Evaluation method for enrichment effect of phenolic amide natural products in Chinese wolfberry

Abstract

The invention discloses a method for evaluating the enrichment effect of phenolic amide natural products in Chinese wolfberry fruits. The method comprises the following steps: step 1, preparing a Chinese wolfberry fruit crude extract containing the phenolic amide natural products by adopting an ethanol extracting solution; carrying out macroporous resin chromatography on the crude extract, and separating to obtain a phenolic amide natural product enriched product; 2, selecting hydrophilic and hydrophobic substances as molecular probes, and evaluating the enrichment effect of the enriched substance in the step 1; according to the method, hydrophilic and hydrophobic substances are selected as combined molecular probes for comprehensively evaluating the enrichment condition of phenolic amide natural products in products obtained through extraction and separation, and the substances are used as indexes for evaluating the separation effect in a combined form.

Description

Technical Field

[0001] The invention relates to the technical field of plant separation and extraction, and in particular to a method for evaluating the enrichment effect of phenolamide natural products in wolfberry. Background Art

[0002] Phenolamides (hereinafter referred to as phenolamides) are a class of secondary metabolites found in the fruits, seeds, leaves, and roots of higher plants. They are widely distributed in plants from the Solanaceae family (e.g., wolfberry and tomato), the Poaceae family (e.g., corn), the Cruciferae family (e.g., Arabidopsis), the Leguminosae family (e.g., soybean), and the Asteraceae family (e.g., sunflower). Phenolamides not only play a key role in physiological processes such as plant defense mechanisms, growth and development, cell wall synthesis, and fruit ripening, but also exhibit great potential for application in the healthcare sector due to their antioxidant, anti-inflammatory, and neuroprotective properties.

[0003] Phenolamides are a class of compounds formed by the covalent linkage of hydroxycinnamic acid and its derivatives (such as p-coumaric acid and caffeic acid) with aromatic amines (such as tyramine) or aliphatic amines (such as spermidine and spermine) via amide bonds. The diversity of hydroxycinnamic acids and their derivatives, the diversity of aromatic or aliphatic amines, and the diversity of amide bond-forming reactions in plants have led to a wide variety of phenolamides in plants, with more than 160 species discovered to date. However, phenolamides are present in low abundance in plant systems (generally less than 0.1% of the total extract), making direct detection or separation extremely difficult. Through enrichment (such as solid-phase extraction, macroporous resin adsorption, or liquid-liquid partitioning), the concentration of the target compound can be increased to a detectable range, avoiding missed detection due to low levels. On the other hand, plant extracts have complex components (such as polysaccharides, pigments, lipids, etc.), and enrichment can selectively remove most interfering substances, providing a "clean" sample for subsequent purification steps such as high-performance liquid chromatography (HPLC).

[0004] There is a lack of effective methods for evaluating the enrichment effect of phenolamides in the prior art, and traditional evaluation methods mostly use a single target molecule to evaluate the enrichment effect. The phenolamide natural products in wolfberry extracts are usually composed of various combinations of molecules such as tyramine, putrescine, spermidine, and spermine as acyl acceptors and hydroxycinnamic acid through covalent bonds. The different carboxyl donors of hydroxycinnamic acid, the diversity of amide bond formation, and the diversity of post-modification (such as glycosylation modification) result in phenolamide substances being hydrophilic and hydrophobic. According to the existing method, the highest abundance is selected as the indicator probe to evaluate the enrichment effect and determine the enrichment parameters, which may cause the polarity of the opposite probe to be diluted or lost during the enrichment. For example, the patent with publication number CN105758977A discloses a method for detecting the quality of black wolfberry fruit or black wolfberry fruit extract, which is determined by high performance liquid chromatography. The chromatographic conditions are: octadecyl bonded phase silica gel is used as the filler, the mobile phase is used for separation, and the black wolfberry phenolamine A component is detected by ultraviolet detector. It selected phenolamine A as a reference substance, but did not specify which substance was selected, nor did it distinguish between the hydrophilic and hydrophobic properties of the selected substance. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present invention provides an evaluation method for the enrichment effect of phenolamide natural products in wolfberry.

[0006] The technical solution adopted by the present invention is: a method for evaluating the enrichment effect of phenolamide natural products in wolfberry, comprising the following steps:

[0007] Step 1: preparing a crude extract of wolfberry fruit containing phenolamide natural products using an ethanol extraction solution; subjecting the crude extract to macroporous resin chromatography, and then separating to obtain an enrichment of the phenolamide natural products;

[0008] Step 2: Select hydrophilic and hydrophobic substances as molecular probes to evaluate the enrichment effect of the enriched material in step 1.

[0009] Furthermore, the hydrophilic substance in step 2 includes one or both of N1,N3-dihydrocaffeoylspermidine and N1,N3-dicaffeoylspermidine.

[0010] Furthermore, the hydrophobic substance in step 2 includes one or both of N-feruloylputrescine and N-caffeoylputrescine.

[0011] Furthermore, the method for selecting the molecular probe in step 2 is as follows:

[0012] The crude extract obtained in step 1 is subjected to ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to obtain the distribution of phenolamide natural products in the crude extract; and a molecular probe is determined based on the distribution of the phenolamide natural products.

[0013] Furthermore, the process of determining the molecular probe according to the distribution of phenolamide natural products is as follows:

[0014] Calculate the peak areas of hydrophilic and hydrophobic substances in the distribution results of phenolamide natural products respectively;

[0015] According to the set threshold, substances with peak areas exceeding the threshold in hydrophilic substances and hydrophobic substances are used as molecular probes.

[0016] Furthermore, the ethanol extract in step 1 is ethanol with a concentration of 65%.

[0017] Furthermore, the preparation process of the crude extract in step 1 is as follows:

[0018] The wolfberry fruit was dried and crushed, and the extract was added at a material-liquid ratio of 1:5, and ultrasonicated to obtain a crude extract solution;

[0019] The crude extract solution is centrifuged to remove the precipitate to obtain the crude extract.

[0020] Furthermore, in step 1, the macroporous resin chromatography adopts gradient elution, and the obtained substance is the enriched product after drying.

[0021] Furthermore, the A and B pumps in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry instrument use aqueous solution and acetonitrile solution respectively.

[0022] Furthermore, formic acid is mixed in both the aqueous solution and the acetonitrile solution; wherein the volume concentration of the formic acid is 0.1%.

[0023] The beneficial effects of the present invention are:

[0024] The present invention selects hydrophilic and hydrophobic substances as combined molecular probes for comprehensively evaluating the enrichment of phenolamide natural products in the products obtained by extraction and separation, and uses them in combination as an indicator for evaluating the separation effect to establish the optimal extraction and separation conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the total ion current diagram of the crude extract of the wolfberry sample in Example 1 of the present invention.

[0026] Figure 2 The peak area statistics of the characteristic phenolamides in wolfberry fruit in Example 1 of the present invention are shown.

[0027] Figure 3 This is the peak electrode statistical result of the characteristic phenolamide in wolfberry fruit in Example 1 of the present invention.

[0028] Figure 4is the molecular structural formula of the probe combination compound used in Example 1 of the present invention.

[0029] Figure 5 These are the results of evaluating and assessing the enrichment effect of phenolamide compounds using combined molecular probes and individual molecular probes in Example 1 of the present invention.

[0030] Figure 6 The results are as follows: the enrichment effect of phenolamide compounds in wolfberry fruits from different production areas was evaluated using the method of Example 1 of the present invention. DETAILED DESCRIPTION

[0031] A method for evaluating the enrichment effect of phenolamide natural products in wolfberry comprises the following steps:

[0032] Step 1: Using ethanol extraction solution to prepare a crude extract of wolfberry fruit containing phenolamide natural products; the crude extract is subjected to macroporous resin chromatography, and then separated to obtain an enrichment of phenolamide natural products; the macroporous resin chromatography adopts gradient elution, and the obtained substance is dried to obtain the enrichment.

[0033] Step 2: Select hydrophilic and hydrophobic substances as molecular probes to evaluate the enrichment effect of the enriched material in step 1.

[0034] The hydrophilic substance includes one or both of N1,N3-dihydrocaffeoylspermidine and N1,N3-dicaffeoylspermidine. The hydrophobic substance includes one or both of N-feruloylputrescine and N-caffeoylputrescine.

[0035] The molecular probe selection method is as follows:

[0036] The crude extract obtained in step 1 is analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to determine the distribution of phenolamide natural products in the crude extract; and a molecular probe is identified based on the distribution of the phenolamide natural products. Pumps A and B of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry utilize an aqueous solution and an acetonitrile solution, respectively. Formic acid is mixed into both the aqueous solution and the acetonitrile solution; the volume concentration of the formic acid is 0.1%.

[0037] The process of determining molecular probes based on the distribution of phenolamide natural products is as follows:

[0038] Calculate the peak areas of hydrophilic and hydrophobic substances in the distribution results of phenolamide natural products respectively;

[0039] According to the set threshold, substances with peak areas exceeding the threshold in hydrophilic substances and hydrophobic substances are used as molecular probes.

[0040] The ethanol extract is 65% ethanol.

[0041] The crude extract was prepared as follows:

[0042] The wolfberry fruit was dried and crushed, and the extract was added at a material-liquid ratio of 1:5, and ultrasonicated to obtain a crude extract solution;

[0043] The crude extract solution is centrifuged to remove the precipitate to obtain the crude extract.

[0044] Example 1

[0045] A method for evaluating the enrichment effect of phenolamide natural products in wolfberry comprises the following steps:

[0046] Step 1: Based on the ultrasound-assisted extraction method, an ethanol solution is used to prepare a crude extract of wolfberry fruit rich in phenolamide natural products; the crude extract is subjected to macroporous resin chromatography, and then separation is performed to obtain an enrichment of phenolamide natural products.

[0047] The extraction method is as follows:

[0048] After drying the wolfberry fruit, crush it into powder. Weigh 50g and add 65% ethanol solution at a solid-liquid ratio of 1:5. Ultrasonic extraction is carried out at 40 kHz and 45°C for 3 hours. Repeat this operation twice.

[0049] Centrifuge to remove precipitate: The extract was then centrifuged at 5000 rpm for 6 min to remove the lower precipitate and collect the supernatant. The supernatant was concentrated using a rotary evaporator at 45°C and 0.1 MPa to obtain a crude extract.

[0050] The chromatography process is as follows:

[0051] The macroporous resin was soaked in anhydrous ethanol for 24 hours in advance, the ethanol was removed, and the resin was repeatedly rinsed with distilled water until it was free of alcohol smell and colorless. The macroporous resin was then loaded into a 3.2 cm × 406 mm chromatography column with a column height of approximately 22 cm.

[0052] Pour the concentrated solution into the macroporous resin column and allow it to slowly fall and adsorb repeatedly until the effluent is clear. Rinse the column with pure water for about 7 to 8 column volumes until the eluate is clear and transparent. Then elute with 35% ethanol solution and collect the eluate.

[0053] The eluate was collected and the solvent was removed using a rotary evaporator at 45°C and 0.1 MPa to obtain a product as an extract sample. The extract sample was placed in a freeze vacuum dryer at a pressure of 0.1 MPa and a temperature of -40°C and freeze-dried for 24 hours. The resulting powder was the enriched phenolamide natural product.

[0054] Step 2: Select hydrophilic and hydrophobic substances as molecular probes to evaluate the enrichment effect of the enriched material in step 1.

[0055] Based on the crude extract, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) was used to analyze the distribution of phenolamide natural products in the crude extract.

[0056] The sample was diluted with a 1:1 H2O:MeOH solution and passed through a 0.22 μm organic filter. UPLC separation was performed using an Agilent 1290infinity-6545 ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer. A proportional valve was used for UPLC separation and subsequent delivery to the mass spectrometer. The sample was directly injected for mass spectrometry analysis. The chromatographic-mass spectrometry conditions and elution schedule are shown in Tables 1 and 2.

[0057] Table 1. Chromatographic conditions

[0058]

[0059]

[0060] Table 2. Elution program

[0061]

[0062] The total ion current of the crude extract of this example was detected by UPLC-Q-TOF / MS. Figure 1 As shown in the figure, it can be seen that for crude extracts, there are complex and diverse compounds in the product. It is difficult to further optimize the extraction and separation scheme by directly targeting all phenolamide natural products. It is necessary to target the target molecular probe among them.

[0063] The process of determining molecular probes based on the distribution of phenolamide natural products is as follows:

[0064] The peak areas of hydrophilic substances and hydrophobic substances in the distribution results of phenolamide natural products are calculated respectively; according to the set threshold, the substances with peak areas exceeding the threshold in the hydrophilic substances and hydrophobic substances are used as molecular probes.

[0065] The peak area calculation results of typical hydrophilic substances are as follows Figure 2 As shown, N1, N3-dihydrocaffeacylspermidine and N1, N3-dicaffeacylspermidine were selected as hydrophilic substance molecular probes according to the situation.

[0066] The peak area calculation results of typical hydrophobic substances are as follows Figure 3 As shown, N-feruloylputrescine and N-caffeoylputrescine were selected as molecular probes of hydrophobic substances according to the situation.

[0067] Therefore, in this embodiment, a combination of N1, N3-dihydrocaffeoylspermidine, N1, N3-dicaffeoylspermidine, N-feruloylputrescine, and N-caffeoylputrescine was used as a combination molecular probe.

[0068] Figure 5 In order to evaluate the enrichment effect of phenolamide compounds using the combined molecular probe determined in the embodiment of the present invention under different eluent ethanol concentrations, N1,N3-dihydrocaffeoylspermidine, N1,N3-dicaffeoylspermidine, N-feruloylputrescine and N-caffeoylputrescine were used as molecular probes respectively. It can be seen that the use of the combined molecular probe is better and more comprehensive than the use of a single probe in evaluating the enrichment effect, that is, it will not cause the loss of a certain type of rare component and has a wider screening effect.

[0069] The present invention uses putrescine and spermidine, two typical representatives with higher abundance, as combined molecular probes to comprehensively evaluate the enrichment of phenolamide natural products in the products obtained by extraction and separation. The combined form is used as an indicator to evaluate the separation effect to establish the optimal extraction and separation conditions.

[0070] The method of Example 1 was used to evaluate the enrichment of phenolamide natural products in wolfberries from different production areas. The results are as follows: Figure 6 It can be seen that the method of the present invention can analyze the differences in phenolamide compounds in wolfberries from different origins and different types.

[0071] The present invention establishes a method for rapidly evaluating the enrichment of phenolamide natural products in wolfberry extract samples using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Combining molecular probes, the method analyzes phenolamide natural products in samples isolated from wolfberry extracts from different origins and species. Combining the UPLC-Q-TOF / MS detection method with molecular probes enables rapid analysis and identification of phenolamide natural compounds in wolfberry plants, revealing differences in phenolamide compounds found in different origins and species. By targeting the target molecular probes for separation, efficient separation of other phenolamide compounds with similar structures and properties can be facilitated through a convenient and rapid screening process, significantly improving experimental efficiency.

Claims

1. A method for evaluating the enrichment effect of phenolamide natural products in wolfberry, characterized in that: The following steps are involved: Step 1: preparing a crude extract of wolfberry fruit containing phenolamide natural products using an ethanol extraction solution; subjecting the crude extract to macroporous resin chromatography, and then separating to obtain an enrichment of the phenolamide natural products; Step 2: Select hydrophilic and hydrophobic substances as molecular probes to evaluate the enrichment effect of the enriched material in step 1.

2. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: The hydrophilic substance in step 2 includes one or both of N1, N3-dihydrocaffeoylspermidine and N1, N3-dicaffeoylspermidine.

3. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: The hydrophobic substance in step 2 includes one or both of N-feruloylputrescine and N-caffeoylputrescine.

4. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: The method for selecting the molecular probe in step 2 is as follows: The crude extract obtained in step 1 is subjected to ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to obtain the distribution of phenolamide natural products in the crude extract; and a molecular probe is determined based on the distribution of the phenolamide natural products.

5. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 4, characterized in that: The process of determining the molecular probe according to the distribution of phenolamide natural products is as follows: Calculate the peak areas of hydrophilic and hydrophobic substances in the distribution results of phenolamide natural products respectively; According to the set threshold, substances with peak areas exceeding the threshold in hydrophilic substances and hydrophobic substances are used as molecular probes.

6. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: The ethanol extract in step 1 is ethanol with a concentration of 65%.

7. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: The preparation process of the crude extract in step 1 is as follows: The wolfberry fruit was dried and crushed, and the extract was added at a material-liquid ratio of 1:5, and ultrasonicated to obtain a crude extract solution; The crude extract solution is centrifuged to remove the precipitate to obtain the crude extract.

8. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 1, characterized in that: In step 1, the macroporous resin chromatography adopts gradient elution, and the obtained substance is the enriched product after drying.

9. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 4, characterized in that: The A and B pumps in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer respectively use an aqueous solution and an acetonitrile solution.

10. The method for evaluating the enrichment effect of phenolamide natural products in wolfberry according to claim 9, characterized in that: The aqueous solution and the acetonitrile solution are both mixed with formic acid, wherein the volume concentration of the formic acid is 0.1%.

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