Separation and purification method of Chia seed polysaccharide with antioxidant activity

Abstract

The invention provides a separation and purification method of Chia seed polysaccharide with antioxidant activity, and relates to the technical field of Chia seed polysaccharide extraction. The separation and purification method of the Chia seed polysaccharide with the antioxidant activity comprises the following steps: step 1, extracting effective substances: selecting 100g of Chia seeds, grinding, extracting for 4 hours by using 0.5 L of n-hexane at 60 DEG C, repeating for three times, merging the obtained extracts, concentrating under reduced pressure, and storing at-20 DEG C to obtain a Chia seed oil sample extract to be detected; step 2, determination of tocopherol; 3, measuring the content of sterol and squalene in the chia seed oil; and step 4, polycyclic aromatic hydrocarbon detection. Through effective extraction of the chia seeds and accurate determination of effective components of the chia seeds, effective utilization of the chia seed polysaccharide can be realized, the utilization efficiency of the chia seeds is improved, and the chia seeds have a very good health-care effect.

Description

Technical Field

[0001] The invention relates to the technical field of chia seed polysaccharide extraction, and in particular to a method for separating and purifying chia seed polysaccharides with antioxidant activity. Background Art

[0002] Chia seeds contain essential alpha-linolenic acid. When mice were fed a diet supplemented with 5% chia seeds, it was found that it could improve the sensitivity of mice to insulin and glucose tolerance; relieve visceral obesity, reduce fatty degeneration of the liver; reduce inflammatory response and fibrosis of the heart and liver without affecting blood lipids and blood pressure. As one of the sources of ALA, chia seeds can induce lipid redistribution and play a key role in heart protection and liver protection.

[0003] Different methods and solvents for extracting chia seed oil will affect its fatty acid content. The most effective method is Soxhlet extraction with acetone as solvent, which can achieve a lipid extraction rate of 95%, 30% higher than the extrusion method, increase the content of bioactive substances, especially phenolic substances and carotenoids, and improve the stability of the oil. The ratio of n-3:n-6 fatty acids is between 3.18-4.18, which is higher than that of other vegetables. Supercritical fluid extraction is more suitable for extracting high-carbon molecular compounds in chia seeds, such as squalene (30 carbon hexaene). Experiments show that fluid extraction at 450 bar for 300 minutes can achieve the best results, with an oil yield of 92.8%. Although the composition of fatty acids varies according to different operating conditions, the ALA content (44.4-64.4%) and linoleic acid (19.6-35.0%) are both high.

[0004] At present, most of the research on chia seeds focuses on lipids or polyphenols, such as fatty acids, tocopherols, caffeic acid, chlorogenic acid, etc. Reyes-Caudillo et al. found that soluble dietary fiber SDF is mainly composed of neutral sugars, and various carbohydrates constitute the structure of the mucus, while uronic acid comes from the uronic acid residues in the mucus polysaccharide chain. Guiotto found that chia seed mucus has good performance as a stabilizer or thickener. Adding 0.75% wt / wt mucus can keep the emulsion stable, form a three-dimensional network structure, reduce the fluidity of oil particles, and avoid stratification of the emulsion during refrigeration. Studies have found that chia seed mucus (CSM) as a wall material for nano-encapsulation of chia seed oil can improve the thermal stability of chia seed oil and can also exist stably at 300°C.

[0005] In the past, there were few studies on chia seeds, and there was even less report on the composition and lipid-lowering activity of chia seed oil. This application took chia seed oil as the main research object, used modern spectral detection technology and HepG2 lipid-lowering model, and systematically studied the composition and lipid-lowering activity of chia seed oil. The purpose is to provide an experimental basis for the development of chia seed oil functional products. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for separating and purifying chia seed polysaccharides with antioxidant activity, which solves the problem that the active ingredients of chia seeds cannot be effectively extracted and determined.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for separating and purifying chia seed polysaccharides with antioxidant activity, comprising the following steps:

[0008] Step 1, extraction of effective substances: 100 g of chia seeds were selected and ground, and then extracted with 0.5 L of n-hexane at 60° C. for 4 hours, repeated three times, and then the obtained extracts were combined, concentrated under reduced pressure, and stored at -20° C. to obtain the chia seed oil sample extract to be tested;

[0009] Step 2, tocopherol determination: weigh 1.0 g of chia seed oil sample, accurate to 0.001 g, place in a 10 ml brown volumetric flask, add n-hexane to dissolve by ultrasonication, after ultrasonic constant volume, pass through a 0.22 μm organic membrane, take 20 μL for injection, and then perform chromatographic determination of tocopherol;

[0010] Step 3, determine the content of sterols and squalene in chia seed oil: accurately weigh 0.2-0.3g of chia seed oil sample, accurate to 0.001g, prepare 1mg / mL of 5α-cholestanol as an internal standard and add it to 0.5mL of chia seed oil sample, add 3mL of 2mol / mL KOH-ethanol solution, 85℃ water bath for 1h, take out and cool, add 2mL of water and 5mL of n-hexane, centrifuge to extract the upper clear liquid, extract the lower layer with 5mL of n-hexane twice, combine the organic liquid, wash with 2mL of water to neutrality, blow dry with nitrogen, add 200μL of BSTFA+TMCS (99:1) silanization reagent, and place in 70℃ water bath for 30min. After taking out and cooling, pass through a 0.22μm organic membrane, wait for injection, injection volume 1μL, and then perform chromatography and mass spectrometry;

[0011] Step 4, detection of polycyclic aromatic hydrocarbons: weigh 0.5g of chia seed oil sample and place it in a 10mL centrifuge tube, add 2mL of petroleum ether and vortex for 1min to dissolve the oil sample, wait for purification, use MIP solid phase extraction to treat the sample, insert the MIP-PAHs polycyclic aromatic hydrocarbons special SPE column into the CNW12 solid phase extraction vacuum device for activation, add 5mL of ethyl acetate and 5mL of petroleum ether to the SPE column in sequence for activation, after the activation, add 2mL of sample to the column, rinse the 10mL centrifuge tube with 3mL of n-hexane, load the column, and control the speed of loading to less than 1mL / min. After loading, continue to add 5mL of petroleum ether to rinse the SPE column to further wash away impurities, add 10mL of ethyl acetate after elution, collect the eluate with a 10mL centrifuge tube, dry with nitrogen, dissolve with acetonitrile ultrasonic for 1min and make up to 1mL, filter the sample with a 0.22μm filter membrane for standby use, and then perform chromatographic determination.

[0012] Preferably, the chromatographic conditions for the determination of tocopherol in step 2 are: chromatographic conditions Sehper i sorbSilica column at 25 cm × 4.6 mm × 5 μm, column temperature 25 ° C, detection wavelength 295 nm, mobile phase n-hexane: isopropanol volume ratio v:v = 98:2, flow rate 1 mL / min;

[0013] Determination: Prepare 1 mg / mL of mixed tocopherol standard in n-hexane solution, and determine the content of α, β, γ and δ-tocopherol and the total tocopherol content in chia seed oil by external standard method.

[0014] Preferably, the chromatographic conditions in step 3 are as follows: Chromatographic conditions: DB-5 column 30m×0.25mm×0.25μm, temperature program 200°C for 1min, 10°C / min to 300°C, keep for 18min, detector and injection port temperature is 290°C. Injection volume 1.0μL, split ratio 120:1, carrier gas: 99.99% helium; carrier gas flow rate: 1mL / min;

[0015] The mass spectrometry conditions in step 3 are: ion source temperature 280° C., transfer line temperature 250° C., ionization mode EI, and molecular ion fragmentation scanning range m / z 50-550.

[0016] Preferably, the determination conditions of the chromatographic determination in step 4 are: chromatographic conditions: LC-PAH chromatographic column of 250 mm×4.6 mm×5 μm; injection volume: 20 μL; column temperature: 25°C; mobile phase: A water, B acetonitrile; gradient elution program: 0-5 min, 50% A; 5-30 min, 50% A rising at a constant speed to 100% A; 30-45 min, 100% A, the detector is a fluorescence detector, the excitation wavelength is 260 nm, and the emission wavelength is 420 nm.

[0017] Preferably, tocopherol is a natural antioxidant and plays an important role in the oxidative stability of oils and fats; squalene can lower cholesterol levels.

[0018] The present invention provides a method for separating and purifying chia seed polysaccharides with antioxidant activity. The method has the following beneficial effects: the present invention can achieve effective utilization of chia seed polysaccharides through effective extraction of chia seeds and accurate determination of their effective components, thereby improving the utilization efficiency of chia seeds and making chia seeds have good health-care effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of tocopherol of the present invention;

[0020] Figure 2 This is a graph showing the effect of the chia seed oil of the present invention on TG, TC, HDL-C and LDL-C levels in the hyperlipidemia model induced by oleic acid. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example:

[0023] like Figure 1-2 As shown, the embodiment of the present invention provides a method for separating and purifying chia seed polysaccharides with antioxidant activity, comprising the following steps:

[0024] Step 1, extraction of effective substances: 100 g of chia seeds were selected and ground, and then extracted with 0.5 L of n-hexane at 60° C. for 4 hours, repeated three times, and then the obtained extracts were combined, concentrated under reduced pressure, and stored at -20° C. to obtain the chia seed oil sample extract to be tested;

[0025] Step 2, tocopherol determination: weigh 1.0 g of chia seed oil sample, accurate to 0.001 g, place in a 10 ml brown volumetric flask, add n-hexane to dissolve by ultrasonication, after ultrasonic constant volume, pass through a 0.22 μm organic membrane, take 20 μL for injection, and then perform chromatographic determination of tocopherol;

[0026] The chromatographic conditions for the determination of tocopherol in the step 2 are as follows: chromatographic conditions: Sehper i sorb Si l ica column at 25 cm × 4.6 mm × 5 μm, column temperature at 25° C., detection wavelength at 295 nm, mobile phase at a volume ratio of n-hexane:isopropanol of v:v=98:2, flow rate at 1 mL / min;

[0027] Determination: Prepare 1 mg / mL mixed tocopherol standard in n-hexane solution, and determine the content of α, β, γ and δ-tocopherol and total tocopherol in chia seed oil by external standard method;

[0028] Step 3, determine the content of sterols and squalene in chia seed oil: accurately weigh 0.2-0.3g of chia seed oil sample, accurate to 0.001g, prepare 1mg / mL of 5α-cholestanol as an internal standard and add it to 0.5mL of chia seed oil sample, add 3mL of 2mol / mL KOH-ethanol solution, 85℃ water bath for 1h, take out and cool, add 2mL of water and 5mL of n-hexane, centrifuge to extract the upper clear liquid, extract the lower layer with 5mL of n-hexane twice, combine the organic liquid, wash with 2mL of water to neutrality, blow dry with nitrogen, add 200μL of BSTFA+TMCS (99:1) silanization reagent, and place in 70℃ water bath for 30min. After taking out and cooling, pass through a 0.22μm organic membrane, wait for injection, injection volume 1μL, and then perform chromatography and mass spectrometry;

[0029] Tocopherol is a natural antioxidant that plays an important role in the oxidative stability of oils and fats. The contents of α-, γ-, δ-tocopherol and total tocopherol are as follows: Figure 1 The results showed that the tocopherol content of chia seed oil was 76.96 mg / kg, of which γ-tocopherol accounted for more than 91%, α-tocopherol accounted for 6.6%, and δ-tocopherol was the lowest, only 1.48 mg / kg. However, β-tocopherol was not detected. The data on total tocopherol content we obtained (76.96±8.47 mg / kg) was quite different from previous studies (238–427 mg / kg), which may be related to the variety, growth environment, processing and storage of chia seeds.

[0030] Squalene is known to be an important factor in lowering cholesterol levels. In this study, the content of squalene in CSO was 226.43 mg / kg, which is much higher than the concentrations found in walnuts, almonds, peanuts, hazelnuts and macadamia nuts by Maguire et al. The range of squalene content in CSO is 50-500 mg / kg, depending on the extraction method used. The high content of squalene in CSO may be an important contribution to the beneficial effects of cancer prevention and health in the human diet.

[0031] The chromatographic conditions in step 3 are as follows: Chromatographic conditions: DB-5 column 30m×0.25mm×0.25μm, temperature program 200℃ for 1min, 10℃ / min to 300℃, keep for 18min, detector and injection port temperature 290℃. Injection volume 1.0μL, split ratio 120:1, carrier gas: 99.99% helium; carrier gas flow rate: 1mL / min;

[0032] The mass spectrometry conditions in step 3 are: ion source temperature 280° C., transfer line temperature 250° C., ionization mode EI, molecular ion fragmentation scanning range m / z 50-550;

[0033] Step 4, detection of polycyclic aromatic hydrocarbons: weigh 0.5g of chia seed oil sample and place it in a 10mL centrifuge tube, add 2mL of petroleum ether and vortex for 1min to dissolve the oil sample, wait for purification, use MIP solid phase extraction to treat the sample, insert the MIP-PAHs polycyclic aromatic hydrocarbons special SPE column into the CNW12 solid phase extraction vacuum device for activation, add 5mL of ethyl acetate and 5mL of petroleum ether to the SPE column in sequence for activation, after the activation, add 2mL of sample to the column, rinse the 10mL centrifuge tube with 3mL of n-hexane, load the column, and control the speed of loading to less than 1mL / min. After loading, continue to add 5mL of petroleum ether to rinse the SPE column to further wash away impurities, add 10mL of ethyl acetate after elution, collect the eluate with a 10mL centrifuge tube, dry with nitrogen, dissolve with acetonitrile ultrasonic for 1min and make up to 1mL, filter the sample with a 0.22μm filter membrane for standby use, and then perform chromatographic determination.

[0034] The determination conditions of the chromatographic determination in the step 4 are as follows: chromatographic conditions: LC-PAH chromatographic column of 250 mm×4.6 mm×5 μm; injection volume: 20 μL; column temperature: 25°C; mobile phase: A water, B acetonitrile; gradient elution program: 0-5 min, 50% A; 5-30 min, 50% A rising at a constant speed to 100% A; 30-45 min, 100% A, the detector is a fluorescence detector, the excitation wavelength is 260 nm, and the emission wavelength is 420 nm.

[0035] Tocopherol is a natural antioxidant that plays an important role in the oxidative stability of oils and fats; squalene can lower cholesterol levels.

[0036] The antioxidant activity of chia seed oil (CSO) was evaluated by two in vitro chemical methods, DPPH and ABTS. As shown in the table below, the IC50 of chia seed oil is 33.94 mg / mL, while the IC50 of catechins is 0.005 mg / mL, which indicates that the DPPH scavenging ability of catechins is much higher than that of chia seed oil. Since the inhibition rate of ABTS is 50%, the IC25 is calculated in this experiment. By comparison, we found that the ability of tocopherol to scavenge ABTS is much higher than that of chia seed oil. Xuan et al. and Scapin et al. also used the DPPH and ABTS methods to study the antioxidant activity of chia seed oil and obtained similar results to ours, but compared with sunflower oil, safflower oil, rapeseed oil and soybean oil, the antioxidant activity of chia seed oil is relatively low.

[0037] As shown in Table 5-4, the toxicity IC25 of chia seed oil to LNcap cells and HepG2 cells are 580μg / mL and 889.68μg / mL (48h), respectively, indicating that CSO is cytotoxic to cells; the antioxidant activity and cytotoxicity comparison table of chia seed oil is as follows:

[0038]

[0039] Chia seed polysaccharides have good lipid-lowering activity. They can induce hepatic steatosis in HepG2 cells by exposing cells to pathophysiological levels of oleic acid (OA) to simulate the excessive lipotoxic influx of free fatty acids in hepatocytes. So far, there have been no reports on the use of oleic acid-induced HepG2 cell lipid-lowering model to study the activity of chia seed oil. Therefore, this paper used this model to evaluate the lipid-lowering function of chia seed oil. Figure 2 As shown: under the action of 0.2mmo l / LOA, the endogenous TG, TC, HDL-C and LDL-C levels of the model group were significantly increased. Chia seed oil had no toxicity to HepG2 cells at a concentration of 500μg / mL. Compared with the model group, the TG and LDL-C levels of the chia seed oil-treated group decreased significantly, with a decrease of 25.8% and 72.9% respectively. However, chia seed oil could not reduce the level of TC content, but increased the level of HDL-C content. Compared with the tocopherol group, the LDL-C level of the chia seed oil (500μg / mL)-treated group was higher. The results of Ayerza et al. showed that rats fed with dietary chia seed powder for 4 weeks could reduce TG and increase HDL-C levels, which is consistent with the research in this article. Therefore, chia seed oil has the potential to be developed into a functional product for lowering TG and LDL-c and increasing HDL-c.

[0040] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for separating and purifying chia seed polysaccharides with antioxidant activity, characterized in that: The following steps are involved: Step 1, extraction of effective substances: 100 g of chia seeds were selected and ground, and then extracted with 0.5 L of n-hexane at 60° C. for 4 hours, repeated three times, and then the obtained extracts were combined, concentrated under reduced pressure, and stored at -20° C. to obtain the chia seed oil sample extract to be tested; Step 2, tocopherol determination: weigh 1.0 g of chia seed oil sample, accurate to 0.001 g, place in a 10 ml brown volumetric flask, add n-hexane to dissolve by ultrasonication, after ultrasonic constant volume, pass through a 0.22 μm organic membrane, take 20 μL for injection, and then perform chromatographic determination of tocopherol; Step 3, determine the content of sterols and squalene in chia seed oil: accurately weigh 0.2-0.3g of chia seed oil sample, accurate to 0.001g, prepare 1mg / mL of 5α-cholestanol as an internal standard and add it to 0.5mL of chia seed oil sample, add 3mL of 2mol / mL KOH-ethanol solution, 85℃ water bath for 1h, take out and cool, add 2mL of water and 5mL of n-hexane, centrifuge to extract the upper clear liquid, extract the lower layer with 5mL of n-hexane twice, combine the organic liquid, wash with 2mL of water to neutrality, blow dry with nitrogen, add 200μL of BSTFA+TMCS (99:1) silanization reagent, and place in 70℃ water bath for 30min. After taking out and cooling, pass through a 0.22μm organic membrane, wait for injection, injection volume 1μL, and then perform chromatography and mass spectrometry; Step 4, detection of polycyclic aromatic hydrocarbons: weigh 0.5g of chia seed oil sample and place it in a 10mL centrifuge tube, add 2mL of petroleum ether and vortex for 1min to dissolve the oil sample, wait for purification, use MIP solid phase extraction to treat the sample, insert the MIP-PAHs polycyclic aromatic hydrocarbons special SPE column into the CNW12 solid phase extraction vacuum device for activation, add 5mL of ethyl acetate and 5mL of petroleum ether to the SPE column in sequence for activation, add 2mL of sample to the column after activation, rinse the 10mL centrifuge tube with 3mL of n-hexane, load the column, and control the speed of loading to less than 1mL / min. After loading, continue to add 5mL of petroleum ether to rinse the SPE column to further wash away impurities, add 10mL of ethyl acetate after elution, collect the eluate in a 10mL centrifuge tube, dry with nitrogen, dissolve and dilute to 1mL with acetonitrile ultrasound for 1min, filter the sample with a 0.22μm filter membrane for standby use, and then perform chromatographic determination.

2. The method for separating and purifying chia seed polysaccharides with antioxidant activity according to claim 1, characterized in that: The chromatographic conditions for the determination of tocopherol in step 2 are as follows: chromatographic conditions: Sehperisorb Silica column at 25 cm × 4.6 mm × 5 μm, column temperature at 25° C., detection wavelength at 295 nm, mobile phase at a volume ratio of n-hexane:isopropanol of v:v=98:2, flow rate at 1 mL / min; Determination: Prepare 1 mg / mL of mixed tocopherol standard in n-hexane solution, and determine the content of α, β, γ and δ-tocopherol and the total tocopherol content in chia seed oil by external standard method.

3. The method for separating and purifying chia seed polysaccharides with antioxidant activity according to claim 1, characterized in that: The chromatographic conditions in step 3 are as follows: Chromatographic conditions: DB-5 column 30m×0.25mm×0.25μm, temperature program 200℃ for 1min, 10℃ / min to 300℃, keep for 18min, detector and injection port temperature 290℃. Injection volume 1.0μL, split ratio 120:1, carrier gas: 99.99% helium; carrier gas flow rate: 1mL / min; The mass spectrometry conditions in step 3 are: ion source temperature 280° C., transfer line temperature 250° C., ionization mode EI, and molecular ion fragmentation scanning range m / z 50-550.

4. The method for separating and purifying chia seed polysaccharides with antioxidant activity according to claim 1, characterized in that: The determination conditions of the chromatographic determination in the step 4 are as follows: chromatographic conditions: LC-PAH chromatographic column of 250 mm×4.6 mm×5 μm; injection volume: 20 μL; column temperature: 25°C; mobile phase: A water, B acetonitrile; gradient elution program: 0-5 min, 50% A; 5-30 min, 50% A rising at a constant speed to 100% A; 30-45 min, 100% A, the detector is a fluorescence detector, the excitation wavelength is 260 nm, and the emission wavelength is 420 nm.

5. The method for using the antioxidant active chia seed polysaccharide according to claim 1, characterized in that: Tocopherol is a natural antioxidant that plays an important role in the oxidative stability of oils and fats; squalene can lower cholesterol levels.

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