Grain amaranth extract based on solid state fermentation of rhizopus oligosporus as well as fermentation method and application of grain amaranth extract

Abstract

The invention discloses an amaranthus hypochondriacus extract based on solid state fermentation of rhizopus oligosporus as well as a fermentation method and application of the amaranthus hypochondriacus extract. According to the invention, rhizopus oligosporus is utilized to carry out solid state fermentation on grain amaranth, and then ethanol is utilized to carry out ultrasonic-assisted extraction, so that the obtained grain amaranth extract has the capability of improving insulin resistance. Experiments prove that the amaranthus hypochondriacus extract can improve insulin resistance of HepG2 and 3T3-L1 cells through IRS-1 / PI3K / Akt / GLUT4 and PPAR gamma / C-EBP alpha / GLUT4 pathways, and compared with an unfermented sample, an oil phase sample obtained after solid state fermentation of rhizopus oligosporus for 3 days has a better effect. The invention provides an important theoretical basis for the amaranthus hypochondriacus in preparation of drugs for regulating insulin resistance.

Description

Technical Field

[0001] The invention belongs to the field of solid-state fermentation, and in particular relates to a grain amaranth extract based on solid-state fermentation of Rhizopus oligosporus, and a fermentation method and application thereof. Background Art

[0002] Insulin resistance (IR) refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons, and the body compensatory secretion of excessive insulin to maintain blood sugar stability. Long-term insulin resistance can easily lead to metabolic syndrome and type 2 diabetes. Currently, the drugs on the market that improve insulin resistance have some side effects. Therefore, exploring natural phytochemicals in food has become a new strategy to improve insulin resistance.

[0003] Amaranth, also known as Yan Lai Hong, Lao Lai Shao, and Tricolor Amaranth, is an annual herbaceous plant belonging to the Amaranthaceae family and the genus Amaranth. It has extremely high nutritional value and can grow under different soil and agricultural climate conditions. It also has the ability to resist heat and drought. In addition to adapting to various climatic conditions, Amaranth also has important nutritional and medicinal properties. Amaranth has significant ameliorative effects on many diseases due to its significant biological and antioxidant activities. Among them, diabetes is considered a metabolic syndrome, and in patients with T2DM, insulin resistance is the core pathophysiological mechanism of T2DM progression. The glycemic index (GI) is a classification of carbohydrates that ranges from 0 to 100, depending on their effect on blood glucose levels within 2 hours after ingestion. Foods that increase insulin levels and blood glucose very slowly generally have low GI (GI < 55). Foods with low GI (e.g., Amaranth, quinoa) have been found to increase glucose intake levels and control weight by controlling appetite. However, most studies on the relationship between grain amaranth and insulin resistance focus on direct use of grain amaranth or extraction with methanol to obtain higher phenolic substances. However, direct use of grain amaranth may lead to anti-nutritional substances such as phytic acid, which may affect its therapeutic effect to a certain extent; at the same time, methanol is prohibited from use in the food industry and has low bioavailability. Existing fermentation technologies are mostly concentrated on grains or beans, and there are no reports on the solid-state fermentation process of grain amaranth and its regulatory effect on insulin resistance. Summary of the invention

[0004] The purpose of the present invention is to provide a grain amaranth extract based on solid-state fermentation of Rhizopus oligosporus and a fermentation method and application thereof. The present invention uses Rhizopus oligosporus to solid-state ferment grain amaranth, and the grain amaranth extract can improve the insulin resistance of HepG2 and 3T3-L1 cells through IRS-1 / PI3K / Akt / GLUT4 and PPARγ / C-EBPα / GLUT4 pathways.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for solid-state fermentation of grain amaranth by using oligosporus Rhizopus, which comprises the following steps:

[0007] (1) preparing Rhizopus oligosporus spores;

[0008] (2) After the amaranth is soaked and sterilized, spores of Rhizopus oligosporus are inoculated for fermentation, and the fermented amaranth is freeze-dried;

[0009] (3) Grinding the freeze-dried amaranth grain obtained in step (2), adding ethanol for ultrasonic-assisted extraction, and obtaining an amaranth grain extract.

[0010] Furthermore, the inoculation amount of oligosporus spores in step (2) is 1×10 4 Spores / g Amaranth grain ~1×10 5 Spores / g of Amaranth grain.

[0011] Furthermore, in step (2), the fermentation temperature is 37°C to 40°C, and the fermentation time is 3 days to 15 days.

[0012] Furthermore, in the step (3), the mass volume ratio of freeze-dried amaranth grains to ethanol is 1:20-25; and the ethanol used has a concentration of 75%.

[0013] In the mass-to-volume ratio described in the present invention, the unit of mass is g, and the unit of volume is mL.

[0014] Furthermore, in step (3), the protection temperature of the ultrasonic-assisted extraction is 40° C., the extraction power is 400 W, the extraction time is 10 minutes to 15 minutes, and the number of extractions is 2 to 3 times.

[0015] Furthermore, in the step (2), the amaranth grain is soaked in distilled water, the mass volume ratio of the amaranth grain to the distilled water is 1:1-2, and the soaking time is 12h-15h.

[0016] The present invention also provides a grain amaranth extract prepared by the method, wherein the total polyphenol content of the grain amaranth extract is not less than 160 mgGAE / 100 g, and the tannin content of the grain amaranth extract is not less than 70 mg / 100 g.

[0017] The present invention also provides application of the amaranth seed extract in preparing medicine for improving insulin resistance.

[0018] Furthermore, the effective dosage of the Amaranthus amaranthus extract is 25 μg / mL to 400 μg / mL.

[0019] The present invention also provides a drug for treating or improving diabetes and / or obesity, wherein the active ingredient comprises a grain amaranth extract with a concentration of 25 μg / mL to 400 μg / mL.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. The present invention utilizes oligosporus Rhizopus to solid-state ferment grain amaranth, samples are taken on the 0th, 1st, 3rd, 5th, 7th, 10th and 15th days of fermentation, crude extraction is performed with 75% ethanol, and the crude extract is freeze-dried. The physicochemical indexes, antioxidant activity and inhibitory effects on α-glucosidase and α-amylase of the crude extracts at different fermentation times are detected, and it is finally determined that the oil phase sample fermented for 3 days has the best effect.

[0022] 2. The present invention selected HepG2 cells as representative cells of the sugar metabolism pathway and used 18 mM glucosamine to establish an insulin resistance model. After adding the grain amaranth extract, the glucose absorption and intracellular glycogen content of IR-HepG2 cells increased (p < 0.05), proving that the grain amaranth extract has an improving effect on the insulin resistance of HepG2 cells.

[0023] 3. The present invention selected 3T3-L1 cells as representative cells of the lipid metabolism pathway, induced preadipocytes into adipocytes, and established an insulin resistance model using 1 μM dexamethasone. After adding the Amaranthus amaranthus extract, it promoted the absorption of glucose by IR-3T3-L1 cells (p < 0.05), proving that the Amaranthus amaranthus extract also improved the insulin resistance of these cells. However, at the same time, the intracellular lipid content and triglycerides in the sample group also increased (both lower than normal cell levels) (p < 0.05).

[0024] 4. The present invention has experimentally confirmed that grain amaranth can improve cellular insulin resistance by regulating the IRS-1 / PI3K / Akt / GLUT4 and PPARγ / C-EBPα / GLUT4 pathways. Moreover, compared with unfermented grain amaranth, the oil phase sample after 3 days of solid-state fermentation with oligosporus Rhizopus has better effect. These conclusions provide an important theoretical basis for the preparation of drugs for regulating insulin resistance using grain amaranth. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The extraction rate of Amaranth seeds at different fermentation days.

[0026] Figure 2The effects of Rhizopus oligosporus fermentation on the physical and chemical indicators of Amaranthus grains; a: the effect of Rhizopus oligosporus fermentation on the total phenol content of Amaranthus grains; b: the effect of Rhizopus oligosporus fermentation on the total flavonoids content of Amaranthus grains; c: the effect of Rhizopus oligosporus fermentation on the tannin content of Amaranthus grains; d: the effect of Rhizopus oligosporus fermentation on the phytic acid content of Amaranthus grains.

[0027] Figure 3 The effect of Rhizopus oligosporus fermentation on the antioxidant activity of Amaranthus grains.

[0028] Figure 4 The effects of Amaranth fermented by Rhizopus oligosporus on the inhibition of α-glucosidase and α-amylase; a: the effect of Amaranth fermented by Rhizopus oligosporus on the inhibition of α-glucosidase; b: the effect of Amaranth fermented by Rhizopus oligosporus on the inhibition of α-amylase.

[0029] Figure 5 The effect of Amaranth fermented with Rhizopus oligosporus on the viability of HepG2 cells.

[0030] Figure 6 To establish the insulin resistance model of HepG2 cells.

[0031] Figure 7 To investigate the effect of fermented Amaranthus amaranthus on insulin resistance in HepG2 cells.

[0032] Figure 8 To study the effect of fermented Amaranthus grain on the glycogen content in HepG2 cells insulin resistance model.

[0033] Fig. 9 To study the effect of fermented Amaranthus grain on the activities of hexokinase and pyruvate kinase in HepG2 cells insulin resistance model.

[0034] Fig.10 mRNA determination of insulin resistance-related genes in HepG2 cells; a: IRS-1 mRNA expression level in HepG2 insulin-resistant cells; b: PI3K mRNA expression level in HepG2 insulin-resistant cells; c: Akt mRNA expression level in HepG2 insulin-resistant cells; d: GLUT4 mRNA expression level in HepG2 insulin-resistant cells.

[0035] Fig.11 Induced differentiation of 3T3-L1 preadipocytes.

[0036] Fig.12 The effect of 3T3-L1 oligosporus fermentation of grain amaranth on 3T3-L1 cell viability.

[0037] Fig.13 To establish the insulin resistance model of 3T3-L1 cells.

[0038] Fig.14 To investigate the effect of fermented Amaranthus amaranthus on glucose absorption in 3T3-L1 cells in an insulin resistance model.

[0039] Fig.15 To investigate the effects of fermented Amaranthus amaranthus on lipids in a 3T3-L1 cell insulin resistance model.

[0040] Fig.16 To investigate the effect of fermented Amaranthus grain on triglyceride content in 3T3-L1 cells.

[0041] Fig.17 Effects of fermented amaranth on fatty acid synthase and acetyl-CoA carboxylase in 3T3-L1 cell insulin resistance model; a: Effects of fermented amaranth on fatty acid synthase in 3T3-L1 cell insulin resistance model; b: Effects of fermented amaranth on acetyl-CoA carboxylase in 3T3-L1 cell insulin resistance model.

[0042] Fig.18 The mRNA of insulin resistance-related genes in T3-L1 cells is determined; a: PPARγ mRNA expression level in 3T3-L1 insulin-resistant cells; b: C-EBPα mRNA expression level in 3T3-L1 insulin-resistant cells; c: GLUT4 mRNA expression level in 3T3-L1 insulin-resistant cells. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0044] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0045] Example 1: Effects of oligosporus fermentation on the antioxidant capacity and physicochemical indexes of Amaranth

[0046] 1. Test methods

[0047] 1. Preparation of fermented amaranth seeds

[0048] In the clean bench, take a 2 cm × 2 cm piece of PDA solid culture medium containing oligosporus Rhizopus into a new PDA culture medium, seal it, and culture it upside down in a closed incubator at 37°C for 7 days to prepare spores.

[0049] Take the plate containing spores, add 5 mL of sterile distilled water, and gently scrape the surface of the solid culture medium with the tip of a pipette, repeat twice. Transfer the mixture of fungi and sterile distilled water into a 50 mL sterile centrifuge tube, and filter it with a 50 mL syringe containing sterile cotton wool to remove the fungal hyphae and obtain a solution containing only fungal spores. Dilute the solution appropriately and count using an electron microscope and a hemocytometer. Dilute the solution to 1×10 according to the counting results. 4 Spores / mL.

[0050] Accurately weigh 20 g of amaranth seeds and mix with 20 mL of distilled water, soak at room temperature for 12 h, and sterilize at 121°C for 20 min. 4 The mixture was inoculated with 100 spores / g of amaranth grain, stirred evenly, sealed, and placed in an incubator at 37°C for fermentation. Samples were taken on the 0th, 1st, 3rd, 5th, 7th, 10th, and 15th days of fermentation, and the fermentation samples were freeze-dried.

[0051] 2. Preparation of fermented amaranth extract

[0052] The freeze-dried amaranth seeds were ground and the powder was weighed. After adding 75% alcohol at a ratio of 1:20 (g:mL), a Φ15 amplitude rod was selected, and the sample was extracted using an ultrasonic cell disruptor under the conditions of a protection temperature of 40°C, a power of 400 W, 3 s of ultrasound, 4 s of intermittent time, and 10 min of ultrasound. Repeat twice. The two extracts were mixed together and centrifuged at 25°C and 7000 r / min for 10 min. The supernatant was taken and filtered with a filter paper with a diameter of 1.1 mm and a suction filter to remove impurities. The filtered solution was evaporated on a rotary evaporator at 60°C and 60 rpm to remove alcohol. It was left to stand at 4°C overnight, and the oil phase and the water phase were separated from the third day of fermentation (the oil phase was too little on the 0th day and could not be separated), and the remaining sample was freeze-dried. The extraction rate calculation formula is as follows:

[0053] Formula (1)

[0054] 3. Determination of physical and chemical indicators

[0055] 3.1 Determination of total phenolic content

[0056] (1) The determination of total phenol content was carried out according to the method of Singleton et al., with appropriate modifications. Weigh 10 mg of gallic acid standard and add 10 mL of distilled water to obtain a 1 mg / mL gallic acid standard solution. Dilute the 1 mg / mL gallic acid standard solution with distilled water to 10 μg / mL, 20 μg / mL, 25 μg / mL, 40 μg / mL and 50 μg / mL. Take 120 μL of gallic acid standard solution of different concentrations in a 96-well plate, add 15 μL of Folin phenol reagent respectively, react for 3 min at 30 rpm in the dark, then add 15 μL of 10% (w / v) sodium carbonate solution and react for 30 min in the dark. Replace the gallic acid standard with 120 μL of distilled water in the blank tube. After the reaction is completed, measure the absorbance at 760 nm and draw a standard curve.

[0057] (2) Weigh 10 mg of the extracted freeze-dried sample, add 1 mL of DMSO to dissolve, and dilute 10 times with distilled water to obtain a 1 mg / mL sample solution. Also add 15 μL of Folin phenol reagent, react at 30 rpm in the dark for 3 min, then add 15 μL of 10% (w / v) sodium carbonate solution, react for 30 min in the dark, and measure the absorbance at 760 nm. Repeat the experiment three times, and the total phenol content in the sample can be calculated using the standard curve. The result is expressed as mg GAE / 100 g DW.

[0058] 3.2 Determination of total flavonoids content

[0059] (1) The determination of total flavonoid content was carried out according to the method of Santas et al., with appropriate modifications. A 1 mg / mL quercetin standard solution was prepared with methanol. The 1 mg / mL standard solution was diluted with methanol to 2.5 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, and 12.5 μg / mL quercetin solutions. First, 112 μL of distilled water was placed in a 96-well plate, followed by 60 μL of DMSO solution and 20 μL of quercetin standard solutions of different concentrations, and finally 4 μL of 1M CH 3 CO 2 K and 4 μL 10% (w / v) aluminum chloride solution. In the dark, the reaction was carried out at 300 rpm for 10 min, and then the reaction was allowed to stand for 30 min. Finally, the absorbance was measured at 415 nm and a standard curve was drawn.

[0060] 2. Dissolve the sample in DMSO and dilute it with methanol to obtain a 1 mg / mL sample solution. Perform the determination in the same way as the standard curve. Repeat the experiment three times and calculate the total flavonoid content in the sample by substituting the absorbance value into the standard curve. The result is expressed as mg QE / 100 g DW.

[0061] 3.3 Determination of tannin and phytic acid content

[0062] The contents of tannin and phytic acid in the samples were determined using detection kits.

[0063] 4. Determination of antioxidant activity

[0064] 4.1 Determination of iron ion reducing ability

[0065] (1) The antioxidant capacity of iron ions was determined by referring to the method of Benzie et al., with appropriate modifications. Weigh 408.2 mg of sodium acetate powder, add 10 mL of distilled water to obtain a 0.3 M sodium acetate solution, and then adjust the solution pH to 3.6 with glacial acetic acid; weigh 6.2 mg of TPTE, add 81 μL of hydrochloric acid and 3919 μL of distilled water to obtain a 0.01 M TPTE solution; weigh 6.5 mg of ferric chloride, add 2 mL of distilled water to obtain a 0.02 M ferric chloride solution. The above three solutions were mixed in a ratio of 10:1:1 to obtain a FRAP solution. Prepare 10 mM FeSO with distilled water. 4 7H 2 O standard solution. Dilute the standard solution with distilled water to 0.1 mM, 0.4 mM, 0.5 mM, 0.8 mM, 1 mM, 1.6 mM, 2 mM, and 2.5 mM FeSO 4 7H 2 O solution. The standard solution, distilled water and FRAP solution were added in a ratio of 1:3:30, respectively, and the absorbance was measured at 593 nm after reacting at room temperature for 30 min in the dark to draw a standard curve.

[0066] (2) After the sample is dissolved in DMSO, it is diluted with distilled water to obtain a 1 mg / mL sample solution. Take 6.25 μL of the sample solution, add 18.75 μL of distilled water and 187.5 μL of FRAP solution, react at room temperature for 30 minutes under dark conditions, and measure the absorbance at 593 nm. Repeat the experiment three times, and the results are expressed in mM Ferrous equivalents.

[0067] 4.2 Determination of oxygen free radical scavenging ability

[0068] (1) Oxygen free radical scavenging ability was determined by referring to the method of Cao et al., with appropriate modifications. Weigh 2.008 g of disodium hydrogen phosphate and 0.718 g of sodium dihydrogen phosphate in a 500 mL conical flask, add 250 mL of distilled water, and adjust the pH to 7.4 to obtain the required phosphate buffer. Then weigh 400 mg of sodium hydroxide and add 10 mL of distilled water to obtain a 1 M sodium hydroxide solution. Weigh 3.3232 mg of sodium fluorescein powder and add it to the prepared 1 M sodium hydroxide solution to obtain a 1 mM sodium fluorescein solution, which is then diluted to 25 nM with phosphate buffer for later use. Accurately weigh 33.89 mg of AAPH reagent and add 5 mL of phosphate buffer to obtain a 25 mM AAPH solution. Weigh 2.5 mg of Trolox and add 10 mL of phosphate buffer to obtain a 1 mM Trolox standard solution. The standard solution was diluted with phosphate buffer to 20 μM, 40 μM, 50 μM, 80 μM, 100 μM, 125 μM, 160 μM, 200 μM, 250 μM, 400 μM, and 500 μM Trolox solutions. 10 μL Trolox solution was taken into a black 96-well plate, and 90 μL fluorescein sodium solution and 100 μL AAPH solution were added respectively. The plate was placed in a preheated microplate reader, and the fluorescence intensity was continuously measured at an emission wavelength of 538 nm and an excitation wavelength of 485 nm. The fluorescence signal was collected every 3 min, and mixed for 15 s before collection. The total measurement was 2 h.

[0069] (2) After the sample is dissolved in DMSO, it is diluted with phosphate buffer to obtain a 1 mg / mL sample solution. Other solutions are added in the same way as the standard curve and the measurement is performed under the same conditions. The experiment is repeated three times and the results are expressed as μM TE / 100 g DW.

[0070] 5. α-Glucosidase inhibition test

[0071] The α-glucosidase inhibitory activity was evaluated following the method outlined by Kim et al. In this assay, the substrate used was 0.5 mM ρ-nitrobenzene-α-d-glucosidase (ρNPG). α-Glucosidase was dissolved in 50 mM potassium phosphate buffer, pH 6.8, at a final concentration of 0.1 U / mL. 50 mg of Amaranthus was used as the sample. During the assay, 20 µL of sample and 40 µL of 500 mM potassium phosphate buffer (pH 6.8) were taken for each sample group and blank group, 20 µL of DMSO (50%) and 40 µL of potassium phosphate buffer were added to the control group, and then 40 µL of α-glucosidase was added to the sample group and control group, and 40 µL of 50 mM potassium phosphate buffer was added to the blank group, and incubated at 37°C for 5 min. Next, 80 µL of ρNPG was added to each centrifuge tube and incubated at 37°C for another 8 min. At the end of the incubation, 250 mM Na2CO3 (400 µL) of cold solution was added to each tube, and the absorbance readings at a wavelength of 405 nm were recorded using the SpectraMax M3. Finally, the percentage of α-glucosidase inhibition activity was calculated using the following formula:

[0072] Formula (2)

[0073] Among them A 0 is the blank absorbance value, A 1 is the absorbance of the sample, A 2 The absorbance value is the control value.

[0074] 6. α-Amylase inhibition test

[0075] The evaluation of α-amylase inhibitory activity followed the method described by Zhong et al. First, starch was dissolved in boiling water to prepare 0.1% soluble starch substrate. Next, α-amylase was dissolved in 50 mM potassium phosphate buffer at pH 6.8. The concentration of grain amaranth extract was 150 mg / mL. In the experiment, 25 µL of sample and 1 mL of 0.1% soluble starch were taken in the sample group and blank group, and 25 µL of potassium phosphate buffer (pH 6.8) and 1 mL of 0.1% soluble starch were added to the control group. Then, 50 µL of α-amylase was added to the sample group and control group, respectively, while 50 µL of concentrated potassium phosphate buffer was added to the blank group. Subsequently, it was incubated at 37°C for 10 min. After that, 1 mL of DNS colorimetric reagent was added to each test tube, and then incubated in a boiling water bath for 5 min. After the water bath incubation, the mixture was cooled on ice for 5 min and the absorbance reading was recorded at 540 nm using a SpectraMax M3 spectrophotometer. The percentage of α-amylase inhibition activity was determined by the following equation:

[0076] Formula (3)

[0077] Among them A 0 is the blank absorbance value, A 1 is the absorbance of the sample, A 2 The absorbance value is the control value.

[0078] 7. Data analysis and processing

[0079] The experimental results were statistically analyzed using IBM SPSS Statistics 22 (IBM, USA). One-way analysis of variance and Duncan test were used for comparison. P < 0.05 was considered statistically significant. The results are expressed as mean ± standard deviation.

[0080] 2. Experimental Results

[0081] 1. Effect of oligosporus fermentation on the extraction rate of Amaranth

[0082] The extraction rate of Amaranth fermented with oligosporus increased with the extension of fermentation time. Among them, the extraction rate of Amaranth without oligosporus fermentation was 2.83%. In the fermentation intervals of 3 d and 15 d, the water phase yield increased significantly, from 2.85% to 23.63%. At the same time, the oil phase yield increased from 1.89% (3 d) to 24.35% (15 d). Figure 1 .

[0083] 2. Effects of oligosporus fermentation on the physicochemical parameters of Amaranth

[0084] 2.1 Effect of Rhizopus oligosporus fermentation on the total phenolic content of Amaranthus seeds

[0085] The total phenolic content (TPC) of grain amaranth showed a significant trend of change during the fermentation process, which was characterized by an increase first and then a decrease. In general, in the unfermented grain amaranth, the TPC content was 151.16 mg GAE / 100 g DW. Subsequently, in the oil phase, TPC reached a peak of 365.97 mg GAE / 100 g DW at 3 d during the fermentation process and continuously decreased to 35.46 mg GAE / 100 g DW at 15 d. A similar trend of change was observed in the aqueous phase, where TPC initially increased to 167.40 mg GAE / 100 gDW at 3 d and then continuously decreased to 20.65 mg GAE / 100 g DW at 15 d. Compared with the unfermented sample, the TPC of the 3 d oil phase was 1.42 times higher (p < 0.05), such as Figure 2As shown in a. Da Costa Maia et al. (Da Costa Maia I, Dos SantosD'Almeida CT et al., 2020) mentioned that solid-state fermentation is generally considered to be an effective method to improve the content of phenolic substances in plants. During fermentation, under the action of enzymes, the level of phenolic substances released from covalent bonds within the plant matrix increases and the structure of the compounds changes.

[0086] 2.2 Effect of Rhizopus oligosporus fermentation on the total flavonoids content of Amaranthus amaranthus seeds

[0087] The total flavonoid content (TFC) also showed a trend of first increasing and then decreasing. In the unfermented grain amaranth, the TFC content was 101.48 mg QE / 100 g DW. In the oil phase, it reached a peak of 226.08 mg QE / 100 g DW at 3 days, and then continuously decreased to 15.90 mg QE / 100 g DW at 15 days. A similar trend was observed in the water phase, where TFC initially increased to 98.61 mg QE / 100 g DW at 3 days and then steadily decreased to 10.54 mg QE / 100 g DW at 15 days. Compared with the unfermented sample, the TFC of the 3-d oil phase increased by 1.23 times (p < 0.05), as shown in Figure 2. Figure 2 Moreover, the TPC and TFC of the oil phase are always better than those of the water phase.

[0088] 2.3 Effect of Rhizopus oligosporus fermentation on tannin content in Amaranth seeds

[0089] The results of tannin content test showed that in the unfermented grain amaranth, the tannin content was 11.04 mg / 100 g DW. Subsequently, in the oil phase on day 3, it reached a peak of 75.26 mg / 100 g DW, and then continuously decreased to 8.04 mg / 100 g DW on day 10. A similar trend was observed in the water phase, where the tannin content initially increased to 11.09 mg / 100 g DW on day 3, and then steadily decreased to 1.32 mg / 100 g DW on day 10. Compared with the unfermented sample, the tannin content of the 3-d oil phase increased by 5.81 times (p < 0.05), as shown in Table 1. Figure 2 c. Moreover, the tannin content of the oil phase is always higher than that of the water phase.

[0090] This is because when the fermentation process begins, the enzymes produced by Rhizopus oligosporus begin to break down the complex compounds present in the grain amaranth. When the enzymes break down the complex components, it will lead to the release of bound tannins, which will cause the initial increase in the level of free tannins. As the fermentation progresses, the enzyme activity in Rhizopus oligosporus reaches its peak, which promotes the release of tannins to the greatest extent, resulting in the highest concentration of free tannins in the fermented product. After reaching the peak, the enzyme activity of Rhizopus oligosporus continues to act on the substrate. Over time, the enzymes will not only break down the complex compounds, but also the released tannins. In addition, some tannins may be chemically modified during the fermentation process. These processes lead to a gradual decrease in the concentration of free tannins in the fermented grain amaranth. Over time, the fermentation process matures and the various reactions reach equilibrium. This may lead to the stabilization of tannin levels.

[0091] 2.4 Effect of Rhizopus oligosporus fermentation on phytic acid content in amaranth seeds

[0092] The phytic acid content in fermented grain amaranth decreased with the extension of fermentation time. The test results showed that the phytic acid content in unfermented grain amaranth was the highest at 41.09 mg / 100 g DW. Subsequently, in the oil phase, the phytic acid content was 20.27 mg / 100 g DW at 3 days, and then continuously decreased to 2.19 mg / 100 g DW at 15 days. A similar trend was observed in the water phase, where the phytic acid content was 15.04 mg / 100 g DW at 3 days, and then steadily decreased to 1.74 mg / 100 g DW at 15 days. Compared with the unfermented sample, the phytic acid content of the oil phase at 15 days decreased by 89.60% (p < 0.05), and the phytic acid content of the water phase at 15 days decreased by 91.76% (p < 0.05). Figure 2 Experiments have shown that, during the fermentation process, endogenous phytase can lead to the degradation of phytic acid, resulting in a decrease in phytic acid content.

[0093] 3. Effect of Rhizopus oligosporus fermentation on the antioxidant activity of Amaranthus amaranth

[0094] The ferric reducing antioxidant capacity (FRAP) experiment highlighted that the oil phase showed the highest antioxidant activity after 3 days of fermentation. In the FRAP test, the antioxidant capacity of unfermented amaranth was 16.28 M Fe 2+ / 100 g DW. With the extension of fermentation time, the antioxidant activity of the water phase and oil phase of grain amaranth showed a significant downward trend. At 3 d and 15 d, the oxidative capacity of the water phase was 19.12 and 1.80 M Fe 2+ / 100 g DW, while the antioxidant capacity of the oil phase was 40.74 and 3.19 M Fe 2+ / 100 g DW. The antioxidant activity of the 3-d oil phase was 1.50 times that of the unfermented sample (p < 0.05) (e.g. Figure 3 as shown).

[0095] The results of oxygen radical absorbance capacity (ORAC) assay showed that the oil phase exhibited the highest antioxidant activity after 3 days of fermentation. In the ORAC experiment, the antioxidant capacity of unfermented grain amaranth was 159.65 and 82.34 mM TE / 100 g DW. Importantly, with the extension of fermentation time, the antioxidant activities of both the aqueous and oil phases of grain amaranth showed a significant downward trend. Among them, at 3 d and 15 d, the activity of the aqueous phase was 131.70 and 38.89 mM TE / 100 g DW, respectively, while the activity of the oil phase was 190.78 and 22.04 mM TE / 100 g DW, respectively. The antioxidant activity of the 3 d oil phase was 1.19 times that of the unfermented sample (p < 0.05) (see Figure 3 ).

[0096] 4. Effect of Rhizopus oligosporus fermentation on the inhibition of α-glucosidase

[0097] The inhibitory activity of 50 mg of Amaranthus amaranthus against α-glucosidase peaked after 3 days of oil fermentation and then decreased. In the range of 3-15 days, the inhibition rate of the water phase was 66.93%-7.21%, while the inhibition rate of the oil phase was 79.67%-7.21%. The α-glucosidase inhibitory activity of the 3-day oil phase was 1.60 times that of the unfermented sample (p < 0.05). Figure 4 a. The experimental results showed that after 3 days of fermentation, the oil phase sample had the greatest inhibitory effect on α-glucosidase.

[0098] 5. Effect of Rhizopus oligosporus fermentation on α-amylase inhibition in Amaranth

[0099] The inhibitory activity of α-amylase, evaluated with 50 mg of Amaranthus seeds, reached a peak after 3 days of oil fermentation and then decreased. In the range of 3-15 days, the inhibition rate of the water phase was 64.99%-8.85%, while the inhibition rate of the oil phase was 90.47%-11.51%. The α-amylase inhibitory activity of the 3-day oil phase was 1.33 times that of the unfermented sample (p < 0.05). Figure 4 The experimental results show that after 3 days of fermentation, the oil phase sample has the greatest inhibitory effect on α-amylase.

[0100] Example 2: Effect of Amaranthus oligosporus fermentation on insulin resistance in HepG2 cells

[0101] 1. Test methods

[0102] 1. HepG2 cell culture

[0103] HepG2 cells were cultured in DMEM high-glucose complete medium (10% fetal bovine serum, 1% streptomycin + penicillin) at 37°C and 5% CO 2 cultured in an incubator.

[0104] 2. CCK-8 cell activity test

[0105] The cytotoxicity of the samples was evaluated by CCK-8 assay. Initially, cells were cultured at 1 × 10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate and incubated in 5% CO 2 The cells were incubated at 37°C for 24 hours to allow them to adhere. Subsequently, the culture medium was replaced with a medium containing different concentrations (12.5-400 μg / mL) of the Amaranthus amaranthus extract prepared in Example 1 and incubated for another 24 hours under the same conditions. After this incubation period, the culture medium was replaced with a solution containing 10% CCK-8 and incubated at 37°C, 5% CO 2 Incubate for further 2 hours in . To reduce the effect of solution color, two additional groups were added: one group had cells and CCK-8 but no sample (control), and the other group had only culture medium and CCK-8 solution but no cells (blank). Cell viability was determined by measuring absorbance at 450 nm using a SpectraMax M3 microplate reader and expressed as a percentage relative to the control. Each experiment was performed three times, and cell viability was calculated according to the provided formula.

[0106] Formula (4)

[0107] 3. Establishment of HepG2 cell insulin resistance model

[0108] The HepG2 cell model of insulin resistance was established according to the method of Wang et al. with slight modifications. HepG2 cell suspension (1×10 5 cells / mL) in 24-well plates for 24 hours. After removing the supernatant and washing with PBS, the cells were starved for 12 hours. Subsequently, different concentrations of glucosamine (1 mM, 10 mM, 18 mm) were added to the culture medium and incubated for 12 or 24 hours. The optimal concentration and culture time of glucosamine to induce insulin resistance were determined by glucose consumption analysis. Untreated HepG2 cells cultured under standard conditions were used as controls.

[0109] 4. Determination of glycogen content

[0110] In order to explore the effect of fermented Amaranthus amaranthus on the insulin resistance model of HepG2 cells, the cells were divided into oil phase groups fermented for 3 days at different concentrations (25, 100, 400 μg / mL), non-fermented group (400 μg / mL), blank group (normal cells), model group (insulin resistance cells) and positive control group (metformin).

[0111] The HepG2 cell insulin resistance model was established according to the experimental protocol described in step 3. After 24 h of sample treatment, the glycogen level was quantitatively determined using a glycogen assay kit according to the instructions.

[0112] 5. Determination of hexokinase and pyruvate kinase

[0113] The HepG2 cell insulin resistance model was established according to the experimental scheme described in step 3, and the HepG2 cells were grouped according to the experimental scheme described in step 4. In order to evaluate the effect of fermented grain amaranth on hexokinase and pyruvate kinase in the HepG2 cell insulin resistance model, the cells were pre-treated according to the instructions of the kit after adding the sample for 24 h, and hexokinase and pyruvate kinase were measured.

[0114] 6. Determination of mRNA of insulin resistance-related genes

[0115] 6.1 RNA extraction

[0116] Establish a cellular insulin resistance model, group the cells, collect the cells of different groups by centrifugation, add 750 µL Buffer RLF, incubate at 15-30℃ for 5 min, and centrifuge at 14000 g for 5 min; place the genomic DNA filter column in a 2 mL collection tube, transfer the supernatant to the filter column, and centrifuge at 14000 g for 2 min; add an equal volume of 70% ethanol to the filtrate; place the adsorption column in a 2 mL collection tube, transfer the mixed solution to the adsorption column, centrifuge at 12000 g for 60 s; add 500 µL Buffer RW1F to the adsorption column, centrifuge at 12000 g for 60 s; add 500 µL Buffer RW2 to the adsorption column, centrifuge at 12000 g for 60 s, and thoroughly dry the residual rinse solution in the adsorption material; transfer the adsorption column to a new RNase-Free centrifuge tube, and add 50 µL RNase-Free ddH 2 O, let stand at room temperature for 2 min, centrifuge at 12000 g for 1 min to obtain RNA solution, and store the eluted RNA solution at -80°C.

[0117] 6.2 RNA integrity and purity determination

[0118] Integrity identification: In general, RNA can be tested for integrity using ordinary agarose gel electrophoresis. If the 28S and 18S bands are bright, clear, and sharp (with clear edges), the RNA is considered to be of good quality. Otherwise, it indicates that the RNA is degraded. The appearance of diffuse flakes or the disappearance of bands indicates that the RNA is severely degraded.

[0119] RNA purity determination: OD of total RNA without contamination 260 / OD 280 It is usually between 1.8 and 2.1. Anything above this range indicates that the sample may be contaminated with protein.

[0120] 6.3 cDNA Synthesis

[0121] Reverse transcription amplification was performed using the SynScript®Ⅲ RT SuperMix for qPCR Reverse Transcription Kit, and the following components were added:

[0122]

[0123] After mixing, incubate at 42℃ for 2 min, then at 60℃ for 5 min, cool on ice immediately, centrifuge briefly, and add the following components:

[0124]

[0125] After mixing, incubate at 25°C for 10 min, 50°C for 30 min, and 85°C for 5 min. Place the reverse transcription product on ice or refrigerate for later use.

[0126] 6.4 Real-time fluorescence quantitative PCR

[0127] Design fluorescent quantitative PCR primers for ISR-1, PI3K, Akt, and GLUT4 related to the glucose metabolism pathway, select β-actin as the internal reference gene, and use 2- ΔΔCT Methods The relative expression of each gene was calculated. After the PCR reaction was completed, the amplification curve and melting curve were confirmed and data analysis was performed. The primer sequences were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequence information is shown in Table 1.

[0128] Table 1 PCR primer sequence information

[0129]

[0130] The cDNA product obtained by reverse transcription was diluted 2 times and used as a qPCR template. It was amplified using ArtiCanCEO SYBR qPCRMix. The components of the amplification system are as follows:

[0131]

[0132] The reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s, for 40 cycles.

[0133] 7. Data analysis and processing

[0134] The experimental results were statistically analyzed using IBM SPSS Statistics 22 (IBM, USA). One-way analysis of variance and Duncan test were used for comparison. P < 0.05 was considered statistically significant. The results are expressed as mean ± standard deviation.

[0135] 2. Experimental Results

[0136] 1. Effect of oligosporus fermentation of Amaranthus amaranthus on HepG2 cell viability

[0137] In order to evaluate the effect of Amaranthus oleraceus on the viability of HepG2 cells, the CCK-8 method was used. The results showed that when Amaranthus oleraceus extract was added in the range of 12.5-400 μg / mL, the cell survival rate was greater than 80%, proving that the sample had no obvious toxic effect on the cells (see Figure 5 ). Therefore, 25, 100 and 400 μg / mL were selected as low, medium and high sample concentrations, respectively, to further study its effect of alleviating insulin resistance in vitro.

[0138] 2. Establishment of HepG2 cell insulin resistance model

[0139] To determine the optimal concentration and treatment time of glucosamine to establish the insulin resistance HepG2 cell model, the effects of different glucosamine concentrations (1, 10, 18, and 20 mM) at different time intervals (9 and 18 h) on glucose concentration were investigated. Figure 6 As shown. A significant reduction in glucose consumption is a key indicator of insulin resistance. The results showed that compared with the blank group (normal cells), except for 1 mM glucosamine treatment, the glucose consumption of HepG2 cells in the other concentrations of glucosamine treatment groups was significantly reduced (p < 0.05). It is worth noting that in the glucosamine treatment group, the cells treated with 18 mM glucosamine for 18 hours had the least glucose consumption, which was 73.14% less than that of the control group (p < 0.05). Therefore, 18 mM glucosamine was selected for 18 hours to induce insulin resistance in HepG2 cells.

[0140] 3. Effects of fermented Amaranth seeds on the insulin resistance model of HepG2 cells

[0141] After establishing the insulin resistance model, IR-HepG2 cells were treated with different concentrations of unfermented amaranth samples (400 μg / mL) and oil phase samples (25, 100 and 400 μg / mL) after three days of fermentation for 24 h, and the glucose consumption of different groups was measured to analyze the effects of the samples on insulin resistance. Among them, the glucose intake of cells in the model group was 3.99 mmol / L / d, and the glucose intake of cells was 7.97, 9.45 and 10.38 mmol / L / d when the concentration of the oil phase samples after three days of fermentation was 25, 100 and 400 μg / mL, respectively. Compared with the model group, the sample groups all promoted the glucose intake of IR-HepG2 cells (p<0.05). Among them, the sample with the addition of the fermented oil phase (400 μg / mL) for three days had the most significant improvement on insulin resistance, and the glucose absorption by cells was 2.60 and 1.05 times that of the model group and the control group (adding the unfermented sample group) (p < 0.05) (e.g. Figure 7 ). These results indicate that Amaranthus can improve the insulin resistance of HepG2 cells.

[0142] 4. Effect of fermented amaranth seeds on glycogen content in HepG2 cell insulin resistance model

[0143] The insulin resistance model was established, and the glycogen content was measured after adding samples to IR-HepG2 cells. Figure 8 As shown in the figure, compared with the normal group, the accumulation of glycogen in the model group decreased by 86.3% (p < 0.05), indicating that the synthesis of glycogen in the cells after modeling decreased significantly. After adding the sample treatment, compared with the model group, when the concentration of the oil phase sample was 25, 100 and 400 μg / mL after three days of fermentation, the glycogen content in the cells increased by 63.43%, 73.79% and 76.59%, respectively, which increased significantly (p < 0.05). At the same time, the glycogen content in the IR-HepG2 cells treated with the oil phase sample after three days of fermentation was 1.38 times that of the unfermented sample. It may be because the grain amaranth improved the absorption of glucose by the cells, and the glucose in the cells was converted into glycogen storage, which further increased the glycogen content.

[0144] 5. Effects of fermented Amaranth seeds on hexokinase and pyruvate kinase in HepG2 cell insulin resistance model

[0145] The insulin resistance model was established, and the HK and PK activities were measured after adding samples to IR-HepG2 cells. Fig. 9 and 10It can be seen that the HK and PK activities in the model group cells decreased by 51.28% and 73.39% (p < 0.05) (compared with the normal group). After adding different concentrations and components of grain amaranth to treat IR-HepG2 cells, the HK and PK activities in the cells of each treatment group were significantly increased (p < 0.05) (compared with the model group). Among them, the oil phase sample concentration of 400 μg / mL after three days of fermentation had the best effect, and the HK and PK activities in the cells increased by 0.79 and 2.13 times (p < 0.05), respectively. At the same time, the HK and PK activities in the IR-HepG2 cells treated with the oil phase sample after three days of fermentation were 1.26 and 1.00 times that of the unfermented samples (p < 0.05). According to the above experimental results, grain amaranth can affect the anaerobic glycolysis and aerobic oxidation of glucose by increasing the activity of HK and PK, thereby promoting the absorption and utilization of glucose by IR-HepG2 cells.

[0146] 6. Determination of mRNA of insulin resistance-related genes in HepG2 cells by fermented amaranth

[0147] The insulin resistance model was established, and the samples were added to IR-HepG2 cells to treat them, and then the mRNA expression of insulin resistance-related genes in HepG2 cells was measured. Fig.10 a-10d shows that the expression levels of IRS-1, PI3K, Akt and GLUT4 in the model group were significantly reduced, only 37.17%, 44.11%, 42.05% and 37.35% of the normal group (p < 0.05). After adding the sample, the expression of each gene mRNA increased to varying degrees. When the concentration of the oil phase sample added three days after fermentation was 100 and 400 μg / mL, the sample had less effect on the mRNA expression of IRS-1 and PI3K genes than Akt and GLUT4. When the concentration of the oil phase sample after three days of fermentation was 400 μg / mL, it had the greatest effect on the expression levels of IRS-1, PI3K, Akt and GLUT4 in IR-HepG2 cells, which increased by 48.91%, 47.12%, 53.93% and 51.59% (p < 0.05), respectively. At the same time, the mRNA expression levels of IRS-1, PI3K, Akt and GLUT4 in IR-HepG2 cells treated with the oil phase sample (400 μg / mL) after three days of fermentation were increased by 5.61%, 5.26%, 17.55% and 31.41% (p < 0.05) compared with the unfermented samples.

[0148] Example 3: Effect of Rhizosporus fermentation on the insulin resistance of 3T3-L1 adipocytes

[0149] 1. Experimental Methods

[0150] 1. Culture and differentiation induction of 3T3-L1 preadipocytes

[0151] Normal 3T3-L1 cells were cultured in DMEM supplemented with 10% newborn calf serum (NCS), streptomycin (100 μg / mL), and penicillin (100 U / mL) at 37°C and 5% CO. 2 Cultivated at concentration.

[0152] 3T3-L1 preadipocytes were cultured at 5 × 10 4 Cells / well were seeded in 24-well plates, and after 3 days of contact inhibition, differentiation was induced with DMEM+10% FBS+1% penicillin / streptomycin containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone (Dex) and 10 μg / mL insulin for 3 days. Subsequently, the cells were cultured in DMEM+10% FBS+1% penicillin / streptomycin medium containing 10 μg / mL insulin for 7 days, and the medium was changed every 2 days. About 90% of the cells differentiated into mature adipocytes and can be used for subsequent experiments. Oil Red O dye was prepared in a ratio of saturated Oil Red O: distilled water = 3:2 (v / v), and the differentiated cells were stained to observe the lipid droplets in the cells.

[0153] 2. CCK-8 cell activity test

[0154] The steps are the same as in Example 2.

[0155] 3. Establishment of 3T3-L1 cell insulin resistance model

[0156] The 3T3-L1 cell model of insulin resistance was established based on a previously described method with slight modifications. After induction of adipogenic differentiation according to the described protocol, the cells were incubated in serum-free medium for 24 h after removal of the supernatant and PBS washing. The medium containing Dex (1 μM) was incubated for 1-4 days, and glucose consumption in the medium was monitored daily. The optimal incubation time for induction of insulin resistance was determined by glucose consumption analysis, and untreated 3T3-L1 cells cultured under standard conditions were used as controls.

[0157] 4. Effects of fermented amaranth seeds on 3T3-L1 cell insulin resistance model

[0158] In order to explore the effect of fermented Amaranthus amaranthus on the insulin resistance model of 3T3-L1 cells, the cells were divided into oil phase groups fermented for 3 days at different concentrations (25, 100, 400 μg / mL), a non-fermented group (400 μg / mL), a blank group (normal cells), a model group (insulin resistance cells) and a positive control group (rosiglitazone).

[0159] The 3T3-L1 cell insulin resistance model was established according to the experimental protocol described. After adding the sample for 24 h, the supernatant was taken and the glucose content in the supernatant was detected. The cells were washed with PBS and fixed by incubation with 4% paraformaldehyde for 30 minutes. After removing the paraformaldehyde, the cells were washed again with PBS and stained with Oil Red O for 30 min at room temperature. After staining, Oil Red O was removed and washed with PBS twice to eliminate background impurities. Lipid cells were captured by 400x microscopy imaging. The cells were then treated with isopropanol to dissolve Oil Red O, and the light absorbance was measured at 510 nm by a microplate reader to quantify lipid accumulation.

[0160] 5. Determination of triglyceride content

[0161] The 3T3-L1 cell insulin resistance model was established according to the experimental protocol described in step 3, and the 3T3-L1 cells were grouped according to the experimental protocol described in step 4. In order to evaluate the intracellular triglyceride (TG) content, the cells were pre-treated according to the instructions of the kit 24 hours after the addition of the sample, and the TG content in the cell lysate was determined.

[0162] 6. Determination of fatty acid synthase and acetyl-CoA carboxylase

[0163] The 3T3-L1 cell insulin resistance model was established according to the experimental scheme described in step 3, and the 3T3-L1 cells were grouped according to the experimental scheme described in step 4. In order to evaluate the effect of fermented grain amaranth on fatty acid synthase and acetyl-CoA carboxylase in the 3T3-L1 cell insulin resistance model, the cells were pre-treated according to the instructions of the kit after adding the sample for 24 h, and fatty acid synthase and acetyl-CoA carboxylase were measured.

[0164] 7. Determination of mRNA of insulin resistance-related genes

[0165] 7.1 RNA extraction

[0166] The steps are the same as in Example 2.

[0167] 7.2 Determination of RNA Integrity and Purity

[0168] The steps are the same as in Example 2.

[0169] 7.3 cDNA Synthesis

[0170] The steps are the same as in Example 2.

[0171] 7.4 Real-time fluorescence quantitative PCR

[0172] Design fluorescent quantitative PCR primers for PPARγ related to lipid metabolism pathway, select β-actin as the internal reference gene, and use 2- ΔΔCTMethods The relative expression of each gene was calculated. After the PCR reaction was completed, the amplification curve and melting curve were confirmed and data analysis was performed. The primer sequences were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequence information is shown in Table 2.

[0173] Table 2 PCR primer sequence information

[0174]

[0175] The rest is the same as Example 2.

[0176] 8. Data analysis and processing

[0177] The experimental results were statistically analyzed using IBM SPSS Statistics 22 (IBM, USA). One-way analysis of variance and Duncan test were used for comparison. P < 0.05 was considered statistically significant. The results are expressed as mean ± standard deviation.

[0178] 2. Experimental Results

[0179] 1. Differentiation induction of 3T3-L1 preadipocytes

[0180] Fig.11 The changes in lipid droplet accumulation in 3T3-L1 preadipocytes after 10 days of induced differentiation and in uninduced cells are shown. The results were observed using a microscope with a magnification of 400× on the 4th, 6th and 10th days. It was observed that the 3T3-L1 preadipocytes without induced differentiation were spindle-shaped or irregular polygonal in shape; with the increase in the induction time, on the 4th day of induction, small round fat droplets began to appear in the cells, the intercellular space gradually increased, and a large number of reticular structures appeared; on the 6th day of induction, the number and size of small fat droplets in the cells increased; on the 10th day of induction, after staining with Oil Red O, it can be observed that the morphology of the intracellular lipid droplets became larger and round, and the lipid droplets were of different sizes and increased in number. After Oil Red staining, a large number of lipid droplets were stained red, the color was deep, and they showed a ring-like shape.

[0181] 2. Effect of oligosporus fermentation of Amaranth on 3T3-L1 cell viability

[0182] According to the above results, after preadipocytes were induced to differentiate into adipocytes, the CCK-8 method was used to evaluate the effect of Amaranthus oleraceus on the viability of 3T3-L1 cells. The results showed that within the range of 12.5-400 μg / mL, the cell survival rate was higher than 80%, indicating that the Amaranthus oleraceus extract within this concentration range had no obvious toxic effect on cells (see Fig.12). Therefore, 25, 100 and 400 μg / mL were selected as low, medium and high sample concentrations, respectively, to further study its in vitro effect of alleviating insulin resistance. This provides a reliable basis for subsequent studies on its regulatory effect on insulin resistance.

[0183] 3. Establishment of 3T3-L1 cell insulin resistance model

[0184] In order to determine the optimal treatment time of dexamethasone to establish the 3T3-L1 cell model of insulin resistance, the glucose uptake by cells was measured within 1-4 days after the addition of dexamethasone, as Fig.13 As shown. A significant reduction in glucose consumption is a sign of a successful model. The results showed that the glucose consumption of all cells induced by 1 µmol / L Dex was significantly reduced compared with the control group (no insulin treatment). The amount of glucose absorbed by the cells showed a trend of first decreasing and then increasing. Among these results, the cells treated with 1 µmol / L Dex for 2 days had the least glucose consumption, which was 77.50% less than the control group (p < 0.05). Therefore, the insulin resistance model of 3T3-L1 cells induced by 1 µmol / L Dex for 2 days was selected.

[0185] 4. Effects of fermented amaranth seeds on 3T3-L1 cell insulin resistance model

[0186] After establishing the insulin resistance model, IR-3T3-L1 cells were treated with different concentrations of unfermented grain amaranth samples (400 μg / mL) and oil phase samples after three days of fermentation (25, 100 and 400 μg / mL) for 24 h, and the glucose consumption and lipid content of each group were measured. The results showed that compared with the model group, the sample groups all promoted the glucose uptake of IR-3T3-L1 cells (p<0.05). Among them, when the concentration of the oil phase sample after three days of fermentation was 25, 100 and 400 μg / mL, the glucose uptake of the cells was 6.17, 9.17 and 12.58 mmol / L / d, respectively. Compared with the model group, the glucose absorption increased significantly (p<0.05). At the same time, the glucose uptake of IR-3T3-L1 cells treated with the oil phase sample (400 μg / mL) after three days of fermentation was 1.22 times that of the unfermented sample (p<0.05) (such as Fig.14 ). It is speculated that Amaranthus can increase the glucose uptake of cells and improve the insulin resistance of 3T3-L1 cells.

[0187] However, the quantitative results of isopropanol on lipids show that ( Fig.15), compared with the model group, the lipid content of the sample group increased to a certain extent. When the concentration of the oil phase sample was 400 μg / mL after three days of fermentation, the oil content increased by 0.17 times compared with the model group (p<0.05). The improvement of insulin resistance is a complex regulatory process, which is affected by various factors such as different regional environments, different age groups and different degrees of insulin resistance, and there are different hypotheses. Experiments have shown that thiazolidinediones enhance the insulin sensitivity of patients with type 2 diabetes by promoting the increase of insulin sensitivity in peripheral adipocytes, thereby causing the redistribution of lipids in blood cells from insulin-responsive organs to peripheral adipocytes. Rosiglitazone belongs to the thiazolidinediones class of drugs. Some studies have shown that the use of rosiglitazone will increase body weight, but it is not significantly higher than the baseline level. This result is consistent with the results of the present invention. Although the sample has increased the lipid content to a certain extent, it is still lower than normal cells. It is speculated that it may be due to the increase in the cell's uptake of glucose, which increases the amount of glucose converted into glycogen and lipids, thereby leading to an increase in the intracellular glycogen content and lipid content.

[0188] 5. Effect of fermented Amaranth seeds on triglyceride content in 3T3-L1 cells

[0189] After establishing the insulin resistance model, the samples were added to the IR-3T3-L1 cells and the triglyceride content was measured. Fig.16 As shown, when the concentration of the oil phase sample was 400 μg / mL after three days of fermentation, the triglyceride content in the cells was 0.22 mmol / gprot, which was 22.26% higher than that in the model group (p < 0.05). Drug intervention using rosiglitazone is one of the effective methods for treating decreased insulin sensitivity, but studies have found that the use of rosiglitazone will increase triglyceride content. The results of this experiment showed that compared with the model group, the triglyceride content in IR-3T3-L1 cells treated with samples and rosiglitazone did increase, but was lower than that in normal cells.

[0190] 6. Effects of fermented amaranth on fatty acid synthase and acetyl-CoA carboxylase in 3T3-L1 cells with insulin resistance

[0191] After establishing the insulin resistance model, the samples were added to the IR-3T3-L1 cells and the activities of ACC and FAS were measured. Fig.17 and 18It can be seen that compared with normal cells, the activities of ACC and FAS in the model group cells decreased. After adding different concentrations and components of grain amaranth to treat IR-3T3-L1 cells, the activities of FAS and ACC in the cells of each treatment group were significantly increased (compared with the model group). When the concentration of the oil phase sample was 25, 100 and 400 μg / mL after three days of fermentation, the FAS activity in the cells increased by 2.78%, 6.07% and 10.68% (p < 0.05), respectively; the ACC activity in the cells increased by 5.08%, 8.98% and 18.80%, respectively, which increased significantly (p < 0.05). At the same time, the FAS and ACC activities in IR-3T3-L1 cells treated with the oil phase sample (400 μg / mL) after three days of fermentation were 1.08 and 1.14 times that of the unfermented samples (p < 0.05). Studies have shown that the increase in cellular triglyceride content is associated with an increase in the rate of lipogenesis and a coordinated increase in the activity of many lipogenic enzymes, including FAS and ACC. Therefore, combined with the results of this experiment, the increase in the activity of ACC and FAS leads to an increase in fatty acid synthesis, which in turn leads to an increase in triglyceride content. However, it is worth noting that the FAS and ACC activities in the sample treatment group are lower than those in normal cells.

[0192] 7. Determination of mRNA of genes related to insulin resistance model in 3T3-L1 cells by fermented amaranth

[0193] After establishing the insulin resistance model, the samples were added to the IR-3T3-L1 cells to treat them, and then the mRNA expression of insulin resistance-related genes in 3T3-L1 cells was measured. Fig.18 a-18c It can be seen that the expression levels of PPARγ, C-EBPα and GLUT4 in the model group cells showed a downward trend, which was only 20.07%, 51.50% and 45.47% of the normal group. After adding the sample, the expression levels of each gene mRNA increased to varying degrees (p < 0.05). Among them, when the concentration of the oil phase sample added for three days of fermentation was 400 μg / mL, it had the greatest effect on the mRNA expression level of the gene in IR-3T3-L1 cells, which was 1.85, 1.52 and 1.52 times that of the model group respectively (p < 0.05). At the same time, the mRNA expression levels of PPARγ, C-EBPα and GLUT4 in IR-HepG2 cells treated with the oil phase sample after three days of fermentation were 1.20, 1.03 and 1.10 times that of the unfermented sample (p < 0.05).

[0194] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for solid-state fermentation of amaranth grains by using oligosporus Rhizopus, characterized in that: The following steps are involved: (1) preparing Rhizopus oligosporus spores; (2) After the amaranth is soaked and sterilized, spores of Rhizopus oligosporus are inoculated for fermentation, and the fermented amaranth is freeze-dried; (3) Grinding the freeze-dried amaranth grain obtained in step (2), adding ethanol for ultrasonic-assisted extraction, and obtaining an amaranth grain extract.

2. The method according to claim 1, characterized in that The inoculation amount of oligosporon spores in step (2) is 1×10 4 Spores / g Amaranth grain ~1×10 5 Spores / g grain of Amaranth.

3. The method according to claim 1, characterized in that In the step (2), the fermentation temperature is 37°C to 40°C, and the fermentation time is 3 days to 15 days.

4. The method according to claim 1, characterized in that In the step (3), the mass volume ratio of freeze-dried amaranth grains to ethanol is 1:20-25; the ethanol used has a concentration of 75%.

5. The method according to claim 1, characterized in that In the step (3), the protection temperature of the ultrasonic-assisted extraction is 40° C., the extraction power is 400 W, the extraction time is 10 to 15 minutes, and the number of extractions is 2 to 3 times.

6. The method according to claim 1, characterized in that In the step (2), the amaranth grain is soaked in distilled water, the mass volume ratio of the amaranth grain to the distilled water is 1:1-2, and the soaking time is 12h-15h.

7. The Amaranth extract prepared by the method according to claims 1 to 6, characterized in that Its total polyphenol content is not less than 160 mg GAE / 100 g, and its tannin content is not less than 70 mg / 100 g.

8. Use of the Amaranthus seed extract according to claim 7 in the preparation of a medicament for improving insulin resistance.

9. The use according to claim 8, characterized in that: The effective dosage of the amaranth seed extract is 25 μg / mL to 400 μg / mL.

10. A drug for treating or improving diabetes and / or obesity, characterized in that: The active ingredient of the drug comprises the Amaranthus amaranthus extract according to claim 7 at a concentration of 25 μg / mL to 400 μg / mL.

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