A method for processing high-activity cereal-derived short-chain alkaloids

Abstract

The application discloses a processing method of high-activity grain source short-chain alkaloids, and belongs to the technical field of modern nutrition food processing. The processing method of high-activity grain source short-chain alkaloids comprises the following steps: placing grain particles in 0-4 DEG C storage for 1-12 weeks and 20-30 DEG C storage for 1-5 days; then placing the grain particles in a buffer solution for 6-16 hours at room temperature, and then placing the grain particles in 20-35 DEG C treatment for 12-48 hours with a humidity of 70-95 %; after the treatment, mixing the grain particles with an alcohol solution at a material weight ratio of 1:5-1:20, and then placing the mixture in a physical field for treatment, centrifugation, and taking supernatant, so that the short-chain alkaloids are obtained. The short-chain alkaloids have an average molecular weight of less than 500, a content of more than 300 mu g / g DW, and a biological accessibility of more than 50 %, and can be widely applied to the fields of health food, cosmetics, medicine and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a processing method of high-activity grain source short-chain alkaloids, and belongs to the technical field of modern nutritional food processing. BACKGROUND

[0002] Alkaloids are nitrogen-containing polyphenolic compounds formed by the dehydration condensation of phenolic acid (hydroxycinnamic acid) and o-aminobenzoic acid, and the basic structure is 5-hydroxy o-aminobenzoic acid and phenyl alkene acid subunit connected by an amide bond. Among them, phenyl alkene acid compounds can be divided into long-chain (C6-C3 structure) and short-chain (C6-C5 structure) according to the length of the chain, and the amide bond can form a resonance system with two aromatic rings through π electrons. Based on this unique chemical structure, short-chain alkaloids exhibit strong activity and efficacy, such as preventing and treating cell dysfunction and human diseases related to oxidative stress and inflammation.

[0003] Therefore, alkaloids as active ingredients have important significance in improving cardiovascular and cerebrovascular diseases, neurodegenerative diseases, metabolic syndrome, etc. At present, short-chain alkaloids in the prior art are mainly obtained through chemical synthesis reaction, but this method often has many disadvantages such as multiple by-products, low purity, serious pollution, high cost, etc. Moreover, existing research has confirmed that the bioavailability of alkaloids is extremely low, only 1-5% of other polyphenolic compounds or drugs, which greatly limits its application in the field of health food, etc.

[0004] Therefore, it is of great application prospect to provide a processing method of high-activity grain source short-chain alkaloids. SUMMARY

[0005] In view of the defects and deficiencies in the prior art, the present application provides a convenient, efficient and safe processing method of high-activity grain source short-chain alkaloids. The method provided by the present application solves the problems of low purity, poor safety and high cost of alkaloids at present, and its application range can be related to the fields of functional food, cosmetics, medicine, etc.

[0006] The first object of the present application is to provide a processing method of high-activity grain source short-chain alkaloids, which comprises the following steps:

[0007] (1) The grain particles are successively stored at 0-4℃ for 1-12 weeks and at 20-30℃ for 1-5 days;

[0008] (2) The grain particles stored in step (1) are soaked in a buffer solution at room temperature for 6-16h, and then treated at 20-35℃ and 70-95% humidity for 12-48h;

[0009] (3) mixing the cereal particles treated in step (2) with alcohol solution at a ratio of 1:5-1:20 (by weight of the material), and then centrifuging the mixture after being treated in a physical field to obtain short-chain alkaloids from the supernatant.

[0010] In one embodiment, the cereal particles in step (1) are one or more of hulled oat, naked oat, buckwheat, quinoa, rye, and wheat.

[0011] In one embodiment, the buffer solution in step (2) is an aqueous solution of one or more of abscisic acid, ascorbic acid, vitamin E, glutathione, and p-coumaric acid.

[0012] In one embodiment, the mass fraction of the solute in the buffer solution in step (2) is 0.5-2%.

[0013] In one embodiment, the alcohol in step (3) is one or more of ethanol, propylene glycol, and butylene glycol.

[0014] In one embodiment, the physical field in step (3) is a microwave field or an ultrasonic field.

[0015] In one embodiment, the microwave field has a power of 200-700 W and a time of 0.5-10 min.

[0016] In one embodiment, the ultrasonic field has a power of 500-2500 W and a time of 2-30 min.

[0017] In one embodiment, the centrifugation condition in step (3) is 5000-8000 g for 10-30 min.

[0018] The second object of the present application is to provide high-activity cereal-derived short-chain alkaloids obtained by the above-mentioned processing method.

[0019] In one embodiment, the short-chain alkaloids have an average molecular weight of <500, a content of >300 μg / g DW, a cellular antioxidant activity of >20 mg / ml, and a bioaccessibility of >50%.

[0020] In one embodiment, the short-chain alkaloids have an average molecular weight of 200-400, a content of 600-900 μg / g DW, a cellular antioxidant activity of >20 mg / ml, and a bioaccessibility of 60-80%.

[0021] The third object of the present application is to provide the use of the above-mentioned short-chain alkaloids in the preparation of health food, cosmetics, and pharmaceutical products.

[0022] The present application has the following advantages:

[0023] (1) The present application makes full use of grain resources, and efficiently develops the product through modern green processing technology, improves the functional activity, and provides a theoretical and technical basis for the development of nutritional and healthy food; and the steps are simple, the reaction conditions are mild, the safety is high, the continuous and low-cost green production is realized, the nutritional quality of modern food is improved, and the manufacturing of nutritional and healthy food is realized.

[0024] (2) The processing method of the high-activity grain source short-chain alkaloid of the present application is to first treat the grain under low temperature and room temperature conditions, use environmental factor stress to improve the enzyme activity related to short-chain alkaloid synthesis, and thus promote the metabolic synthesis of alkaloid content.

[0025] (3) The product prepared by the present application has strong antioxidant activity and bioavailability, improves the nutritional value and health value of the processed product, has the effects of preventing and treating diseases and maintaining human health, can be used as raw materials for healthy food, cosmetics and medicines, has important significance for improving health level, and has broad market prospect and economic benefit. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the liquid chromatogram of the short-chain alkaloid in Example 1 (the arrow marks the short-chain alkaloid).

[0027] Figure 2 is the mass spectrum of the short-chain alkaloid in Example 1. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application and are not used to limit the present application.

[0029] Test method involved in the present application

[0030] 1. Determination of short-chain alkaloid

[0031] The content of short-chain alkaloid is quantified by HPLC external standard method, and the chromatographic conditions are as follows: Agilent 1200 HPLC system, Shodex C-18 column (4.6*250mm, 5um), column temperature 40℃, flow rate 1mL / min, mobile phase A is 1‰ formic acid aqueous solution, B is acetonitrile; elution gradient: 0min, 20%B; 25min, 40%B; 30min, 20%B; 40min, 20%B, detection wavelength: 340nm; the alkaloid is identified by comparing the retention time and ultraviolet absorption spectrum data of the standard and the sample, the standard concentration gradient is set to: 3.125-100ug / mL, and the result is expressed as ug / gDW (sample dry weight).

[0032] The average molecular weight of short-chain alkaloids was determined using Q-TOF-MS (quadrupole high-resolution mass spectrometry) with the following parameters: detection voltage 1800 V, desolvation temperature 400 °C, cone voltage 30 V, capillary voltage 3.5 kV, scan range 20-2000 m / z, and negative ionization mode.

[0033] 2. Cellular antioxidant activity

[0034] Using human liver cancer cells (HepG2) as a model, AAPH and DCFH-DA were used as free radical inducers and fluorescent probes, respectively, to effectively reflect the ability of antioxidant components to scavenge free radicals during cell uptake. After incubating DCFH-DA working solution and DMEM medium containing samples of different concentrations for 1 hour, the medium was removed and washed with PBS buffer, and 100 μL of oxidative culture medium was added. The 96-well plate was then immediately placed in a fluorescence microplate reader to read the fluorescence values.

[0035] The calculation formula is as follows: Cellular antioxidant activity (mg / mL) = 1 - (∫SA / ∫CA), where: ∫SA - peak area of ​​the sample group, ∫CA - peak area of ​​the control group.

[0036] 3. Biological accessibility

[0037] The bioavailability of short-chain alkaloids was determined using Englyst in vitro simulation analysis. Oat alkaloids were digested with a mixture of enzymes (pancreatic α-amylase, saccharifying enzyme, and invertase) under simulated in vitro conditions (pH 5.2, 37℃). The content of oat alkaloids or phenolic acids was determined by high-performance liquid chromatography (HPLC), and the calculation formula is as follows:

[0038] Biological accessibility (%) = [(C 小肠 ×V 小肠 / (C 消化前 ×V 消化前 )]×100,

[0039] Among them, C 小肠 —The concentration (μg / mL) of oat alkaloids or phenolic acids in the intestinal fluid after in vitro digestion, V 小肠 —Volume of intestinal fluid after in vitro digestion (mL); C 消化前 —Concentration (μg / mL) of oat alkaloids or phenolic acids in the solution before in vitro digestion, V 消化前 —Volume (mL) of the solution before in vitro digestion.

[0040] Example 1

[0041] A method for processing highly active cereal-derived short-chain alkaloids, the method comprising the following steps:

[0042] Take 100 g of intact clean oat particles and store them at 4 °C for 4 weeks and at 30 °C for 2 days; then soak them in an aqueous solution of ascorbic acid with a mass fraction of 2% at room temperature for 12 h, and then store them at 30 °C and 70% humidity for 24 h; mix them with an ethanol solution at a material weight ratio of 1:20, and then place them in an ultrasonic field at 800 W for 50 min; centrifuge (5000 g, 30 min), and take the supernatant, which is the short-chain alkaloid.

[0043] After measurement, the short-chain alkaloid obtained in this embodiment has an average molecular weight of 354, a content of 809 μg / g DW, a cell antioxidant activity of 31 mg / ml, and a bioavailability of 61%.

[0044] Example 2

[0045] A processing method of a high-activity grain-derived short-chain alkaloid, the method comprising the following steps:

[0046] Take 100 g of intact clean oat particles and store them at 4 °C for 4 weeks and at 30 °C for 2 days; then soak them in an aqueous solution of ascorbic acid with a mass fraction of 2% at room temperature for 12 h, and then store them at 30 °C and 70% humidity for 24 h; mix them with an ethanol solution at a material weight ratio of 1:20, and then place them in an ultrasonic field at 800 W for 50 min; centrifuge (5000 g, 30 min), and take the supernatant, which is the short-chain alkaloid.

[0047] After measurement, the short-chain alkaloid obtained in this embodiment has an average molecular weight of 354, a content of 809 μg / g DW, a cell antioxidant activity of 31 mg / ml, and a bioavailability of 61%.

[0048] Example 3

[0049] A processing method of a high-activity grain-derived short-chain alkaloid, the method comprising the following steps:

[0050] Take 100 g of intact clean oat particles and store them at 4 °C for 4 weeks and at 30 °C for 2 days; then soak them in an aqueous solution of ascorbic acid with a mass fraction of 2% at room temperature for 12 h, and then store them at 30 °C and 70% humidity for 24 h; mix them with an ethanol solution at a material weight ratio of 1:20, and then place them in an ultrasonic field at 800 W for 50 min; centrifuge (5000 g, 30 min), and take the supernatant, which is the short-chain alkaloid.

[0051] After measurement, the short-chain alkaloid obtained in this embodiment has an average molecular weight of 354, a content of 809 μg / g DW, a cell antioxidant activity of 31 mg / ml, and a bioavailability of 61%.

[0052] Comparative Example 1

[0053] Referring to Example 1, the only difference is that the low temperature treatment is omitted, and the intact clean oat kernels are directly placed at 30°C, and other conditions are unchanged, to prepare the short-chain alkaloids.

[0054] It is determined that the short-chain alkaloids obtained in this comparative example have an average molecular weight of 475, a content of 282 μg / g DW, a cell antioxidant activity of 8 mg / ml, and a bioaccessibility of 43%.

[0055] Comparative Example 2

[0056] Referring to Example 1, the only difference is that the 2% ascorbic acid aqueous solution is directly replaced by an aqueous solution, and other conditions are unchanged, to prepare the short-chain alkaloids.

[0057] It is determined that the short-chain alkaloids obtained in this comparative example have an average molecular weight of 530, a content of 105 μg / g DW, a cell antioxidant activity of 11 mg / ml, and a bioaccessibility of 32%.

[0058] Comparative Example 3

[0059] Referring to Example 1, the only difference is that the 2% ascorbic acid aqueous solution is replaced by a 2% citric acid aqueous solution, and other conditions are unchanged, to prepare the short-chain alkaloids.

[0060] It is determined that the short-chain alkaloids obtained in this comparative example have an average molecular weight of 219, a content of 78 μg / g DW, a cell antioxidant activity of 6 mg / ml, and a bioaccessibility of 25%.

[0061] Comparative Example 4

[0062] Referring to Example 1, the only difference is that the 2% ascorbic acid aqueous solution is replaced by a 10% ascorbic acid aqueous solution, and other conditions are unchanged, to prepare the short-chain alkaloids.

[0063] It is determined that the short-chain alkaloids obtained in this comparative example have an average molecular weight of 305, a content of 143 μg / g DW, a cell antioxidant activity of 9 mg / ml, and a bioaccessibility of 32%.

[0064] The above examples provided are not intended to limit the scope of the present application, and the described steps are not intended to limit the order of execution. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for processing high-activity cereal-derived short-chain alkaloids, characterized by, The method comprises the following steps: (1) placing the cereal grains in 0-4℃ for 1-12 weeks, and 20-30℃ for 1-5 days; (2) placing the cereal grains preserved in step (1) in a buffer solution for 6-16 h at room temperature, and then treating them at 20-35℃ and 70-95% humidity for 12-48 h; The buffer solution is a solution formed by using one or more of solutes abscisic acid, ascorbic acid, and glutathione; The mass fraction of the solutes in the buffer solution is 0.5-2%; (3) mixing the cereal grains treated in step (2) with an alcohol solution at a material weight ratio of 1:5-1:20, and then treating them in a physical field, centrifuging, and taking the supernatant to obtain short-chain alkaloids; The physical field is a microwave field or an ultrasonic field.

2. The method of claim 1, wherein, The cereal grains in step (1) are one or more of hulled oats, naked oats, buckwheat, quinoa, rye, and naked millet.

3. The method of claim 1 wherein, The alcohol in step (3) is one or more of ethanol, propylene glycol, and butylene glycol.

4. The method of processing according to claim 1, wherein, The power of the microwave field is 200-700W, and the time is 0.5-10 min.

5. The method of processing according to claim 1, wherein, The power of the ultrasonic field is 500-2500W, and the time is 2-30 min.

6. High-activity cereal-derived short-chain alkaloids obtained by the processing method of any one of claims 1-5.

7. The high active, grain-derived short chain alkaloid of claim 6, wherein, The short-chain alkaloids have an average molecular weight of <500, a content of >300 μg / g DW, a cellular antioxidant activity of >20 mg / ml, and a bioavailability of >50%.

8. Use of the high-activity cereal-derived short-chain alkaloids of any one of claims 6 or 7 in the preparation of functional foods, cosmetics, and pharmaceutical products.

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