WO2022105726A1

WO2022105726A1 - Production method for lycopene-containing granules with efficient antioxidation effect

Info

Publication number: WO2022105726A1
Application number: PCT/CN2021/130819
Authority: WO
Inventors: 冯鹏; 王连安; 王颖; 周亚杰; 钱一帆; 冯敏
Original assignee: 中科健康产业集团股份有限公司; 南京中科药业有限公司; 中科健康产业集团江苏药业有限公司
Priority date: 2020-11-18
Filing date: 2021-11-16
Publication date: 2022-05-27
Also published as: CN112569192A

Abstract

A production method for lycopene-containing granules with an efficient antioxidation effect. The method comprises: extracting Ganoderma lucidum spore oil from Ganoderma lucidum spore powder having a broken sporoderm rate of 90%-92% by means of a CO₂ supercritical extraction process to obtain degreased Ganoderma lucidum spore powder; and mixing the degreased Ganoderma lucidum spore powder and lycopene oil resin at a weight ratio of (1-2):(1-2), and performing spray granulation on same. In a granular product prepared by using the method, the effective component content is high, the lycopene content is more than 6%, and the polysaccharide content is more than 0.8%; and the product has good stability and has an efficient antioxidation effect.

Description

A kind of production method of lycopene-containing granules with high-efficiency antioxidant effect

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for producing lycopene-containing granules with high-efficiency antioxidant effect.

Background technique

Lycopene is a functional edible natural pigment, belonging to an important carotenoid, widely present in fruits and vegetables, especially in tomatoes with the highest content. Lycopene is a straight-chain hydrocarbon composed of 11 conjugated carbon-carbon double bonds, the molecular formula is C ₄₀ H ₅₆ , and the pure crystalline product is dark red. Lycopene is widely distributed in various organs and tissues of the human body, and exists in a cis configuration. The cis configuration and the trans configuration can be converted into each other according to different environments. But almost all lycopene derived from natural plants is in the trans configuration, which is the most thermostable.

Oxidative stress damage refers to tissue damage caused by excessive production of reactive oxygen species and reactive nitrogen radicals in the body when the body is subjected to harmful stimuli, or the weakening of the enzyme system for scavenging oxidative free radicals. Oxidative stress damage is an important factor leading to human skin aging. It damages cells and tissues, which can lead to degeneration of body functions, and is also the main factor causing nerve damage in cardiovascular and cerebrovascular diseases. Proper supplementation of antioxidants can alleviate oxidative stress damage in the body. In recent years, most of the research on new antioxidative drugs has focused on natural products that can be extracted from the daily diet, because these products have few side effects. Natural antioxidants have always been a research hotspot in the preventive medicine and health care industry for various middle-aged and elderly chronic diseases such as coronary heart disease, hyperlipidemia, diabetes, and neurodegenerative diseases.

The antioxidant effect of lycopene is mainly manifested in that it can quench singlet oxygen and scavenge free radicals. The rate constant of scavenging singlet oxygen is 100 times that of antioxidant vitamin E and more than 2 times that of β-carotene. It has important physiological functions in the prevention and treatment of prostate cancer, lung cancer, and regulation of immunity, and has become a focus of international research on functional food ingredients. Hypochlorous acid can lead to tissue oxidation in cardiovascular diseases by modifying proteins, deoxyribonucleic acid, and ribonucleic acid, while lycopene has the effect of scavenging hypochlorous acid, which can reduce the body damage caused by hypochlorous acid. Both animal experiments and population studies have shown that lycopene has a protective effect on the nervous system. Lycopene can reduce the expression of inflammatory factors by inhibiting related inflammatory pathways, reduce the level of inflammation in the body, and inhibit the apoptosis pathway, reducing the loss of nerve cells and apoptosis. It can improve the antioxidant capacity of nerve cells, inhibit the production of reactive oxygen species, and exert a neuroprotective effect. The combination of lycopene and other substances can enhance its antioxidant effect, and its clinical use has bright prospects.

Because lycopene contains a large number of unsaturated structures, it is easily degraded by oxidation under the action of light, heat and oxygen, and is prone to degradation and isomerization during processing and storage, resulting in reduced physiological activity, and lycopene is a kind of Fat-soluble carotenoids are insoluble in water, and these characteristics greatly limit its promotion and application. At present, there are mainly two kinds of raw materials for lycopene, namely, semi-fluid raw materials (oleoresin) dissolved in fat-soluble solvents and granular embedded solid raw materials. The granular embedded raw material after macromolecular embedding has a great advantage in stability, and will not be damaged by strong acid in gastric juice after taking, so that its bioavailability is improved.

At present, lycopene products cover the fields of medicine, health care and cosmetics. There are a variety of injection drugs with lycopene as the main agent, which are used to prevent ultraviolet burns, remove stains, protect the skin and adjuvant cancer treatment; there are All kinds of health care products with lycopene as the main component are mainly used for anti-oxidation, anti-aging, enhancing immunity, regulating blood lipids and preventing cancer and anti-cancer; some supplements with lycopene are added to jam, meat products, In dairy products and beverages, lycopene is used as a preservative and added to edible oil to prevent the oxidative rancidity of the oil and prolong the shelf life of the oil.

At present, the preparations containing lycopene are mainly soft capsules, which are made by mixing lycopene oleoresin with soybean oil, safflower oil and other edible oils, and filling soft capsules. However, the lycopene product of this dosage form has poor stability. The embedding process is also widely used in lycopene products, including hard capsules, granules, etc., but many excipients are used, and the content of active ingredients is low, generally not more than 3%.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the above-mentioned deficiencies, and provide a method for producing lycopene-containing particles with high-efficiency antioxidant effect. The lycopene-containing granules obtained by the method have high content of effective components, convenient preparation and high stability.

The object of the present invention is achieved in the following ways:

A production method of lycopene-containing particles with high-efficiency antioxidant effect, comprising the following steps:

Take Ganoderma lucidum spore powder with a wall breaking rate of 90-92%, extract Ganoderma lucidum spore oil through CO ₂ supercritical extraction process to obtain degreasing Ganoderma lucidum spore powder, and combine the defatted Ganoderma lucidum spore powder with lycopene oleoresin in a ratio of 1-2:1 -2 weight ratio mixing spray granulation.

The above-mentioned Ganoderma lucidum spore powder is the Ganoderma lucidum spore powder erupted from Red Ganoderma lucidum. Defatted Ganoderma lucidum spore powder can still see the complete shape of Ganoderma lucidum spores under a 600x microscope.

The above-mentioned CO ₂ supercritical extraction process is as follows: the extraction temperature is 35～36° C., the extraction pressure is 25～28Mpa, the extraction time is 6～7 hours, the CO ₂ flow rate is 400～600L/h, and the sample loading volume is 38kg each time.

The preferred _CO2 supercritical extraction process is as follows: the extraction temperature is 35°C, the extraction pressure is 25Mpa, the extraction time is 7 hours, and the _CO2 flow rate is 500L/h.

The above mixing method is as follows: mixing lycopene oleoresin with a little absolute ethanol to obtain lycopene oleoresin alcoholate to increase its fluidity. Put the defatted Ganoderma lucidum spore powder into the fluidized bed granulator, in the fluidized bed granulator, the lycopene oleoresin alcoholate is atomized from the nozzle and sprayed onto the fluidized bed layer by compressed air, and it is in a fluidized state On the broken wall of Ganoderma lucidum spore powder, make it wet and mix well, and flow granulation.

The conditions used for spray granulation are: the atomization speed of the nozzle is 40-100 mL/min, the atomization particle size is 10-12 μm, and the one-time sample loading of the fluidized bed is 8-10 kg. Preferably, the atomization speed of the nozzle is 100 mL/min, the atomization particle size is 10 μm, the one-time sample loading of the fluidized bed is 8 kg, and flow granulation is carried out. The conditions can be fully mixed, and after granulation, the prepared granules are placed in a cool and dry place to dry in the shade.

Preferably, the mixing weight ratio of the above-mentioned defatted Ganoderma lucidum spore powder and lycopene oleoresin is 1:1.

Mixing in this ratio can increase the lycopene content to the maximum amount, and the particles are uniform.

Compared with the prior art, the beneficial effects of the present invention are: the granule product prepared by the method of the present invention has high content of active ingredients, content of lycopene > 6%, content of polysaccharide > 0.8%, good stability, and efficient anti-oxidation. effect.

Description of drawings

Figure 1 is a diagram showing the protective effect of lycopene particles on the oxidative degradation of BSA caused by AAPH according to an embodiment of the present invention.

Figure 2 is a graph showing the protective effect of Example lycopene particles on U87 cells from oxidative damage.

Among them, L. lycopene granules; Q. quercetin; ^* P < 0.05, ^** P < 0.01 compared with the control group; ^# P < 0.05 ^{, ##} ^P < 0.01 compared with the injury group.

Fig. 3 is a graph showing the effect of lycopene particles on the content of 8-OH-dG in an embodiment.

Among them, CK. control group; HH ₂ O ₂ treatment group; H+L 1.1 μmol·L ^-1 lycopene particle protection group; H+L 5.5 μmol·L ^-1 lycopene particle protection group; H+L10. 10μmol·L ^-1 lycopene particle protection group; H+Q5.5μmol·L ^-1 quercetin protection group.

Detailed ways

The present invention will be further explained below through specific examples: wherein, lycopene oleoresin is purchased from Chenguang Biotechnology Group Co., Ltd., is a dark red liquid oil, and its lycopene content is greater than 12%.

Example 1

Take the Ganoderma lucidum spore powder with a wall breaking rate of 90-92%, and extract the Ganoderma lucidum spore oil with _CO2 supercritical extraction process to obtain the degreasing Ganoderma lucidum spore powder. The _CO2 supercritical extraction process is as follows: the extraction temperature is 35°C, and the extraction pressure is 25Mpa , the extraction time was 7 hours, the flow rate of CO ₂ was 500 L/h, and the amount of each sample was 38 kg. Defatted Ganoderma lucidum spore powder can still see the complete shape of Ganoderma lucidum spores under a 600x microscope. Defatted Ganoderma lucidum spore powder and lycopene oleoresin were taken in a weight ratio of 1:1.

First put the defatted Ganoderma lucidum spore powder into the fluidized bed granulator, mix the lycopene oleoresin with a little anhydrous ethanol, and then spray the lycopene oleoresin mixed with the ethanol from the nozzle and spray it to the On the fluidized bed layer, the broken-wall Ganoderma lucidum spore powder in fluidized state is wetted, fully mixed, and flow granulated. The sample loading capacity of the bed is 8kg at a time; the prepared granules are placed in a cool and dry place to dry in the shade.

Three batches of samples were made in parallel, the contents of lycopene in the obtained products were 8.36%, 8.29%, 8.28%, and the contents of polysaccharides were 1.23%, 1.25%, and 1.22, respectively. They are 1#, 2#, and 3# products (hereinafter referred to as lycopene particles).

Comparative Example 1

Take the Ganoderma lucidum spore powder with a wall breaking rate of 90-92%, and extract the Ganoderma lucidum spore oil with _CO2 supercritical extraction process to obtain the degreasing Ganoderma lucidum spore powder. The _CO2 supercritical extraction process is as follows: the extraction temperature is 35°C, and the extraction pressure is 25Mpa , the extraction time was 7 hours, the flow rate of CO ₂ was 500L/h, and the sample loading was 38kg each time. Defatted Ganoderma lucidum spore powder can still see the complete shape of Ganoderma lucidum spores under a 600x microscope. The defatted Ganoderma lucidum spore powder and the lycopene oleoresin are fully mixed in a weight ratio of 1:1, and conventional dextrin and other auxiliary materials are added for granulation, and the obtained granule is a 4# product.

Comparative Example 2

Take the Ganoderma lucidum spore powder with a wall breaking rate of 90-92%, and extract the Ganoderma lucidum spore oil with _CO2 supercritical extraction process to obtain the degreasing Ganoderma lucidum spore powder. The _CO2 supercritical extraction process is as follows: the extraction temperature is 35°C, and the extraction pressure is 25Mpa , the extraction time was 7 hours, the flow rate of CO ₂ was 500 L/h, and the amount of each sample was 38 kg. Defatted Ganoderma lucidum spore powder can still see the complete shape of Ganoderma lucidum spores under a 600x microscope. Defatted Ganoderma lucidum spore powder and lycopene oleoresin were taken in a weight ratio of 9:1.

First put the defatted Ganoderma lucidum spore powder into the fluidized bed granulator, mix the lycopene oleoresin with a little anhydrous ethanol, and then spray the lycopene oleoresin mixed with the ethanol from the nozzle and spray it to the On the fluidized bed layer, the broken-wall Ganoderma lucidum spore powder in the fluidized state is wetted, fully mixed, and flow granulated. The one-time sample loading of the chemical bed is 8kg; the prepared granules are placed in a cool and dry place to dry in the shade. The obtained granules are 5# products.

Comparative Example 3

Take Ganoderma lucidum spore powder with a wall breaking rate of 90-92%, and use _CO2 supercritical extraction process to extract Ganoderma lucidum spore oil to obtain degreased Ganoderma lucidum spore powder. The _CO2 supercritical extraction process is: the extraction temperature is 40°C, and the extraction pressure is 30Mpa , the extraction time was 5 hours, the flow rate of CO ₂ was 500 L/h, and the amount of each sample was 38 kg. Defatted Ganoderma lucidum spore powder and lycopene oleoresin were taken in a weight ratio of 1:1.

Put the defatted Ganoderma lucidum spore powder into the fluidized bed granulator, mix the lycopene oleoresin with a little anhydrous ethanol, and then spray the lycopene oleoresin mixed with the ethanol from the nozzle and spray it to the flow through the compressed air. On the fluidized bed layer, the broken-wall Ganoderma lucidum spore powder in a fluidized state is wetted, fully mixed, and flow granulated. The sample loading capacity of the bed is 8kg at a time; the prepared granules are placed in a cool and dry place to dry in the shade. The obtained granule is 6# product.

The above-mentioned 1#-6# lycopene granule product is subjected to lycopene content stability test and polysaccharide content stability, and the specific results are shown in Table 1 and Table 2:

Table 1 Stability data of lycopene content

样品编号Sample serial number	0月0	3月March	6月June	12月December
1#1#	8.36％8.36%	8.35％8.35%	8.33％8.33%	8.30％8.30%
2#2#	8.29％8.29%	8.28％8.28%	8.28％8.28%	8.25％8.25%
3#3#	8.28％8.28%	8.27％8.27%	8.26％8.26%	8.26％8.26%
4#4#	3.26％3.26%	3.25％3.25%	3.24％3.24%	3.22％3.22%
5#5#	1.27％1.27%	1.27％1.27%	1.26％1.26%	1.26％1.26%
6#6#	7.84％7.84%	7.80％7.80%	7.72％7.72%	7.45％7.45%

Table 2 Polysaccharide content stability data

样品编号Sample serial number	0月0	3月March	6月June	12月December
1#1#	1.23％1.23%	1.24％1.24%	1.22％1.22%	1.20％1.20%
2#2#	1.25％1.25%	1.24％1.24%	1.22％1.22%	1.22％1.22%
3#3#	1.22％1.22%	1.21％1.21%	1.22％1.22%	1.20％1.20%

4#4#	1.55％1.55%	1.55％1.55%	1.56％1.56%	1.54％1.54%
5#5#	1.82％1.82%	1.80％1.80%	1.81％1.81%	1.80％1.80%
6#6#	1.20％1.20%	1.17％1.17%	1.16％1.16%	1.16％1.16%

The determination methods of lycopene and polysaccharide content are as follows:

1. Determination of lycopene content of the product by high performance liquid chromatography

1. Pre-treatment of the sample to be tested:

Weigh 2g of the sample to be tested and fully grind it. After mixing evenly, accurately weigh 100mg and place it in a 100mL brown volumetric flask. Add 20mL of 4% ammonia buffer solution and ultrasonicate it in a 55°C water bath for 20min, shaking it every 5min. After room temperature, use a stable tetrahydrofuran solution to dilute to volume, stir on a magnetic stirrer for 20 min, and then let stand for 5 min. After the solution is clear, draw 1.0 mL of supernatant and place it in a 10 mL brown volumetric flask; finally, use a volume ratio of 90:10. Dichloromethane and methanol solutions were adjusted to the mark to obtain the solution to be tested.

The preparation method of the 4% ammonia buffer solution is as follows: firstly, 143 mL of ammonia water is added to 1 L of purified water and mixed evenly, and then the pH is adjusted to 9.8 with 85% phosphoric acid, thus obtaining the 4% ammonia buffer solution. The stable tetrahydrofuran solution was prepared by dissolving 0.25g BHT in 1L tetrahydrofuran. The mobile phase solution was a dichloromethane and methanol solution with a volume ratio of 90:10.

2. Prepare standard curve solution:

(1) Preparation of standard stock solution: Precisely weigh 6.0 mg of lycopene standard and place it in a 25 mL brown volumetric flask, dissolve with 22.5 mL of dichloromethane, and then dilute to the mark with methanol to prepare 0.2 mg/mL tomato Red pigment standard stock solution, the bottle is filled with nitrogen and sealed, and stored at 0°C for future use;

(2) Preparation of standard curve solution: when used for detection, take the above 0.2mg/mL lycopene standard stock solution, respectively take 40μL, 80μL, 120μL, 160μL, 200μL into five 1.0mL quantitative bottles, respectively. The standard curve solutions with concentrations of 4 μg/mL, 8 μg/mL, 12 μg/mL, 16 μg/mL and 20 μg/mL were prepared with mobile phase solutions of dichloromethane and methanol with a volume ratio of 90:10, respectively.

3. Separation and detection of lycopene in the liquid to be tested by high performance liquid chromatography:

The standard curve solution was injected under chromatographic conditions, and the concentration was taken as the abscissa and the peak area as the ordinate to obtain the standard curve equation; The sample was injected under the same chromatographic conditions, separated by a reverse-phase high-efficiency separation chromatographic column, and detected by a UV detector, and the sample to be tested was qualitatively determined by the retention time; according to the above standard curve equation, the retention time and peak area were used to calculate the sample to be tested. lycopene content.

Wherein, the chromatographic conditions are:

Chromatographic column: C18 column

Column temperature: 30℃

Flow rate: 0.6～0.8mL/min

Mobile phase: dichloromethane:methanol in a volume ratio of 90:10

UV detector detection wavelength: 472nm

Injection volume: 5 μL.

4. Calculation of the content of lycopene in the sample to be tested:

In the formula: X——the content of lycopene in the sample, %;

ρ——The concentration of lycopene in the sample calculated according to the standard curve, μg/mL;

V——dilution factor of the sample;

m——Weighing sample size of the sample, g.

Calculate the content of lycopene in the sample according to the standard curve equation, and combine with the quality of the sample to obtain the content of lycopene in the sample to be tested.

2. Detection of polysaccharide content of the product by anthrone sulfate method

1 Preparation of sample solution

Take about 1g of this product powder, accurately weigh it, put it in a 100mL volumetric flask, heat 90ml of water, extract it in a boiling water bath for 2h, cool it to room temperature, dilute it with water and make up to the mark. Filter, accurately measure 2.0ml of the subsequent filtrate, add 30ml of ethanol, shake well, place at 4°C for 12h, take out the centrifuge, pour off the supernatant, dissolve the precipitate in water, shake well, and make up to 100ml, which is the sample solution.

2 Preparation of reference solution

Prepare 100mg/L glucose solution, accurately measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution, put them in 10mL volumetric flasks, add water to 2.0mL, and then accurately add anthrone sulfate. Solution (accurately weigh 0.1 g of anthrone, add 100 mL of 80% sulfuric acid solution to dissolve, shake well) 6 mL, shake well, heat in a boiling water bath for 15 minutes, take out, shake well, cool in ice water for 15 minutes, take the corresponding reagent as Blank, according to the UV-Vis spectrophotometry test, measure the absorbance at the wavelength of 625nm, draw the standard curve with the absorbance as the ordinate and the concentration as the abscissa.

3 Determination of polysaccharide content

Precisely measure 2.0mL of the test solution, put it in a 10mL test tube with a stopper, and follow the method under the preparation of the reference solution, starting from "adding 6mL of anthrone sulfate solution", measure the absorbance according to the law, and read it from the standard curve. The amount of glucose in the sample solution, calculated and obtained.

In the formula: X--------- polysaccharide content in the sample, g/100g;

m ₁ -------- the mass of glucose in the sample solution, mg;

m--------the mass of the sample, mg;

V ₁ --------Sample processing volume, mL;

V ₂ -------- Volume of sample solution used to precipitate polysaccharide, mL;

V ₃ -------- Precipitated polysaccharide volume, mL;

V ₄ ----- Determination of polysaccharide solution volume, mL.

Calculate the content of polysaccharide in the sample according to the standard curve, and combine the quality of the sample to obtain the content of polysaccharide by calculation.

Test Example 1

1.1 Materials

The product of Example 1 of the present invention (hereinafter referred to as lycopene particles), quercetin, lycopene oleoresin, defatted Ganoderma lucidum spore powder, AAPH (2,2'-azobisisobutylamidine dihydrochloride), Agarose and 8-OH-dG standards were purchased from Sigma, deoxyribose from BioRad, sodium dodecyl sulfonate (SDS) from Serva, and malignant glioma cells U87 from Lanzhou University School of Life Sciences, DMEM medium was purchased from Gibco in the United States, newborn bovine serum was produced by Hangzhou Sijiqing Company, and other reagents were of domestic analytical grade. Evolution 201 UV-Vis spectrophotometer (ThermoScientific, USA), benchtop refrigerated high-speed centrifuge (Allegra 64R Beckman), _CO2 incubator (Precision Scientific, USA), ultra-clean workbench (Sujing Group, Jiangsu), inverted microscope (Olympus Corporation, Japan), P/ACEMDQ type capillary electrophoresis apparatus (Beckman Coulter, USA).

1.2 Methods

1.2.1 Determination of reducing power

Antioxidants can reduce Fe ³⁺ to Fe ²⁺ , and form a soluble blue complex KFe[Fe(CN) ₆ ] with potassium ferricyanide with maximum light absorption at 700 nm. Therefore, the stronger the reducing power, the larger the measured absorbance value. Different concentrations (1, 10, 100 μmol·L ^-1 ) of lycopene particle sample solution, 10 μmol·L ^-1 lycopene oleoresin and 10 μmol·L ^-1 degreasing Ganoderma lucidum spore powder were taken 1 mL each, and added 0.2 mol pH=6.6 2.5 mL each of phosphate buffer solution and 1% potassium ferricyanide (K ₃ Fe(CN) ₆ ) solution were mixed well, placed in a 50°C water bath for 20 min, added 2.5 mL of 10% trichloroacetic acid, and the mixture was 4000 r · min ^-1 centrifugation for 10 min. Take 2.5 mL of supernatant, add 2.5 mL of distilled water and 1 mL of 0.1% ferric chloride solution. Absorbance was detected at 700 nm after standing for 10 min.

1.2.2 Determination of hydroxyl radical scavenging ability

Using the deoxyribose degradation method, 1160 μL of pH=6.5 phosphate buffered solution (PBS), 580 μL of EDTA-Na ₂ ( ^{1 mmol·L -1} ) solution and 380 μL of FeCl ₃ ( ^{1 mmol·L -1} ) solution were added to the system in sequence, and the system was shaken. Shake well. 750 μL of deoxyribose solution (20 mmol·L ^-1 ), 100 μL of 30% hydrogen peroxide solution and antioxidants of various concentrations were added in sequence, and 30 μL of ascorbic acid (10 ^{mmol·L -1} ) solution was used to initiate the Fenton reaction and shake well. The reaction mixture was placed in a 50°C water bath for 30 min, taken out and cooled to room temperature, and then 500 μL of 2.8% trichloroacetic acid (TCA) solution was added to terminate the reaction, and 500 μL of 1% chromogenic reagent thiobarbituric acid (TBA) was added to the solution. 100°C water bath for 30min. After cooling to room temperature, the absorbance was detected at 532 nm. Clearance rate (%)=[1-A ₁ /A ₀ ]×100 (A ₀ is the absorbance value without scavenger; A ₁ is the absorbance value with scavenger added).

1.2.3 Determination of superoxide anion scavenging ability

Take 0.5 mL of each sample with different concentrations, add 4.43 mL of Tris-HCl (50 mmol·L ^-1 ) buffer with pH=8.2, add 70 μL of pyrogallol solution (10 ^{mmol·L -1} ), time it immediately and shake it up quickly, The absorbance at the corresponding 325nm was detected every 30s after the reaction started, until 4.5min. In the control tube, 70 μL of hydrochloric acid (10 ^{mmol·L −1} ) was used to replace the pyrogallol solution. Superoxide anion scavenging rate (%)=[1-(A ₁ -A ₂ )/A ₀ ]×100 (A ₀ is the absorbance without scavenger; A ₁ is the absorbance with scavenger added; A ₂ is absorbance without pyrogallol).

1.2.4 Inhibition of lipid peroxidation

The microsomes were extracted by thiobarbituric acid colorimetry, referring to the method of Liu Guoan et al., and diluted to 0.3～0.5g·L ^-1 . Take 300 μL of microsomes and add 100 μL of PBS and 300 μL of distilled water, then add 100 μL of samples with different concentrations, and replace the positive control with distilled water. Start the reaction with 200 μL Vc/Fe ²⁺ , incubate at 37°C for 1 h, add 1 mL of 20% TCA to stop the reaction, mix with 1.5 mL of 0.67% TBA, bath in boiling water for 1 h, centrifuge for 30 min, take the supernatant and measure the absorbance at 532 nm .

1.2.5 Detection of protective effect on protein oxidative degradation

Take 200 mL of BSA (5 mg·L ^-1 ) and add 400 μLAAPH (50 mmol·L ^-1 ) to form the damage model reaction system. The control and test groups added 5, 50, 500 μmol·L ^-1 lycopene particles and 50 μmol·L ^-1 quercetin were incubated at 37°C for 24 h, and 200 μL 4% BHT was added to stop the reaction. SDS-PAGE electrophoresis, staining, destaining and photographing were routinely performed.

1.2.6 Cell Culture

Tumor cells U87 were cultured in DEME complete medium containing 10% newborn bovine serum, streptomycin (100μg·.L ^-1 ) and penicillin (100U·mL ^-1 ), and were cultured in a 5% CO ₂ saturated humidity incubator (37°C), cells in logarithmic growth phase were used for experiments.

1.2.7 MTT colorimetric assay to detect the protective effect of lycopene on U87 cell injury induced by H ₂ O ₂

U87 cells in logarithmic growth phase were taken and seeded in 96-well plates at a concentration of 1×10 ⁵ ·mL ^-1 , 100 μL per well, and incubated for 24 h. In the protection group, lycopene particles of different concentrations were added to make the final concentration 0.5 , 1, 5, 25μmol·L ^-1 , after 2 hours of incubation, H ₂ O ₂ with a final concentration of 100 μmol·L ^-1 was added; the control group and the injury group were added DMEM complete medium containing 10% newborn calf serum and the final concentration 100 μmol·L ^-1 of H ₂ O ₂ , incubate for 24 h, add 20 μL MTT (5 mg · L ^-1 ) to each well, incubate at 37°C for 4 h, remove the supernatant, add 150 μL DMSO, shake for 10 min to dissolve the crystals, and use a microplate reader The absorbance value (A) of each well was measured at 490 nm wavelength, and the cell viability was expressed as a percentage of the control group. Cell growth rate (%)=(A ₁ -A ₀ )/(A - A ₀ )×100, where A ₀ is the absorbance value of the blank group; A is the absorbance value of the control group; A ₁ is the absorbance value of the experimental group.

1.2.8 Capillary electrophoresis to detect the protective effect of lycopene on DNA damage in U87 cells

U87 cells in the logarithmic growth phase were taken and seeded in 6-well plates at a concentration of 1×10 ⁵ ·mL ^-1 , 100 μL per well, and incubated for 24 hours. In the protection group, different concentrations of lycopene were added to make the

final concentration

1 , 5, 10μmol·L ^-1 , incubated for 2h, and added H ₂ O ₂ with a final concentration of 100μmol·L ^-1 ; the control group and the injury group were respectively added with DEME complete medium containing 10% bovine serum and the final concentration was 100μmol·L -1 L ^-1 of H ₂ O ₂ was incubated for 24h, DNA was extracted with Tiangen DP304-02 DNA extraction kit, DNA purity was determined, pre-loading was performed, and capillary electrophoresis was performed. Detection conditions: 25°C, 20kV, injection for 20s; detection for 10min; pH 9.0 boric acid-sodium hydroxide (10mmol·L ^-1 ) as electrode buffer. Results The detected peak area was brought into the linear regression equation y=1245x-50176, and the correlation coefficient R ² =0.966 to obtain the 8-OH-dG content (x is the 8-OH-dG standard concentration, y is the peak area).

1.2.9 Data processing

All experiments were repeated three times, and the experimental results were expressed as the mean ± standard deviation. The t-test was used to analyze the significance of differences, and P<0.05 was considered statistically significant.

2 Results and Analysis

2.1 Reducing power of lycopene particles

Quercetin is one of the common dietary antioxidants, and the activity of lycopene granules was compared with quercetin as a reference. Table 1 shows the reducing power of lycopene particles with different concentrations. It can be seen that with the increase of the concentration, the reducing power is also increasing, which is positively correlated within the test range, and the absorbance value reaches 0.0975 at 100 μmol·L ^-1 . ·L ^-1 quercetin has weaker activity than quercetin.

Table 3 Reducing power of lycopene particles

2.2 The free radical scavenging effect of lycopene particles

OH ^· and O ₂ ^{- ·} are the most representative free radicals, which can be produced in almost all aerobic organisms, while OH ^· is the most active and aggressive reactive oxygen species in organisms, and their abnormal production can damage important Such as protein, DNA and other biological macromolecules, causing damage to the body. The scavenging ability of lycopene particles to OH ^· and O ₂ ^-· was expressed as the concentration when the scavenging rate reached 50% - the half scavenging concentration (EC ₅₀ ), and the results are shown in Table 2. The EC ₅₀ of lycopene particles for OH ^· was 0.03 mol·L ^-1 , while that of quercetin was 0.08 μmol·L ^-1 . The EC ₅₀ of lycopene granules to remove O ₂ ^-· was 72.63 μmol·L ^-1 , while that of quercetin was 183.52 μmol·L ^-1 , indicating that lycopene granules had strong activity.

Table 4 The ability of lycopene particles to scavenge free radicals

2.3 Inhibitory effect of lycopene particles on lipid peroxidation

Table 5 shows that lycopene granules have inhibitory effect on lipid peroxidation in rat liver microsomes, the half-inhibitory concentration (IC ₅₀ ) is 22.53μmol·L ^-1 , and its inhibitory effect is stronger than that of quercetin (IC ₅₀ is 29.14μmol·L -1 ) L ^-1 ) was strong and showed good antioxidant activity.

Table 5 Inhibitory effect of lycopene particles on lipid peroxidation

2.4 Example 1 Protective effect of lycopene particles on protein oxidative damage

Figure 1 shows the results of the protective effect of lycopene granules and quercetin on AAPH-induced BSA oxidative damage. Compared with the control, after the action of BSA and AAPH at 37°C, the bands of SDS-PAGE gel electrophoresis were obviously lighter, indicating that the bovine serum albumin was oxidatively degraded. After adding different concentrations of lycopene particles to the system, the degradation of BSA was inhibited as the concentration increased. According to the depth of the bands, it can be seen that 5μmol·L ^-1 lycopene particles have a certain protective effect, and 50μmol·L ^-1 has a stronger protective effect; while the results of 500μmol·L ^-1 and 50μmol·L ^-1 groups Similar to the control group, all have strong protective effect and the band is similar to the control group. At the same time, at the same concentration, 50μmol·L ^-1 quercetin treatment group had a weaker inhibitory effect on protein degradation, indicating that lycopene granules in this system had a stronger protective effect than quercetin.

2.5 The protective effect of lycopene granules on U87 cell injury induced by H ₂ O ₂

H ₂ O ₂ is an oxidative metabolite of organisms and is also a reactive oxygen species. U87 cells were injured with 100 μmol·L ^-1 H ₂ O ₂ or pretreated with different concentrations of lycopene, and the protective effect of lycopene was evaluated by observing cell viability.

It can be seen from Figure 2 that when quercetin and 0.5, 1, 5 μmol·L ^-1 lycopene particles acted on U87 cells alone, they did not have much effect on the cell growth rate, and there was no significant difference compared with the control. However, 25μmol·L ^-1 lycopene particles had a certain growth inhibitory effect on U87 cells, and the growth rate was 76.60%. 100 μmol·L ^-1 H ₂ O ₂ can lead to a sharp decrease in cell growth rate to 66.28%. After pretreatment with the sample to be tested, 0.5μmol·L ^-1 lycopene particles had no protective effect, while quercetin, 1μmol·L ^-1 and 5μmol·L ^-1 lycopene particles increased cell activity, among which The protective effect of ¹ μmol·L ^-1 lycopene granules was very weak, and there was no significant difference compared with the H ₂ O ₂ injury group. Compared with the same concentration of quercetin (5μmol·L ^-1 ), the protective ability of lycopene particles was stronger, and the The difference between the injured group was very significant. The cell activity of the high-dose lycopene granule protection group (54.79%) was even lower than that of the H ₂ O ₂ injury group, which not only had no protective effect, but also showed a certain pro-oxidative effect.

2.6 Protective effect of lycopene granules on cellular DNA oxidative damage

When the free radicals in the body are excessive, they will attack the DNA and eventually cause oxidative damage. Among the various DNA oxidative damage products that have been found, guanine is the most vulnerable to oxidative damage. This is because it has a higher energy level molecular orbital. It is easy to form modified nucleosides and chemically stable 8-OH-dG. A large number of studies have shown that 8-OH-dG can be used as a more sensitive and stable biomarker to reflect the oxidative damage of DNA by endogenous and exogenous factors. Figure 3 shows the effect of lycopene particles on H ₂ O ₂ -induced DNA damage in U87 cells detected by CE. The peak area is proportional to the content of 8-OH-dG, that is, the higher the content, the more serious the DNA oxidative damage. Compared with the control group, after adding H ₂ O ₂ to damage the cells, the peak area increased and the 8-OH-dG content increased. 1 μmol·L ^-1 lycopene particles had no effect on this injury, while the peak area decreased when the concentration was 5 μmol·L ^-1 , and the concentration of 8-OH-dG decreased, showing a significant protective effect; in contrast, 10 μmol·L -1 L ^-1 also had a certain protective effect, but it was not as strong as the 5μmol·L ^-1 group. Likewise, the anti-DNA damage activity of quercetin was not as strong as that of lycopene granules.

Claims

A method for producing lycopene-containing granules with high-efficiency antioxidant effect, characterized in that the method comprises the following steps: Ganoderma lucidum spore powder with a wall-breaking rate of 90-92%, extracting Ganoderma lucidum spore oil through a CO2 supercritical extraction process The defatted Ganoderma lucidum spore powder is obtained, and the defatted Ganoderma lucidum spore powder and the lycopene oleoresin are mixed and sprayed for granulation in a weight ratio of 1-2:1-2.
The method for producing lycopene-containing particles with high-efficiency antioxidant effect according to claim 1, wherein the weight ratio of defatted ganoderma lucidum spore powder and lycopene oleoresin is 1:1.
The production method of lycopene-containing granules with high-efficiency antioxidant effect according to claim 1, characterized in that the spray granulation is: after mixing lycopene oleoresin with a little absolute ethanol, lycopene oleoresin alcoholate is obtained, Using a fluidized bed granulator, the lycopene oleoresin alcoholate is atomized from a nozzle by compressed air and sprayed onto the broken-wall Ganoderma lucidum spore powder in a fluidized state on the fluidized bed, so that it is wetted and fully mixed , flow granulation.
The production method of lycopene-containing granules with high-efficiency antioxidant effect according to claim 1, characterized in that the conditions used in spray granulation are: nozzle atomization speed of 40-100 mL/min, atomization particle size of 10-12 μm , the fluidized bed sample loading is 8-10kg.
The production method of lycopene-containing granules with high-efficiency antioxidant effect according to claim 4, characterized in that the conditions used in spray granulation are: nozzle atomization speed 100 mL/min, atomization particle size 10 μm, fluidized bed A loading sample is 8kg.
The production method of lycopene-containing granules with high-efficiency antioxidant effect according to claim 1, characterized in that the Ganoderma lucidum spore powder is the Ganoderma lucidum spore powder erupted from Ganoderma lucidum.
The method for producing lycopene-containing particles with high-efficiency antioxidant effect according to claim 1, characterized in that the CO 2 supercritical extraction process is: the extraction temperature is 35-36°C, the extraction pressure is 25-28Mpa, and the extraction time is 35-36°C. For 6 to 7 hours, the flow rate of CO 2 is 400 to 600 L/h.
The production method of lycopene-containing particles with high-efficiency antioxidant effect according to claim 7, characterized in that the CO 2 supercritical extraction process is: the extraction temperature is 35°C, the extraction pressure is 25Mpa, and the extraction time is 7 hours, The CO 2 flow was 500 L/h.