A method for efficiently preparing flavonoid components from peanut stems and leaves and its application

Abstract

The present invention relates to active ingredient separation and extraction technology, and specifically to a highly efficient preparation method and application of flavonoids from peanut stems and leaves. A crude peanut stem and leaf extract is subjected to gradient elution separation by high-speed countercurrent chromatography using a stationary phase, sequentially employing a first mobile phase, a second mobile phase, and a third mobile phase, to obtain daidzein and formononetin. The stationary phase comprises an upper phase of n-hexane, ethyl acetate, methanol, and water (5:4.8-5.2:1.9-2.1:7.6-8.4), the first mobile phase comprises a lower phase of n-hexane, ethyl acetate, methanol, and water (5:4.8-5.2:2.9-3.1:6.6-7.4), and the third mobile phase comprises a lower phase of n-hexane, ethyl acetate, methanol, and water (5:4.8-5.2:4.8-5.2:4.8-5.2). The present invention enables high-speed and efficient extraction of daidzein and formononetin.

Description

Technical Field

[0001] The invention relates to active ingredient separation and extraction technology, and in particular to a high-efficiency preparation method and application of flavonoid components in peanut stems and leaves. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Peanut stems and leaves are an agricultural byproduct of peanut production. Currently, with the exception of a small portion used as medicine, animal feed, or fuel, most are discarded, resulting in a significant waste of resources. According to the inventors' research, peanut stems and leaves contain a variety of functional active ingredients, including flavonoids, phenolic acids, and terpenes. The isolation and purification of these active ingredients primarily utilizes traditional separation techniques, such as macroporous resins, silica gel column chromatography, and gel column chromatography, which are costly, time-consuming, and inefficient. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art, the present invention aims to provide a method for efficiently preparing flavonoid components from peanut stems and leaves and its application, which can extract daidzein and formononetin at a high speed and high efficiency.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In one aspect, a method for efficiently preparing flavonoid components from peanut stems and leaves comprises the following steps:

[0007] The peanut stems and leaves are subjected to heating reflux extraction using 94-96% ethanol to obtain a crude peanut stem and leaf extract;

[0008] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:1.9-2.1:7.6-8.4, let stand until complete equilibrium, take the upper phase as the stationary phase, and take the lower phase as the first mobile phase;

[0009] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:2.9-3.1:6.6-7.4, let stand until complete equilibrium, and remove the lower phase as the second mobile phase;

[0010] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:4.8-5.2:4.8-5.2, let it stand until it is completely balanced, and remove the lower phase as the third mobile phase;

[0011] The stationary phase is used to perform gradient elution separation on the crude extract of peanut stems and leaves through high-speed countercurrent chromatography to obtain daidzein and formononetin; wherein, during the gradient elution separation process, the first mobile phase, the second mobile phase, and the third mobile phase are used in sequence.

[0012] The present invention utilizes high-speed countercurrent chromatography (HSCCC), which features solvent conservation, short separation time, and high efficiency, effectively resolving issues such as irreversible sample adsorption and denaturation associated with conventional column chromatography. However, studies have shown that when isolating daidzein and formononetin from crude peanut stem and leaf extracts, the peak resolution of extraction using a single stationary phase and mobile phase is poor, making it difficult to effectively separate and extract daidzein and formononetin. Therefore, the present invention utilizes the aforementioned stationary phase along with a first, second, and third mobile phases for gradient elution separation, effectively isolating daidzein and formononetin.

[0013] In addition, molecular docking technology has revealed that both daidzein and formononetin have sleep-promoting activity. Therefore, another aspect of the present invention provides a use of flavonoids from peanut stems and leaves in the preparation of a sleep-promoting drug, wherein the flavonoids from peanut stems and leaves are daidzein and / or formononetin.

[0014] The beneficial effects of the present invention are:

[0015] The present invention uses high-speed countercurrent chromatography to separate flavonoid components from peanut stems and leaves. A solvent system of n-hexane / ethyl acetate / methanol / water (5:4.8-5.2:1.9-2.1:7.6-8.4, 5:4.8-5.2:2.9-3.1:6.6-7.4, 5:4.8-5.2:4.8-5.2:4.8-5.2, v / v) is used for gradient elution. Multiple injections are performed for enrichment and purification to obtain two compounds, which are identified as daidzein and formononetin by mass spectrometry and nuclear magnetic resonance spectroscopy. Molecular docking analysis shows that both daidzein and formononetin have good affinity for two protein receptors associated with insomnia, primarily through hydrogen bonding interactions, and have potential sleep-promoting activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0017] Figure 1 This is a high performance liquid chromatogram of the peanut stem and leaf extract in Example 1 of the present invention;

[0018] Figure 2This is a high-speed countercurrent chromatogram of the peanut stem and leaf extract in Example 1 of the present invention;

[0019] Figure 3 This is a two-dimensional planar image (2D) of the docking of daidzein extracted from an embodiment of the present invention with GABRA1;

[0020] Figure 4 This is a 2D diagram of the docking of daidzein extracted from an embodiment of the present invention and FOS;

[0021] Figure 5 This is a 2D diagram of the docking of formononetin extracted from an embodiment of the present invention and GABRA1;

[0022] Figure 6 This is a 2D diagram of the docking of formononetin extracted in the examples of the present invention and FOS. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0024] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0025] In view of the fact that it is difficult to extract flavonoids from peanut stems and leaves with high speed and high efficiency with the existing technology, the present invention proposes a method for efficiently preparing flavonoids from peanut stems and leaves and its application.

[0026] A typical embodiment of the present invention provides a method for efficiently preparing flavonoid components from peanut stems and leaves, comprising the following steps:

[0027] The peanut stems and leaves are subjected to heating reflux extraction using 94-96% ethanol to obtain a crude peanut stem and leaf extract;

[0028] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:1.9-2.1:7.6-8.4, let stand until complete equilibrium, take the upper phase as the stationary phase, and take the lower phase as the first mobile phase;

[0029] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:2.9-3.1:6.6-7.4, let stand until complete equilibrium, and remove the lower phase as the second mobile phase;

[0030] Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:4.8-5.2:4.8-5.2, let it stand until it is completely balanced, and remove the lower phase as the third mobile phase;

[0031] The stationary phase is used to perform gradient elution separation on the crude extract of peanut stems and leaves through high-speed countercurrent chromatography to obtain daidzein and formononetin; wherein, during the gradient elution separation process, the first mobile phase, the second mobile phase, and the third mobile phase are used in sequence.

[0032] In some embodiments, the elution time of the first mobile phase is 55 to 65 hours, the elution time of the second mobile phase is 55 to 65 hours, and the elution time of the third mobile phase is 55 to 65 hours.

[0033] In one or more embodiments, a fraction between 115 and 130 min and a fraction between 190 and 205 min are collected.

[0034] In some embodiments, the stationary phase, the first mobile phase, the second mobile phase, and the third mobile phase are ultrasonically degassed before use.

[0035] In some embodiments, the crude extract of peanut stems and leaves is added to the stationary phase and the first mobile phase in equal amounts and dissolved by ultrasonication.

[0036] In some embodiments, peanut stems and leaves are dried, crushed, and then added to 94-96% ethanol by volume.

[0037] In one or more embodiments, the solid-liquid ratio of the dried and crushed peanut stems and leaves to 94-96% ethanol by volume is 1:18-22, g / mL.

[0038] In some embodiments, absorbance is detected at 250 nm.

[0039] Another embodiment of the present invention provides a use of flavonoid components in peanut stems and leaves in the preparation of sleep-aiding drugs, wherein the flavonoid components in the peanut stems and leaves are daidzein and / or formononetin.

[0040] In some embodiments, the sleep-aiding drug comprises an active ingredient and a pharmaceutical excipient, wherein the active ingredient is the flavonoids found in the stems and leaves of peanuts. The pharmaceutical excipient comprises a pharmaceutical carrier and / or an excipient. The excipient may include a binder, a filler, or the like. The pharmaceutical excipient may be present in an amount of 1% to 99% by weight.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0042] Example

[0043] Materials and reagents: Peanut stems and leaves, Shandong Jinsheng Grain and Oil Group Co., Ltd.; Daidzein and formononetin, purity ≥98%, Shanghai Yuanye Biotechnology Co., Ltd.; Acetonitrile, chromatographic grade, Sweden Opson; Formic acid, n-hexane, ethanol, ethyl acetate, and methanol, analytical grade, Tianjin Komiou Chemical Reagent Co., Ltd.

[0044] Instruments: High-speed Chinese medicine grinder (YF-150-500g), Ruian Yongli Pharmaceutical Machinery Co., Ltd.; electronic balance (ME204 / 02), Mettler-Toledo Instrument Co., Ltd.; rotary evaporator (R-3), BUCHI, Switzerland; high-performance liquid chromatograph (Agilent 1220), Agilent, USA; high-speed countercurrent chromatograph (TBF), Shanghai Tongtian Biotechnology Co., Ltd.; freeze dryer (Scientz-1ON), Ningbo Xinzhi Biotechnology Co., Ltd.; nuclear magnetic resonance spectrometer (VANCEⅢHD), Bruker, Germany; triple quadrupole mass spectrometer (Xevo TQ-XS), Waters, USA; constant flow pump (TBP5002), Shanghai Tongtian Biotechnology Co., Ltd.; automatic fraction collector (BS-100N), Shanghai Huxi Analytical Instrument Factory Co., Ltd.

[0045] Sample Preparation: Wash peanut stems and leaves, air-dry, and then crush. Extract with 95% ethanol at a solid-liquid ratio of 1:20 (g / mL) and heat under reflux for 1 hour three times. Filter the extract, concentrate under reduced pressure, and evaporate until the alcohol is gone. Freeze-dry the crude peanut stem and leaf extract and place it in a desiccator for later use.

[0046] Determination of the distribution coefficient: Select the HSCCC solvent system according to the distribution coefficient (K value) of the target compound, and analyze the effect of different solvent systems on the K value. Use high performance liquid chromatography to determine the K value of the target compound. Prepare solvent systems of different proportions in test tubes, shake the test tubes to completely balance them, let them stand for separation, take 3 mL of the upper and lower phases of the solvent system, add appropriate amount of sample and ultrasonically dissolve them. After complete balance, take the upper and lower phase solutions for HPLC detection, and calculate the K value of the target sample according to the distribution coefficient formula: K = A U / A L , where A U is the peak area of the target compound in the upper phase, A L is the peak area of the target compound in the lower phase.

[0047] Preparation of the two-phase solvent system and sample solution: The HSCCC solvent system consisted of n-hexane / ethyl acetate / methanol / water in a volume ratio of 5:5:2:8, 5:5:3:7, and 5:5:5:5 (v / v) in a separatory funnel. The mixture was shaken thoroughly and allowed to stand for 30 minutes to allow for complete equilibrium. An appropriate amount of sample was added to equal amounts of the upper and lower phases and dissolved by sonication to prepare the sample solution.

[0048] High-speed countercurrent chromatography separation: Using the head-to-tail elution mode of HSCCC, the target compounds were separated using the optimal solvent system. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. Ultrasonic degassing was performed, and the cold hydrazine was turned on and the temperature was 25°C. The stationary phase was pumped into the chromatographic column at a flow rate of 30 mL / min. Once completely filled, the speed was adjusted to 800 rpm in the forward direction. The mobile phase began to equilibrate at a flow rate of 5 mL / min until hydrodynamic equilibrium was achieved. The sample solution was manually injected into the chromatographic column through the injection valve. The UV detector was set to continuously monitor the absorbance at 250 nm, and the recorder was turned on to record the chromatographic data. The eluent was continuously collected by an automatic collector, and each peak fraction was collected into a reagent bottle according to the chromatogram. After the desired peak was eluted, the speed and elution were stopped, and the components in the chromatographic column were collected in a measuring cylinder using a vacuum pump. The collected fractions were concentrated under reduced pressure to obtain flavonoid components.

[0049] HPLC analysis: Column: Phenomenex C18 column (250×4.6mm, 5μm); Mobile phase: A-0.1% formic acid in water, B-acetonitrile; Gradient elution conditions: 0-30 min, 95-50% A, 5-50% B; 30-45 min, 50-23% A, 50-77% B; 45-47 min, 23-95% A, 77-5% B; 47-55 min, 95% A, 5% B; Flow rate: 1 mL / min; Column temperature: 25°C; Detection wavelength: 250 nm; Injection volume: 10 μL. The HPLC chromatogram of peanut stem and leaf extract is shown below. Figure 1 shown.

[0050] Structural identification: by ESI-MS, 1 H and 13 The isolated compounds were identified by C NMR spectroscopy. ESI-MS (positive and negative ion modes) mass spectrometric analysis was performed on an Agilent 6520Q-TOF, and NMR spectra were recorded on a Bruker AV-400 spectrometer. DMSO-d6 was used as the solvent and tetramethylsilane (TMS) was used as the internal standard. Chemical shifts (δ) are expressed in ppm, and coupling constants (J) are reported in Hz.

[0051] Molecular docking of compounds with target proteins: 3D structure files of the compounds were downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) in SDF format to establish a ligand database. Proteins associated with insomnia were selected as docking receptor proteins. The target protein 3D structure was downloaded from the PDB database (http: / / www.rcsb.org) in PDB format. Moe was used to remove water molecules and residues, add polar hydrogens, and perform energy optimization to screen for active sites. The small molecule ligand database was docked with the selected receptor proteins, and the results were measured using the Alpha triangle placement method. The London scoring function was used to evaluate the docking affinity and compare the docking. The conformations were studied using LigandInteractions.

[0052] Results and Analysis:

[0053] Choice of solvent system for countercurrent chromatography:

[0054] A K value range of 0.5 to 2 is optimal for HSCCC separations. The K value is positively correlated with separation time. Higher K values may produce excessively broad peaks and prolonged elution times, while lower K values may result in poor peak resolution. Different solvent systems were used for separation, and the K values of the target compounds in the samples were measured to select the optimal solvent system. The results are shown in Table 1, comparing different ratios of n-hexane / ethyl acetate / methanol / water solvent systems. Comprehensive analysis indicated that no single system was suitable, so a gradient elution method was considered.

[0055] Table 1 K values of target compounds in different solvent systems

[0056]

[0057] High-speed countercurrent chromatography separation and preparative purification:

[0058] A gradient solvent system of n-hexane / ethyl acetate / methanol / water (5:5:2:8, 5:5:3:7, 5:5:5:5, v / v) was selected for high-speed countercurrent chromatography separation of peanut stem and leaf extracts. The separation chromatogram is shown in FIG. Figure 2As shown. In the head-to-tail elution mode, the upper phase of n-hexane / ethyl acetate / methanol / water (5:5:2:8, v / v) was used as the stationary phase, followed by the lower phase of n-hexane / ethyl acetate / methanol / water (5:5:2:8, v / v) as the mobile phase for 1 hour; the lower phase of n-hexane / ethyl acetate / methanol / water (5:5:3:7, v / v) was used as the mobile phase for 1 hour; and the lower phase of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v) was used as the mobile phase until the end, with a total separation time of 4 hours. After gradient separation, two main peaks, peak 1 and peak 2, were obtained by HPLC determination, with a purity of more than 98%. The target compound was enriched using the same method to obtain a sample of sufficient quality for further structural identification.

[0059] Compound structure identification and analysis:

[0060] Compound 1: white powder, ESI-MS m / z 254.85 [M+H] + , molecular formula is C 15 H 10 O4; the NMR data are shown in Table 2, and compound 1 is identified as daidzein, and its structural formula is shown below.

[0061]

[0062] Compound 2: white powder, ESI-MS m / z 268.89 [M+H] + , molecular formula is C 16 H 12 O4; the NMR data are shown in Table 2, and compound 2 is identified as formononetin, and its structural formula is shown below.

[0063]

[0064] Table 2 Compounds 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data

[0065]

[0066] Molecular docking of compounds:

[0067] The 3D structures of daidzein and formononetin were downloaded from the PubChem database. The insomnia-related proteins GABRA1 (PDB ID: 6HUK) and FOS (PDB ID: 1A02) were selected as docking receptor proteins. Affinity was used as the evaluation metric. The greater the absolute value of affinity, the more stable the binding between the ligand and receptor, and the higher the likelihood of interaction. The results are shown in Table 3. It is generally believed that a binding energy less than 0 indicates spontaneous binding between the two, and the minimum binding energy is ≤ -5.0 kJ·mol -1 This indicates that the docking effect between the molecule and the protein is good, and the minimum binding energy of daidzein and formononetin with the two receptor proteins is less than -5.0 kJ·mol -1 , with a strong binding ability. That is, both daidzein and formononetin may play a sleep-promoting role through these two targets and are potential active compounds.

[0068] Table 3 Docking results of compounds and receptor proteins

[0069]

[0070] From the two-dimensional plane diagram of compound-protein interaction relationship ( Figure 3-6 ) shows that in the optimal docking score conformation, daidzein forms hydrogen bonds with GABRA1's THR207; in FOS, it primarily forms hydrogen bonds with GLY531 and THR533. Formononetin forms hydrogen bonds with GABRA1's ARG180 and THR207; in FOS, it primarily forms hydrogen bonds with GLN180 and SER402. Therefore, the primary interaction force is hydrogen bonding, and it is speculated that hydrogen bonding may enhance the compound's receptor binding ability, thereby increasing its activity.

[0071] GABRA1, a γ-aminobutyric acid receptor, plays a key regulatory role in insomnia, and changes in FOS levels can, to a certain extent, reflect the severity of insomnia. Docking results indicate that both daidzein and formononetin have strong affinities for both protein receptors, primarily through hydrogen bonding interactions, suggesting potential sleep-promoting activity. Molecular docking, as a computer-assisted screening technique, provides a rapid and efficient method for identifying target compounds with specific structures and activities.

[0072] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for efficiently preparing flavonoid components from peanut stems and leaves, characterized by: The following steps are involved: Peanut stems and leaves were subjected to heating reflux extraction using 94-96% ethanol to obtain a crude peanut stem and leaf extract. Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:1.9-2.1:7.6-8.4, let stand until complete equilibrium, take the upper phase as the stationary phase, and take the lower phase as the first mobile phase; Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8-5.2:2.9-3.1:6.6-7.4, let stand until complete equilibrium, and remove the lower phase as the second mobile phase; Mix n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:4.8~5.2:4.8~5.2:4.8~5.2, let it stand until it is completely balanced, and remove the lower phase as the third mobile phase; The stationary phase is used to perform gradient elution separation on the crude extract of peanut stems and leaves through high-speed countercurrent chromatography to obtain daidzein and formononetin; wherein, during the gradient elution separation process, the first mobile phase, the second mobile phase, and the third mobile phase are sequentially used; The flavonoid components are daidzein and formononetin.

2. The method for efficiently preparing flavonoids from peanut stems and leaves as claimed in claim 1, characterized in that: The stationary phase is an upper phase obtained by mixing n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:2:8 and allowing to stand until completely balanced. The first mobile phase is a lower phase obtained by mixing n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:2:8 and allowing to stand until completely balanced. The elution time of the first mobile phase is 1 h. The second mobile phase is a lower phase obtained by mixing n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:3:7 and allowing to stand until completely balanced. The elution time of the second mobile phase is 1 h. The third mobile phase is a lower phase obtained by mixing n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:5:5 and allowing to stand until completely balanced. The total separation time is 4 h.

3. The method for efficiently preparing flavonoid components from peanut stems and leaves as claimed in claim 2, characterized in that: The fractions from 115 to 130 min and 190 to 205 min were collected.

4. The method for efficiently preparing flavonoid components from peanut stems and leaves as claimed in claim 1, characterized in that: The stationary phase, the first mobile phase, the second mobile phase and the third mobile phase were ultrasonically degassed before use.

5. The method for efficiently preparing flavonoids from peanut stems and leaves as claimed in claim 1, characterized in that: Equal amounts of the peanut stem and leaf extract were added to the stationary phase and the first mobile phase and dissolved by ultrasonication.

6. The method for efficiently preparing flavonoids from peanut stems and leaves as claimed in claim 1, characterized in that: The peanut stems and leaves are dried, crushed, and then added to 94-96% ethanol by volume.

7. The method for efficiently preparing flavonoids from peanut stems and leaves as claimed in claim 6, characterized in that: The solid-liquid ratio of the dried and crushed peanut stems and leaves to 94-96% ethanol by volume is 1:18-22 g / mL.

8. The method for efficiently preparing flavonoids from peanut stems and leaves as claimed in claim 1, characterized in that: The absorbance was detected at 250 nm.

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