols in the samples. The pretreatment process and chromatographic analysis method for phytosterols are optimized to establish a simple, reproducible, and accurate GC-MS method for phytosterol analysis.

Description

Technical Field

[0001] The invention relates to a method for detecting phytosterols, in particular to a method for detecting phytosterols in peony pollen and stamens. Background Art

[0002] Peony is an ornamental and medicinal plant unique to my country. It has a cultivation history of more than 2,000 years in my country, and its planting area is nearly one million mu. The whole peony is a treasure. The utilization of its roots, stems, leaves, flowers and seeds has a certain research foundation, and it has shown good prospects in the food, medical, health care and other industries. However, the annual output of peony pollen is estimated to reach thousands of tons, but due to the lack of research and development efforts, a large amount of resources are wasted.

[0003] Pollen is the male reproductive cell on the plant body and the essence of the plant. It is considered to be a new type of green and healthy natural health product and tonic medicine raw material. It has significant therapeutic effects in regulating metabolism, improving the body's immunity, lowering blood lipids, anti-cancer, and preventing prostate diseases. Among them, flavonoids, fatty acids, and especially phytosterols are considered to be its main pharmacologically active ingredients. There are many reports on flavonoids and fatty acid components in pollen, but there are fewer studies on the determination of its main active ingredient, phytosterols. Phytosterols are a natural product with multiple physiological functions such as lowering blood cholesterol, preventing and treating prostate hypertrophy, enhancing immunity, anti-cancer, and hormone-like.

[0004] Since the analysis of plant sterols is relatively complicated, the methods in the literature vary greatly. In addition, there are more reports on sterols in oil samples, but fewer reports on sterol detection in other food samples, and even fewer reports on pollen samples. Summary of the invention

[0005] In order to make up for the deficiencies in the above fields, the main purpose of the present invention is to provide a method for detecting the phytosterol content in peony pollen and stamens with simple operation, good repeatability, high accuracy and sensitivity.

[0006] The method for detecting phytosterols in peony pollen and stamens provided by the present invention comprises the following steps:

[0007] (1) Hydrolysis: adding internal standards to peony pollen and stamen samples respectively, and adding potassium hydroxide-90% ethanol aqueous solution at a solid-liquid ratio of 1:15 to 1:35 g / mL respectively, mixing, cooling to room temperature after water bath, and obtaining peony pollen and stamen reaction solutions respectively;

[0008] (2) Extraction: Pour the peony pollen and stamen reaction solutions into a separating funnel respectively, add water and n-hexane for extraction, let stand in the separating funnel to separate the layers, and collect the organic layer extracts of the peony pollen and stamens;

[0009] (3) Washing: Wash the peony pollen and stamen extracts with water until neutral, dehydrate with anhydrous sodium sulfate, place in a rotary evaporator, concentrate, and blow dry with nitrogen to obtain a dry sample;

[0010] (4) Derivatization: Add a derivatizing agent and pyridine to the dried sample for silanization derivatization, vortex mix, and blow dry with nitrogen after water bath; add n-hexane to make up the volume, filter the membrane, and obtain the peony pollen and stamen sample solutions;

[0011] (5) Preparation of standard substances: preparing a series of standard solutions containing both an internal standard and a plurality of plant sterols to be tested, performing silylation derivatization according to the method of step (4), and performing gas chromatography-mass spectrometry; using the concentration of the sterol standard substance as the abscissa and the ratio of its peak area to the internal standard peak area as the ordinate to obtain an internal standard standard working curve;

[0012] (6) GC-MS / MS analysis: The sample solution and the standard substance were respectively injected into a gas chromatograph for analysis, and the sample was qualitatively analyzed by the standard substance, retention time, NIST14 mass spectrum library and related literature; the content of phytosterol in the peony pollen and stamen samples was calculated using the peak area ratio of the phytosterol in the sample solution to the internal standard corresponding to the standard curve.

[0013] Preferably, in the potassium hydroxide-90% ethanol aqueous solution in step (1), the concentration of potassium hydroxide in the 90% ethanol aqueous solution is 3 to 6 mol / L.

[0014] Preferably, the extraction in step (2) is: shaking extraction in a separatory funnel for 3 to 5 minutes.

[0015] Preferably, in step (2), the volume ratio of the potassium hydroxide-90% ethanol aqueous solution, water, and n-hexane is 1:1:2 to 3:1:5 mL / mL / mL.

[0016] Preferably, the derivatizing agent in step (4) is N,O-bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane); the mass volume ratio of the sample to the derivatizing agent is 2:1 to 1:2 mg / μL.

[0017] Preferably, the conditions for the GC-MS / MS analysis in step (6) are as follows: chromatographic column: HP-5MS; heating program: 200°C for 1 min, heating to 265°C at 5°C / min, holding for 2 min, then heating to 283°C at 3°C / min, holding for 5 min, heating to 300°C at 2°C / min, holding for 10 min; split ratio: 15:1; injection port temperature: 300°C; injection volume: 1 μL; carrier gas is high-purity helium, and its flow rate is set at: 1.0 mL / min; EI ion source, electron energy: 70 eV, transmission line temperature: 300°C, ion source temperature: 230°C, quadrupole temperature: 150°C, mass scanning range: 40-650 m / z.

[0018] Preferably, the peony pollen sample needs to be subjected to a wall-breaking treatment before hydrolysis; and the peony stamen sample does not need to be subjected to a wall-breaking treatment before hydrolysis.

[0019] Preferably, the best time to harvest the peony pollen is the color-revealing stage, and the best time to harvest the peony stamens is the hard bud stage; if both are harvested at the same time, the color-revealing stage is selected.

[0020] Preferably, the peony variety is 'Fengdan'.

[0021] After the qualitative analysis in step (6), if the chemical structure of the plant sterols in the sample does not contain a methylene side chain or an ethylene side chain or a 9,19-cyclopropane structure, the step (1) of the sample sterol analysis is changed to an acid-base hydrolysis step: adding a hydrochloric acid-ethanol solution to the sample, mixing, and cooling to room temperature after a water bath; then adding a potassium hydroxide-90% ethanol aqueous solution, mixing, and cooling to room temperature after a water bath to obtain a reaction solution, and then performing the above steps (2) to (6) to perform sterol detection.

[0022] The present invention takes the pollen of the main oil-producing peony variety 'Fengdan' with the largest planting area as the research object, and studies the main active ingredients of its pollen. In order to ensure the accurate detection of the sterol components of peony pollen, the present invention adopts GC-MS for detection, and simultaneously investigates the influence of alkaline hydrolysis and acid-base hydrolysis on the extraction of sterols in the sample, and optimizes the plant sterol pretreatment process and chromatographic analysis method, aiming to establish a simple, reproducible and highly accurate plant sterol analysis method. In addition, since pollen will produce a large amount of stamens (the part after the anthers are depollened) during the collection process, the two may have similar active ingredients, so the present invention conducts parallel research on them for the first time, laying a foundation for the excavation of peony resources. At the same time, the present invention also investigates the influence of different periods and temperature difference wall breaking on its plant sterol content. It is intended to provide a basis for the scientific detection of peony pollen and stamens, and also provide a reference for the accurate detection of sterol components of other foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] For purposes of illustration and not limitation, the present invention will now be described in terms of preferred embodiments thereof, with particular reference to the accompanying drawings, in which:

[0024] Figure 1 These are the pollen and stamens of 'Fengdan' harvested at different times.

[0025] Figure 2 GC-MS chromatograms of phytosterols from pollen and stamens of 'Fengdan' after different pretreatments; (A) Alkaline hydrolysis extraction of pollen (B) Acid-base hydrolysis extraction of pollen (C) Alkaline hydrolysis extraction of stamens (D) Acid-base hydrolysis extraction of stamens; chromatographic peaks with the same molecular weight are marked with the same color.

[0026] Figure 3 This is the chemical structural formula of phytosterols in pollen and stamens of 'Fengdan'.

[0027] Figure 4 This is the possible metabolic mechanism catalyzed by isofucosterol, 24-methylenecholesterol, and cycloartenolic acid.

[0028] Figure 5 Flow chart for sterol analysis of samples. DETAILED DESCRIPTION

[0029] It should be noted that those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0030] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0031] Example 1: Detection of phytosterols in peony pollen and stamens (acid-base hydrolysis method)

[0032] 1. Sample collection

[0033] Sampling was carried out before 10 am, and anthers of 'Fengdan' (peony planting area in Changzhi City, Shanxi Province) at different stages were collected, dried under natural ventilation at room temperature (20-25℃), and then sieved repeatedly to separate the anthers into pollen and stamens ( Figure 1 ), mechanically crushed, passed through an 80-mesh sieve, dried in a vacuum drying oven for 3 days, and the completely dried pollen and stamens were sealed and stored in a brown glass desiccator.

[0034] 2. Acid-base hydrolysis extraction of phytosterols

[0035] (1) Hydrolysis: Accurately weigh 0.3 g of each pollen and stamen sample, place in a 16 mL glass stoppered centrifuge tube, add 0.6 mg / mL internal standard (1 mL for pollen and 0.2 mL for stamen), add 2 mol / L hydrochloric acid-ethanol (5 mL), vortex mix, place in a water bath at 80°C for 60 min, shake every 10 min, and cool to room temperature. Add 10 mL of potassium hydroxide-90% ethanol aqueous solution (4 mol / L), vortex mix, keep constant temperature at 80°C for 60 min, shake every 10 min, and cool to room temperature to obtain a reaction solution.

[0036] (2) Extraction: Pour the reaction solution into a separatory funnel, add 10 mL of water, and then add 20 mL of n-hexane three times for extraction for about 3 min each time. Allow to stand in a separatory funnel to separate the layers and collect the extract.

[0037] (3) Washing: Add 100 mL of water and wash the extract 3 times until it is neutral. Dehydrate it with anhydrous sodium sulfate and place it in a rotary evaporator. Concentrate it under reduced pressure at 32°C to 2 mL. Use a pipette to transfer the liquid to a stoppered glass test tube and blow it dry with nitrogen.

[0038] (4) Derivatization: Add 500 μL of derivatization agent (N,O-bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane)) and pyridine (1:1, v / v), vortex mix, place in a 75°C water bath for 40 min, and blow dry with nitrogen. Add chromatographic n-hexane to make up to volume (pollen to 5 mL, stamen to 1 mL), pass through a 0.22 um membrane, and place in a sample vial.

[0039] 3. GC-MS / MS analysis of silanized phytosterols

[0040] The analysis system is a GC7890A / MS5975C gas chromatograph-mass spectrometer, and the chromatographic column is HP-5MS (30m×0.25mm×0.25m, Agilent). The heating program is as follows: 200°C for 1 min, then increase the temperature to 265°C at 5°C / min, and maintain for 2 min, then increase the temperature to 283°C at 3°C / min, and maintain for 5 min, and then increase the temperature to 300°C at 2°C / min, and maintain for 10 min. Split ratio 15:

[0041] 1. The injection port temperature was 300°C, the injection volume was 1 L, the carrier gas was high-purity helium, and its flow rate was set at 1.0 mL / min; EI ion source, electron energy was 70 eV, transmission line temperature was 300°C, ion source temperature was 230°C, quadrupole temperature was 150°C, and mass scanning range was 40-650 m / z.

[0042] 4. Establishment of standard curve

[0043] The quantitative method of phytosterols adopts the internal standard curve method. 5α-cholestane, stigmasterol, β-sitosterol were purchased from Beijing Wokai Biotechnology Co., Ltd.; campesterol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 24-methylenecholesterol was purchased from Sigma. The standard substance is prepared into a series of standard solutions of different concentrations so that the sterol detection content in the sample is within the linear range: β-sitosterol concentration is 0.05, 0.2, 0.8, 1.6, 3.2, 6.4 mg / mL; campesterol concentration is 0.02, 0.05, 0.1, 0.2, 0.5, 1 mg / mL; stigmasterol concentration is 0.005, 0.01, 0.02, 0.05, 0.1, 0.3 mg / mL; 24-methylene-cholesterol concentration is 0.02, 0.1, 0.5, 2, 4, 8 mg / mL, of which the internal standard (5α-cholestane) concentration is 0.12 mg / mL. Silylation derivatization is performed according to the derivatization in step 3, and gas chromatography-mass spectrometry is performed. The concentration of the sterol standard is used as the horizontal coordinate, and the ratio of its peak area to the internal standard peak area is used as the vertical coordinate to obtain the internal standard standard working curve. The correlation coefficient r 2 >0.997, indicating good linearity. Other trace components in the sample were quantified based on their structures and similarities with the standard, as shown in Table 1.

[0044] Table 1 Standard curve and linear range of phytosterols

[0045]

[0046] 5. Identification of Phytosterol Structure

[0047] The present invention uses GC-MS / MS to analyze the phytosterol components of pollen and stamens of 'Feng Dan' under acid-base hydrolysis. Although there are many types of phytosterols and they are highly similar, most of the sterols are well separated, such as Figure 2 As shown, Figure B is the result of acid-base hydrolysis extraction of pollen, and Figure D is the result of acid-base hydrolysis extraction of stamens.

[0048] Through GC-MS / MS analysis, a variety of phytosterol isomers were detected in the pollen and stamens of 'Fengdan' under acid-base hydrolysis, which brought great difficulties to the structural identification of phytosterol compounds. The phytosterol compounds were repeatedly identified through standard substances, retention time, NIST14 mass spectrum library and literature. Finally, under this method, a total of 16 phytosterol compounds were identified in the pollen and stamens of 'Fengdan', as shown in Table 2. They are: cholesterol (cholesterol, 1), ergosta-5,23-dien-3β-ol (ergosta-5,23-dien-3β-ol, 2), 24-methylenecholesterol (24-methylenecholesterol, 3), campasterol (campasterol, 4), stigmasterol (stigmasterol, 5), 24-methyldesmosterol (24-methyldesmosterol, 6), △ 5,23 -Stigmasterol (△ 5,23 -stigmastadienol, 7), clerosterol, 8), obtusifoliol, 9, β-sitosterol, 10, β-amyrin, 11, isofucosterol, 12, parkeol, 13, △ 5,24 -Stigmasterol (△ 5,24 -stigmastadienol, 14), lanosterol (lanosterol, 15), cycloartenol (cycloartenol, 16). Among them, 13 kinds of phytosterols were identified in pollen (1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16) under acid-base hydrolysis, and 16 kinds of phytosterols were identified in stamens (1-16). There are a large number of isomers, and their chemical structures are as follows Figure 3 As shown above, it can be seen that the pollen and stamens of 'Fengdan' are rich in phytosterols, which lays the foundation for the realization of its active functions.

[0049] Table 2 Silylation identification of phytosterol components in pollen and stamens of 'Fengdan'

[0050]

[0051]

[0052] Note: a: Relative retention time, i.e. the ratio of the chromatographic peak to the elution time of β-Sitosterol (25.99 min). b: Base peak.

[0053] 6. Quantitative analysis of phytosterols

[0054] As shown in Table 3, 13 phytosterols were identified in the pollen of Fengdan by the acid-base hydrolysis method, and their contents were 24-methylenecholesterol>24-methyldesmosterol>ergosta-5,23-dien-3β-ol>campasterol. The types of phytosterols in the stamens were more than those in the pollen. 16 sterols were detected by this method, and their contents were β-sitosterol>campasterol>24-methyldesmosterol>24-methylenecholesterol. Among them, the three newly added sterol components in the stamens were △ 5,23 -stigmastadienol, clerosterol, obtusifoliol, these sterol contents are between 10.25-38.29mg / 100g. Although the stamens have more sterol types, the total sterol content of pollen (2965.62mg / 100g) is about 3 times that of stamens (1037.14mg / 100g). This is mainly because the content of 24-methylenecholesterol (1134.83mg / 100g) and 24-methyldesmosterol (1203.17mg / 100g) in pollen is relatively high, which is about 8 times the content of 24-methylenecholesterol (135.59mg / 100g) and 24-methyldesmosterol (158.55mg / 100g) in stamens. Through the detection of sterol components in pollen and stamens of 'Fengdan', it is known that the stamens have richer sterol types than pollen, and the content difference of the main sterol components is small, which may have a wider range of pharmacological activities. The total sterol content of pollen is much higher than that of stamens, and the main sterol components 24-methylenecholesterol and 24-methyldesmosterol account for about 80% of the total sterol content, which is a good raw material for the development and utilization of 24-methylenecholesterol and 24-methyldesmosterol.

[0055] Table 3 Phytosterol content in pollen and stamens of 'Fengdan' under acid-base hydrolysis method (mg / 100g, n=3)

[0056]

[0057] Example 2: Detection of phytosterols in peony pollen and stamens (alkaline hydrolysis method)

[0058] On the basis of Example 1, the hydrolysis operation of 2. Acid-base hydrolysis extraction of phytosterols (1) was changed to 2. Alkaline hydrolysis extraction of phytosterols (1) Hydrolysis: Accurately weigh 0.3 g of pollen and stamen samples, respectively, and place them in 16 mL glass stoppered centrifuge tubes, respectively add 0.6 mg / mL 5α-cholestane internal standard (pollen 1 mL, stamen 0.2 mL), add 4 mol / L potassium hydroxide-90% ethanol aqueous solution (10 mL), vortex mix, water bath at 80°C for 60 min, shake every 10 min, cool to room temperature, and obtain a reaction solution. The other steps are the same as in Example 1.

[0059] 1. Identification of phytosterol structure

[0060] The present invention uses GC-MS / MS to analyze the phytosterol components of pollen and stamens of 'Feng Dan' under alkaline hydrolysis. Although there are many types of phytosterols and they are highly similar, most of the sterols are well separated, such as Figure 2 As shown, Figure A is the result of pollen alkaline hydrolysis extraction, and Figure C is the result of stamen alkaline hydrolysis extraction.

[0061] In the GC-MS / MS analysis, the phytosterol compounds were repeatedly identified by comparing the standard, retention time, NIST14 mass spectral library and literature. Finally, under this method, a total of 11 phytosterol compounds were identified in the pollen and stamens of 'Fengdan', as shown in Table 2. They are: cholesterol (cholesterol, 1), 24-methylenecholesterol (24-methylenecholesterol, 3), campasterol (campasterol, 4), stigmasterol (stigmasterol, 5), obtusifoliol (obtusifoliol, 9), β-sitosterol (β-amyrin, 10), isofucosterol (isofucosterol, 12), △ 5,24 -Stigmasterol (△ 5,24 -stigmastadienol, 14), lanosterol (lanosterol, 15), cycloartenol (cycloartenol, 16). Among them, under alkaline hydrolysis, 6 kinds of phytosterols were identified in pollen (3, 5, 10, 11, 12, 16), and 11 kinds of phytosterols were identified in stamens (1, 3, 4, 5, 9, 10, 11, 12, 14, 15, 16). There are a large number of isomers, and their chemical structures are as follows Figure 3 As shown above, it can be seen that the pollen and stamens of 'Fengdan' are rich in phytosterols, which lays the foundation for the realization of its active functions.

[0062] 2. Quantitative analysis of phytosterols

[0063] Table 4 shows that the contents of the six phytosterols in the pollen of Fengdan are 24-methylenecholesterol>β-sitosterol>cycloartenol. The types of phytosterols in the stamens are more than those in the pollen, with 11 types detected, and their contents are 24-methylenecholesterol>β-sitosterol>campasterol>isofucosterol. Among them, the five newly added sterol components in the stamens are cholesterol, lanosterol, campasterol, △ 5,24 -stigmastadienol, obtusifoliol, except for campasterol (155.36mg / 100g), the other sterols are in the range of 9.04-15.89mg / 100g. In addition, the sterol content of the same sterols in the stamens is higher than that in the pollen (except 24-methylenecholesterol). Although the stamens have more sterol types, the total sterol content of pollen (3009.57mg / 100g) is more than 3 times that of the stamens (910.44mg / 100g). This is mainly because the 24-methylenecholesterol in the pollen is as high as 2857.69mg / 100g, which is more than 8 times that of the 24-methylenecholesterol in the stamens (348.28mg / 100g). Through the detection of sterol components in pollen and stamens of 'Fengdan', it can be known that the stamens have richer sterol types than pollen, and the difference in the content of the main sterol components is small, which may have a wider range of pharmacological activities. The total sterol content of pollen is much higher than that of stamens, and the main sterol component 24-methylenecholesterol accounts for 95% of the total sterol content, which is a good raw material for the development and utilization of 24-methylenecholesterol.

[0064] Table 4 Phytosterol content in pollen and stamens of 'Fengdan' under alkaline hydrolysis method (mg / 100g, n=3)

[0065]

[0066]

[0067] 3. Comparison of phytosterol extraction methods (alkaline hydrolysis and acid-base hydrolysis)

[0068] Alkaline hydrolysis and acid-base hydrolysis were used to determine the phytosterols in pollen and stamens of 'Fengdan', as shown in Tables 3 and 4. Whether it is pollen or stamens, there are large differences in the types of sterols and the contents of some sterols determined by the two methods, but the total sterol content is similar. It can be found from the structure of sterol compounds that there are a large number of sterol isomers in pollen and stamens, and the content of each isomer under the two detection methods is quite different. We took the stamens with the most sterol types as the research object, and summarized the contents of phytosterol isomeric compounds measured in the stamen samples of the three periods, and the results are shown in Table 5. It can be found that the ratio of the total content of three groups of isomer sterols is close to 1 under the two detection methods. Based on the similarity of structure and the closeness of total content, it can be concluded that the pollen and stamens of 'Feng Dan' will degrade isofucansterol, 24-methylene cholesterol, and cycloartenol under strong acid conditions, and partially isomerize into isomers, so that more sterol components are detected in the pollen and stamens of 'Feng Dan' under acid-base hydrolysis, but compared with alkaline hydrolysis, the total sterol content is almost the same. It is further speculated that plant sterols containing methylene side chains, ethylene side chains and 9,19-cyclopropane structures may also undergo rearrangement reactions under strong acid conditions and isomerize into isomers. Its possible reaction mechanism is as follows Figure 4 shown.

[0069] That is, for the pollen, stamens or other sterol samples containing these three groups (methylene side chain, ethylene side chain and 9,19-cyclopropane structure), alkaline hydrolysis can only be used to detect sterols. However, if the sterol components in the sample do not contain these three groups, acid-base hydrolysis may be more suitable for the detection of its sterol components. Figure 5 shown.

[0070] Table 5 Content of phytosterol isomers in the pistil of 'Fengdan' under alkaline hydrolysis and acid-base hydrolysis (mg / 100g, n=3)

[0071]

[0072]

[0073] Note: nd: the content is too low to be detected. Ratio: total sterol content by alkaline hydrolysis / total sterol content by acid-base hydrolysis.

[0074] Example 3: Optimization of the Detection Method of Phytosterols in Peony Pollen and Stamens

[0075] Since the analysis of phytosterols is relatively complex, the literature methods vary greatly, and there are more reports on sterols in oil samples, fewer reports on sterol detection in other food samples, and even fewer reports on pollen samples. In order to ensure the accuracy of the results of phytosterol determination in peony pollen and stamens, we optimized the key parameters for the extraction and analysis of phytosterols, and the optimization aspects are as follows. The other steps are the same as in Example 1.

[0076] 1. Optimization of phytosterol extraction method

[0077] On the basis of Example 1, the key parameters in operation 2, extraction of phytosterols, were optimized. Only one operation step was changed for each sterol detection, and the other steps remained the same as in Example 1. The operation steps investigated were as follows:

[0078] (1) Hydrolysis: ① Alkaline hydrolysis extraction: no acid was added, and the concentrations of the alkali were 1, 2, 4, and 6 moL / L. ② Acid-base hydrolysis extraction: the acid concentration remained unchanged, and the concentrations of the alkali were 2, 4, 6, and 8 moL / L. Under both methods, when the alkali concentration was 4 moL / L, the total sterol content detected was the highest for both pollen and stamens, which was a more appropriate concentration for alkaline hydrolysis and acid-base hydrolysis. See Tables 6 and 7.

[0079] (2) Extraction: ① Extraction in a separatory funnel for 3 minutes. ② Vortex extraction in a centrifuge tube for 1 minute. ③ Ultrasonic extraction in a centrifuge tube for 10 minutes. The investigation of these three extraction methods shows that vortex extraction is prone to emulsification and is difficult to separate. Ultrasonic extraction has low extraction efficiency. Extraction in a separatory funnel has high extraction efficiency and faster separation.

[0080] (3) The amount of derivatization agent (150 μL, 250 μL, 300 μL) showed that when the derivatization agent was 150 μL, the pollen sterol peak was not completely derivatized, while when the derivatization agent was 250 μL and 300 μL, the derivatization was complete. Therefore, the amount of derivatization agent was 250 μL.

[0081] Table 6 Effect of different alkali concentrations on the sterol content of pollen and stamens of 'Fengdan' under alkaline hydrolysis (mg / 100g, n=3)

[0082]

[0083] Note: 1: Alkali concentration. 2: Low content not detected. 3: Total sterol content.

[0084] Table 7 Effects of different alkali concentrations on the sterol content of pollen and stamens of 'Fengdan' under acid-base hydrolysis (mg / 100g, n=3)

[0085]

[0086]

[0087] Note: 1: Alkali concentration. 2: Low content not detected. 3: Total sterol content.

[0088] 2. Optimization of GC-MS / MS analysis method for silanization of phytosterols

[0089] Based on Example 1, the key parameters in operation 3, GC-MS / MS analysis of phytosterol silanization, were optimized, and only one operation step was changed for each sterol detection, and the other steps remained the same as in Example 1. The aspects investigated were as follows:

[0090] (1) Inlet temperature (280℃, 300℃). The results show that the separation effect of some main peaks is better when the injection temperature is 300℃ than when it is 280℃.

[0091] (2) Split ratio (10:1, 15:1, 20:1, 40:1). When the split ratio is 10:1, the sample volume is too large, the main peak is too high, and the separation effect of some peaks is poor. When the split ratio is 40:1 and 20:1, some small peaks are not identified. Overall, the split ratio of 15:1 is more appropriate.

[0092] (3) Flow rate (1 mL / min, 1.2 mL / min, 1.5 mL / min). When the flow rate was 1.5 mL / min, the separation effect of some peaks was poor. When the flow rates were 1 mL / min and 1.2 mL / min, the separation effect of sterol peaks was better. The final flow rate was 1 mL / min.

[0093] (4) Elution gradient. Since the phytosterols in the pollen and stamens of 'Feng Dan' have similar structures and multiple components, the elution time is relatively concentrated, and the elution gradient needs to be optimized. In order to separate the internal standard from other components, the separation temperature is set at 265-270°C. In order to optimize the separation of peaks 2, 3, 4 and peaks 10, 11, 12 (such as Figure 2 ), the heating rate is 2-3℃ / min, and the temperature is set at 280-285℃.

[0094] Example 4: Detection of phytosterols in pollen and stamens of peony flowers after wall breaking and at different flowering periods

[0095] 1. Sample collection

[0096] Sampling was carried out before 10 am, and anthers of 'Fengdan' (peony planting area in Changzhi City, Shanxi Province) at different stages were collected, dried under natural ventilation at room temperature (20-25℃), and then sieved repeatedly to separate the anthers into pollen and stamens ( Figure 1 ), mechanically crushed, passed through an 80-mesh sieve, dried in a vacuum drying oven for 3 days, and the completely dried pollen and stamens were sealed and stored in a brown glass desiccator.

[0097] 2. Pollen and stamen wall breaking

[0098] The collected pollen and stamens of 'Fengdan' were broken by temperature difference method: appropriate amount of pollen and stamen powder were weighed and wrapped in multiple layers of tin foil, and placed in a -70℃ refrigerator for 24 hours. After taking out, they were quickly placed in a 95℃ water bath for 10 minutes for temperature difference breaking.

[0099] 3. Detection of phytosterols

[0100] Based on Example 1 and Example 2, it can be known that the pollen and stamens of 'Fengdan' will cause the sterol components 24-methylenecholesterol, cycloartenol, and isofucosterol to rearrange under strong acid conditions, and partially isomerize into isomers. Therefore, we use the alkaline hydrolysis method (Example 2) to detect the sterol components in the sample. In addition, wall breaking may make the sterol components in the sample better detected, and the sterol content of pollen and stamens at different flower development stages may also be different, so we investigated this. Wall breaking and different harvesting periods (hard bud stage, color exposure stage, initial opening stage, such as Figure 1 The results of phytosterol content in peony pollen and stamens of ) are shown in Tables 8 and 9.

[0101] Table 8 Phytosterol content of 'Fengdan' pollen before and after cell wall breaking at different times under alkaline hydrolysis (mg / 100g, n=3)

[0102]

[0103] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05). nd: The content is too low to be detected.

[0104] Table 9 Phytosterol content of 'Fengdan' flower stamens before and after wall breaking at different times under alkaline hydrolysis (mg / 100g, n=3)

[0105]

[0106]

[0107] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05). nd: The content is too low to be detected.

[0108] During the structural identification, we detected 6 phytosterol components in the pollen of Fengdan, of which 2 sterol components (stigmasterol and β-amyrin) were identified in peony pollen for the first time. In the stamens of Fengdan, 11 phytosterols were detected. Although the sterol types are richer than those of pollen, they are used for phytosterol research for the first time. Compared with peony pollen, the stamens of Fengdan have 5 sterol components (stigmasterol, β-amyrin, lanosterol, △ 5,24 -stigmastadienol, obtusifoliol) were identified for the first time; compared with other pollen varieties, there are two sterol components (△ 5,24 -stigmastadienol, obtusifoliol) was identified for the first time.

[0109] Comparison before and after cell wall breaking shows that cell wall breaking increases the sterol content of pollen in Fengdan at different stages, but has little effect on the sterol content of stamens. Therefore, cell wall breaking is suitable for the detection of pollen sterols, while stamen sterols do not require cell wall breaking. It can be seen that the detection of sterol components does not always require sample cell wall breaking, but depends on the sample.

[0110] By analyzing the phytosterol content of pollen and stamens of 'Feng Dan' at the hard bud stage, coloring stage, and initial opening stage, it can be found that the best time to harvest pollen of 'Feng Dan' is the coloring stage, and the best time to harvest stamens is the hard bud stage, but if both are harvested at the same time, the coloring stage is more suitable considering the total sterol content. The present invention provides a basis for the reasonable harvesting time of pollen and stamens of 'Feng Dan'.

[0111] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting phytosterols in peony pollen and stamens, comprising the following steps: (1) Hydrolysis: adding internal standards to peony pollen and stamen samples respectively, and adding potassium hydroxide-90% ethanol aqueous solution at a solid-liquid ratio of 1:15 to 1:35 g / mL respectively, mixing, cooling to room temperature after water bath, and obtaining peony pollen and stamen reaction solutions respectively; (2) Extraction: Pour the peony pollen and stamen reaction solutions into a separating funnel respectively, add water and n-hexane for extraction, let stand in the separating funnel to separate the layers, and collect the organic layer extracts of the peony pollen and stamens; (3) Washing: Wash the peony pollen and stamen extracts with water until neutral, dehydrate with anhydrous sodium sulfate, place in a rotary evaporator, concentrate, and blow dry with nitrogen to obtain a dry sample; (4) Derivatization: Add derivatization agent and pyridine to the dried sample for silylation derivatization, vortex mix, and blow dry with nitrogen after water bath; Add n-hexane to make up the volume, filter the membrane, and obtain the peony pollen and stamen sample test solutions respectively; (5) Preparation of standard substances: preparing a series of standard solutions containing both an internal standard and a plurality of plant sterols to be tested, performing silylation derivatization according to the method of step (4), and performing gas chromatography-mass spectrometry; using the concentration of the sterol standard substance as the abscissa and the ratio of its peak area to the internal standard peak area as the ordinate to obtain an internal standard standard working curve; (6) GC-MS / MS analysis: The sample solution and the standard substance were respectively injected into a gas chromatography-mass spectrometer for analysis, and the sample was qualitatively analyzed by the standard substance, retention time, and NIST14 mass spectrum library; the content of phytosterol in the peony pollen and stamen samples was calculated by using the peak area ratio of phytosterol in the sample solution to the internal standard corresponding to the standard curve; The 11 phytosterols in the peony pollen and stamens are cholesterol, 24-methylene cholesterol, campesterol, stigmasterol, phytosterol, β-sitosterol, β-amyrin, isofucansterol, △ 5,24 -Stigmasterol, lanosterol, cycloartenol.

2. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: In the potassium hydroxide-90% ethanol aqueous solution in step (1), the concentration of potassium hydroxide in the 90% ethanol aqueous solution is 3 to 6 mol / L.

3. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The extraction in step (2) is as follows: extracting by shaking in a separatory funnel for 3 to 5 minutes.

4. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The volume ratio of the potassium hydroxide-90% ethanol aqueous solution, water and n-hexane in step (2) is 1:1:2 to 3:1:

5.

5. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The derivatizing agent in step (4) is N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane; the mass volume ratio of the sample to the derivatizing agent is 2:1 to 1:2 mg / μL.

6. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The conditions for the GC-MS / MS analysis in step (6) are as follows: chromatographic column: HP-5MS; temperature program: 200°C for 1 min, then increase the temperature to 265°C at 5°C / min, maintain for 2 min, then increase the temperature to 283°C at 3°C / min, maintain for 5 min, increase the temperature to 300°C at 2°C / min, maintain for 10 min; split ratio: 15:1; injection port temperature: 300°C; injection volume: 1 μL; carrier gas is high-purity helium, and its flow rate is set at: 1.0 mL / min; EI ion source, electron energy: 70 eV, transmission line temperature: 300°C, ion source temperature: 230°C, quadrupole temperature: 150°C, mass scanning range: 40-650 m / z.

7. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The peony pollen sample needs to be subjected to a wall-breaking treatment before hydrolysis; the peony stamen sample does not need to be subjected to a wall-breaking treatment before hydrolysis.

8. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The harvesting period of the peony pollen is the color-revealing stage, and the harvesting period of the peony stamen is the hard bud stage; if both are harvested at the same time, the color-revealing stage is selected.

9. The method for detecting phytosterols in peony pollen and stamens according to claim 1, characterized in that: The peony variety is Fengdan.

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