Diglycerol linolenate galactoside compound in pepper as well as preparation method and application of diglycerol linolenate galactoside compound

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a diglycerol linolenate galactoside compound in pepper as well as a preparation method and application of the diglycerol linolenate galactoside compound. The preparation method comprises the following steps: (1) taking red peppers to prepare pretreated red peppers; (2) adding the pretreated red peppers into an ethanol solution, performing ultrasonic extraction, and collecting first filtrate and first filter residues; (3) adding an ethanol solution into the first filter residue, carrying out ultrasonic extraction, combining the first filtrate and the second filtrate, and concentrating to obtain a crude extract; (4) separating the crude extract by adopting high-speed counter-current chromatography to obtain a primary purified product; and (5) further separating and purifying the primary purified product by adopting high performance liquid chromatography, and identifying to obtain the diglycerol linolenate galactoside compound. The purity of the diglycerol linolenate galactoside compound obtained by the preparation method is as high as 99.3%, and the diglycerol linolenate galactoside compound has a remarkable scavenging effect on DPPH free radicals, shows good antioxidant activity and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a diglyceride linolenic acid galactoside compound in chili peppers, its preparation method, and its application. Background Technology

[0002] Chili peppers (Capsicum annuum L.) belong to the genus Capsicum in the family Solanaceae. They are a plant widely cultivated and consumed globally. Chili peppers are not only an important vegetable but also an indispensable ingredient in condiments worldwide, giving food its unique color, aroma, and flavor.

[0003] In addition, chili peppers also possess certain medicinal value. Traditional Chinese medicine believes that chili peppers are pungent and hot in nature, affecting the heart and spleen meridians. They have the effects of warming the middle jiao (spleen and stomach), dispelling cold, regulating qi and promoting digestion, and invigorating the spleen and stomach. They are often used to treat abdominal pain, vomiting, dysentery, diarrhea, and frostbite caused by spleen and stomach deficiency. Modern research further indicates that the bioactivity of chili peppers mainly stems from their rich content of various functional components, such as capsaicin, vitamins, and various phenolic and glycoside compounds. Although the bioactivity of chili peppers is widely recognized, further research is needed to determine which specific monomeric compounds exert their effects. Current research focuses primarily on pungent components such as capsaicin, while studies on the various glycoside compounds abundant in red bell peppers are relatively limited. Therefore, isolating and preparing high-purity monomeric compounds from the complex matrix of chili peppers has significant scientific and applied value.

[0004] Based on the above objectives, this invention uses fresh red bell peppers as raw materials and employs modern separation techniques such as high-speed countercurrent chromatography and high-performance liquid chromatography to directionally separate and prepare a high-purity diglyceride linoleic acid galactoside compound from chili peppers, providing a scientific basis for the development of new functional foods and drugs. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for preparing diglyceride linolenic acid galactoside compounds from chili peppers.

[0006] A second objective of this invention is to provide a diglyceride linolenic acid galactoside compound for chili peppers.

[0007] A third objective of this invention is to provide the application of a diglyceride linolenic acid galactoside compound from chili peppers in the preparation of antioxidant drugs.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a diglyceride linolenic acid galactoside compound from chili peppers, the method specifically comprising the following steps: (1) Take red chili peppers, wash and crush them to obtain pretreated red chili peppers; (2) Add the pretreated red peppers to an ethanol solution and extract by ultrasonication; filter, centrifuge, and collect the first filtrate and the first filter residue; (3) Then add ethanol solution to the first filter residue and extract by ultrasonication; after filtration and centrifugation, collect the second filtrate, combine the first and second filtrates, and evaporate and concentrate to crude extract. (4) The crude extract obtained in step (3) is separated by high-speed countercurrent chromatography, and the fraction of the target compound is collected to obtain the primary purified product. (5) The primary purified product obtained in step (4) was further separated and purified by high performance liquid chromatography. After freeze drying, the diglyceride linoleate galactoside compound was identified.

[0009] Further, in step (2), the concentration of the ethanol solution is 90-95 v / v; the ratio of the amount of pretreated red pepper to ethanol solution is 5 kg: 8-12 L; the temperature of the ultrasonic extraction is 45-55℃; and the time of the ultrasonic extraction is 55-65 min.

[0010] Further, in step (3), the concentration of the ethanol solution is 90-95 v / v; the ratio of the first filter residue to the ethanol solution is 5 kg: 4-6 L; the temperature of the ultrasonic extraction is 45-55 °C; and the time of the ultrasonic extraction is 45-65 min.

[0011] Further, the separation conditions for high-speed countercurrent chromatography in step (4) are as follows: the volume ratio of methanol:petroleum ether:n-hexane:water in the high-speed countercurrent chromatography solvent system is 5.5:3:5:2; the flow rate is 1.4-1.8 mL / min; the injection volume is 0.5-1.4 g; the rotation speed is 800-900 r / min; and the detection wavelength is 250-260 nm.

[0012] Furthermore, the separation conditions for high-performance liquid chromatography in step (5) are: C 18 Chromatographic column; the volume ratio of methanol to water in the mobile phase of high performance liquid chromatography is (90-95):8; the flow rate is 3-7 mL / min; the detection wavelength is 450-470 nm.

[0013] A diglyceride linolenic acid galactoside compound for chili peppers was prepared according to the above-described method for preparing a diglyceride linolenic acid galactoside compound for chili peppers.

[0014] Furthermore, the chemical structural formula of the diglyceride linolenic acid galactoside compound is as follows: The above describes the application of a diglyceride linolenic acid galactoside compound from chili peppers in the preparation of antioxidant drugs.

[0015] The beneficial technical effects of this invention are as follows: This invention uses fresh red bell peppers as raw material and employs modern separation techniques such as high-speed countercurrent chromatography and high-performance liquid chromatography to directionally separate diglyceride linolenic acid galactoside compounds with a purity of up to 99.3%. Experimental results show that the separated diglyceride linolenic acid galactoside compounds have a significant scavenging effect on DPPH free radicals, exhibiting excellent antioxidant activity. This discovery provides strong experimental evidence for its application as a natural antioxidant, making it a promising candidate for use in the food and cosmetic industries. Attached Figure Description

[0016] Figure 1 The detection spectra are obtained by high performance liquid chromatography at 254 nm and 460 nm. Figure 2 The separation chromatogram is obtained by high-speed countercurrent chromatography. Figure 3 The high-performance liquid chromatography (HPLC) spectra of effluent I at 254 nm and 460 nm are shown. Figure 4 The high-performance liquid chromatography (HPLC) spectra of the compound at 254 nm and 460 nm are shown. Figure 5 The structural formula of the diglyceride linolenic acid galactoside compound; Figure 6 The results of the scavenging effect of diglyceride linolenic acid galactoside on DPPH free radicals are shown in the figure. Detailed Implementation

[0017] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0018] Example 1 Preparation and high-performance liquid chromatography analysis of crude extracts from fresh red bell peppers (1) Take 5 kg of fresh red bell peppers, wash and crush them to obtain pretreated red peppers; (2) Add the pretreated red peppers to 10L of 95v / v% ethanol solution and extract by ultrasonication at 50℃ for 60min; filter, centrifuge, and collect the first filtrate and the first filter residue; (3) Then add 95v / v% ethanol solution to the first filter residue; the ratio of the first filter residue to the ethanol solution is 1kg:1L; ultrasonic extraction at 50℃ for 60min; after filtration and centrifugation, collect the second filtrate, combine the first filtrate and the second filtrate, and evaporate and concentrate to a dark red crude extract. (4) To determine the main target components and the wavelength for high-speed countercurrent separation of the dark red crude extract, high-performance liquid chromatography (HPLC) was used for further analysis. A small amount of crude extract was dissolved in methanol and injected. The HPLC analysis conditions were as follows: column: Agilent C18 (250 mm × 4.6 mm, 5 µm); mobile phase B was acetonitrile, and mobile phase C was ultrapure water. Gradient elution: 0-30 min, 80%-95% B; 30-40 min, 95%-95% B; flow rate: 1.0 mL / min; detection wavelength: 460 nm; column temperature: 30℃. The HPLC analysis results are as follows: Figure 1 As shown.

[0019] The results are as follows Figure 1 The image shows the chromatograms obtained by high-performance liquid chromatography (HPLC) at 254 nm and 460 nm. Figure 1 a is the chromatogram detected by high performance liquid chromatography at 460 nm. Figure 1 b is the chromatogram detected by high-performance liquid chromatography at 254 nm. Figure 1 As can be seen from point a, multiple chromatographic peaks were observed after 20 minutes at a wavelength of 460 nm in the visible light region, with peak 1 being the highest and the others being lower. Peak 1 was thus identified as the target for subsequent separation. Figure 1 As shown in b, peak 1 also has weak absorption at 254 nm, which can be used as the wavelength for high-speed countercurrent separation.

[0020] Example 2 Optimization of different solvent systems in high-speed countercurrent chromatography (HSCCC) To establish an efficient HSCCC separation method, a suitable two-phase solvent system must first be screened. This study evaluated the method by measuring the K values ​​of the target compound in several solvent systems with different ratios; the results are shown in Table 1.

[0021] Table 1. K values ​​for solvent systems with different ratios The results are shown in Table 1, which presents the K values ​​for several solvent systems with different ratios. As can be seen from Table 1, system 2 (volume ratio of methanol: petroleum ether: n-hexane: water is 5.5:3:5:2) has the best K value of 1.17, indicating that the target compound has a balanced distribution behavior between the stationary phase and the mobile phase in this system. Therefore, it was selected as the optimal solvent system for subsequent HSCCC separation.

[0022] Example 3 Separation efficiency and condition optimization of high-speed countercurrent chromatography (HSCCC) (1) According to the optimal solvent system determined in Example 1 (methanol:petroleum ether:n-hexane:water = 5.5:3:5:2, v / v), the mixture was allowed to stand overnight, and after separation of the two phases, it was ultrasonically degassed for 30 min. The upper phase was used as the stationary phase and the lower phase as the mobile phase; the instrument was rotated forward at 850 r / min, the temperature was 25℃, and the detection wavelength was 254 nm. After the instrument system was balanced, a certain amount of the dark red crude extract prepared in Example 1 was dissolved in 20 mL of the lower phase and injected, while a high-speed countercurrent chromatogram was collected. To improve the preparation efficiency, a two-injection mode was adopted. About 110 min after the first injection, 20 mL of the sample solution was injected again. The separation chromatogram of the high-speed countercurrent chromatogram is shown in the figure. Figure 2 The effluent I portion (110-120 min and 275-285 min) was received, and its purity was determined by high performance liquid chromatography. The same components were combined. The high performance liquid chromatograms of effluent I at 254 nm and 460 nm are shown in Figure 3. Based on the retention time and effluent color, it was inferred to be the target compound.

[0023] The results are as follows Figure 2 The figure shows the separation chromatogram detected by high-speed countercurrent chromatography. The results are as follows: Figure 3 The figure shows the high performance liquid chromatography (HPLC) spectra of eluent I at 254 nm and 460 nm. A main peak is shown at both 254 nm and 460 nm wavelengths, and its retention time is consistent with the target peak in the crude extract. It is preliminarily speculated that this fraction I is the target compound.

[0024] (2) By optimizing the two key parameters of mobile phase flow rate and sample injection volume, the optimal process conditions for HSCCC separation were determined to achieve a balance between high efficiency and high purity. The results are shown in Tables 2 and 3.

[0025] Table 2. Effect of different flow rates on the separation of compounds at an injection volume of 1.0 g. Table 3. Separation effect of different injection volumes on compounds at a flow rate of 1.6 mL / min The results are shown in Table 2, illustrating the effect of different flow rates on the separation of the compounds. The table shows that different flow rates have a relatively small effect on the stationary phase retention rate (P-value), but a significant effect on the purity of the separated products. At a flow rate of 1.6 mL / min, not only was a high stationary phase retention rate maintained (87.5%), but the product purity also reached its highest level (83.20%), thus this was determined to be the optimal flow rate.

[0026] The results are shown in Table 3, which illustrates the effect of different injection volumes on the separation of the compounds. The table shows that when the injection volume is 0.5–1.4 g, the purity of the compounds remains above 82% with little variation; therefore, 1.4 g is selected as the optimal injection volume.

[0027] Example 4 To further improve the purity of the target compound, this experiment utilized preparative high-performance liquid chromatography (HPLC) to further purify the crude product obtained from high-speed countercurrent chromatography (HSC). The preparative HPLC conditions were: C18 column (10 mm I.D. * 250 mm, 5 μm); flow rate 5 mL / min; mobile phase: methanol-water (92:8, v / v); detection wavelength: 460 nm. Fractions were manually collected based on the elution time of the main peak in the preparative HPLC chromatogram. After freeze-drying, a deep red, flocculent, or powdery compound was successfully obtained. To accurately assess the purity of the prepared compound, analytical HPLC was used for detection at 254 nm and 460 nm. The results are as follows: Figure 4 As shown.

[0028] The results are as follows Figure 4 The image shows the chromatograms of the compound detected by high-performance liquid chromatography (HPLC) at 254 nm and 460 nm. HPLC analysis revealed a compound purity of 99.3%. These results demonstrate that a high-purity diglyceride linolenic acid galactoside monomer was successfully isolated and prepared using a purification strategy combining high-speed countercurrent chromatography and HPLC, achieving a purity meeting high-level scientific research standards.

[0029] Experimental Example 1 Nuclear magnetic resonance (NMR) technology was used to analyze and identify the structure of the compound from Example 4, which was obtained by high-speed countercurrent chromatography and preparative liquid chromatography, to determine its chemical structure. The NMR H-s spectra of the compound are shown in Table 4; the NMR C-s spectra of the compound are shown in Table 5.

[0030] Table 4. H-NMR Spectrum Information of Compounds Table 5. C-NMR spectrum information of compounds The results are shown in Tables 4 and 5, which present the NMR H-spectrum and NMR C-spectrum information of the compound. Analysis of Tables 4 and 5 successfully resolved the molecular structure of the compound, confirming it as diglyceride linolenic acid galactoside, with the following chemical structural formula: Figure 5 As shown.

[0031] Experimental Example 2 Determination of the antioxidant activity of diglyceride linolenic acid galactoside To evaluate the bioactivity of the diglyceride linolenic acid galactoside compound obtained in Example 4, its antioxidant capacity was determined using a DPPH free radical scavenging assay, with β-carotene as a positive control. The specific experimental steps are as follows: (1) Preparation of DPPH solution: Accurately weigh 2.05 mg of DPPH and add ethanol to make up to 25 mL.

[0032] (2) Sample solution preparation: Accurately weigh 2.03 mg of the high-purity diglyceride linolenic acid galactoside monomer prepared in Example 4, dissolve it in ethanol and then serially dilute it to different concentrations (0.13-0.90 mmol / L); accurately weigh 2.04 mg of the reference standard β-carotene, dissolve it in ethanol and then serially dilute it to different concentrations (0.19-1.33 mmol / L).

[0033] (3) Determination of DPPH free radical scavenging ability: Take the same volume of sample solution and DPPH solution, mix them well, and place them in the dark for 30 min. Measure the absorbance value A1 at 517 nm. Measure the absorbance value A2 when the sample solution is mixed with ethanol. Measure the absorbance value A0 when the DPPH solution is mixed with ethanol as a blank group. Use β-carotene of the same concentration as a control group. Set up three replicates for each group. The scavenging rate is calculated as follows: DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%. The results of the scavenging effect of diglyceride linolenic acid galactoside on DPPH free radicals are as follows: Figure 6 As shown.

[0034] The results are as follows Figure 6 The figure shown is a graph illustrating the scavenging effect of diglyceride linolenic acid galactoside on DPPH free radicals. Figure 6 It was found that diglyceride linolenic acid galactoside has a significant scavenging effect on DPPH free radicals. Within the studied concentration range, the DPPH free radical scavenging rate of this compound increased with increasing diglyceride linolenic acid galactoside concentration. When the concentration was greater than 0.90 mmol / L, its free radical scavenging rate was not significantly different from that of the classic natural antioxidant β-carotene at the same concentration. This result demonstrates that diglyceride linolenic acid galactoside, successfully isolated from red bell pepper, is an effective free radical scavenger, and its excellent antioxidant activity lays a solid theoretical foundation for its application as a natural antioxidant.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for preparing a diglyceride linolenic acid galactoside compound from chili peppers, characterized in that, The preparation method specifically includes the following steps: (1) Take red chili peppers, wash and crush them to obtain pretreated red chili peppers; (2) Add the pretreated red peppers to an ethanol solution and extract by ultrasonication; filter, centrifuge, and collect the first filtrate and the first filter residue; (3) Then add ethanol solution to the first filter residue and extract by ultrasonication; after filtration and centrifugation, collect the second filtrate, combine the first and second filtrates, and evaporate and concentrate to crude extract. (4) The crude extract obtained in step (3) is separated by high-speed countercurrent chromatography, and the fraction of the target compound is collected to obtain the primary purified product. (5) The primary purified product obtained in step (4) was further separated and purified by high performance liquid chromatography. After freeze drying, the diglyceride linoleate galactoside compound was identified.

2. The method for preparing a diglyceride linolenic acid galactoside compound from chili peppers according to claim 1, characterized in that, In step (2), the concentration of the ethanol solution is 90-95 v / v; the ratio of the amount of pretreated red pepper to ethanol solution is 5 kg: 8-12 L; the temperature of the ultrasonic extraction is 45-55℃; and the time of the ultrasonic extraction is 55-65 min.

3. The method for preparing a diglyceride linolenic acid galactoside compound from chili peppers according to claim 1, characterized in that, In step (3), the concentration of the ethanol solution is 90-95 v / v; the ratio of the first filter residue to the ethanol solution is 5 kg: 4-6 L; the temperature of the ultrasonic extraction is 45-55 °C; and the time of the ultrasonic extraction is 45-65 min.

4. The method for preparing a diglyceride linolenic acid galactoside compound from chili peppers according to claim 1, characterized in that, The separation conditions for high-speed countercurrent chromatography in step (4) are as follows: the volume ratio of methanol:petroleum ether:n-hexane:water in the high-speed countercurrent chromatography solvent system is 5.5:3:5:2; the flow rate is 1.4-1.8 mL / min; the injection volume is 0.5-1.4 g; the rotation speed is 800-900 r / min; and the detection wavelength is 250-260 nm.

5. The method for preparing a diglyceride linolenic acid galactoside compound from chili peppers according to claim 1, characterized in that, The separation conditions for high performance liquid chromatography in step (5) are: C 18 Chromatographic column; the volume ratio of methanol to water in the mobile phase of high performance liquid chromatography is (90-95):8; the flow rate is 3-7 mL / min; the detection wavelength is 450-470 nm.

6. A diglyceride linolenic acid galactoside compound for chili peppers, characterized in that, The compound was prepared according to any one of claims 1-5.

7. The diglyceride linolenic acid galactoside compound in chili peppers according to claim 6, characterized in that, The chemical structural formula of the diglyceride linolenic acid galactoside compound is as follows: 。 8. The application of the diglyceride linolenic acid galactoside compound in chili peppers as described in claim 6 in the preparation of antioxidant drugs.

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