Application of fermented wolfberry clear juice freeze-dried powder in relieving caenorhabditis elegans metabolism-related aging induced by high glucose

Abstract

The invention discloses an application of fermented wolfberry clear juice freeze-dried powder in relieving caenorhabditis elegans metabolism-related aging induced by high glucose, and belongs to the technical field of biological medicines. According to the application, through a kluyveromyces marxianus fermentation process, the problem of sugar interference in fresh Chinese wolfberry primary pulp is solved, core anti-aging active ingredients are reserved, fermented Chinese wolfberry clear juice freeze-dried powder is mixed with a 50-100 mM high-sugar solution at the concentration of 1.53-7.65 mg / mL and then acts on N2-type nematodes at the L3-L4 stage, and the anti-aging effect is achieved. The life of high-glucose-induced metabolism-related senescent nematodes is remarkably prolonged, lipofuscin deposition is reduced, the ROS level is reduced, the SOD enzyme activity is improved, and expression of age-1 and sir-2.1 genes is down-regulated by inhibiting abnormal activation of an IIS pathway. The invention proves that the fermented lycium barbarum clear juice freeze-dried powder can slow down glucose-induced nematode metabolism-related aging to a certain extent, and a new strategy is provided for anti-aging intervention in a high-glucose environment. The invention also discloses application of the fermented wolfberry clear juice freeze-dried powder in preparation of food and daily chemical products for relieving metabolism-related aging induced by high glucose.

Description

Technical Field

[0001] The present invention belongs to the field of biology and medicine technology, and specifically relates to an application of fermented wolfberry juice freeze-dried powder in high-sugar-induced Caenorhabditis elegans metabolism-related aging, and as a food and daily chemical product for alleviating high-sugar-induced metabolism-related aging. Background Art

[0002] Caenorhabditis elegans ( Caenorhabditis elegans ) As a model organism, it has significant advantages in evaluating the study of high glucose-induced metabolism-related aging. First, the life cycle of nematodes is short (about 2-3 weeks), and the aging process is highly similar to that of mammals, which can quickly reflect the impact of a high sugar environment on aging. Secondly, the genetic background of nematodes is clear and the genome is completely sequenced, which facilitates the study of the role of specific genes in metabolic disorders and aging through gene editing technology. In addition, the high sugar-induced nematode model can simulate the metabolic disorders caused by a high sugar diet in humans, such as insulin resistance, abnormal lipid metabolism, and increased oxidative stress, providing an ideal platform for studying the molecular mechanisms of metabolism-related aging.

[0003] As a traditional Chinese medicinal material, wolfberry has multiple biological activities such as antioxidant, anti-inflammatory, and immunomodulatory. In recent years, it has received extensive attention in the field of anti-aging and metabolic regulation. However, the efficacy verification of wolfberry does not involve high-sugar stress scenarios, which may be because the high sugar content of wolfberry (reducing sugar ≥ 15%) limits its application in high-glucose-induced metabolic-related aging models. The high-sugar environment itself will aggravate the metabolic disorder state, and the high-sugar components in wolfberry may further increase the metabolic burden of the model and interfere with the experimental results. In addition, most current studies focus on a single active ingredient in wolfberry, and lack a systematic study of the overall active ingredient group of wolfberry. Therefore, the present invention develops a low-sugar fermented wolfberry juice freeze-dried powder product by fermentation, and uses a high-glucose-induced metabolic-related aging Caenorhabditis elegans model to verify its efficacy, providing more comprehensive technical support for the application of fermented wolfberry juice freeze-dried powder in alleviating high-sugar-induced nematode metabolic-related aging. Summary of the invention

[0004] The purpose of the present invention is to provide a fermented wolfberry juice freeze-dried powder for use in metabolism-related aging. To achieve the above purpose, the technical methods adopted by the present invention are as follows: The present invention provides an application of fermented wolfberry juice freeze-dried powder in alleviating high-sugar-induced metabolism-related aging of Caenorhabditis elegans.

[0005] Preferably, the Kluyveromyces marxianus fermentation process is used to eliminate the interference of sugars in fresh wolfberry puree and retain the core anti-aging active ingredients.

[0006] Preferably, the administration of a fermented wolfberry juice freeze-dried powder aqueous solution at a concentration of 1.53-7.65 mg / mL prolongs the lifespan of nematodes with high sugar-induced metabolism-related aging.

[0007] Preferably, the administration of a fermented wolfberry juice freeze-dried powder aqueous solution at a concentration of 1.53-7.65 mg / mL reduces the accumulation of lipofuscin in nematodes with high sugar-induced metabolism-related aging.

[0008] Preferably, the administration of a fermented wolfberry juice freeze-dried powder aqueous solution at a concentration of 1.53-7.65 mg / mL reduces the level of ROS in nematodes with high sugar-induced metabolism-related aging.

[0009] Preferably, the administration of a 7.65 mg / mL aqueous solution of the fermented wolfberry juice freeze-dried powder increases the SOD content in nematodes with high sugar-induced metabolism-related aging.

[0010] Preferably, the administration concentration of 7.65 mg / mL of the fermented wolfberry juice freeze-dried powder aqueous solution inhibits the abnormal activation of the IIS pathway of nematodes in the high-sugar-induced metabolism-related aging, and downregulates the lifespan-related age-1 and sir-2.1 Gene expression.

[0011] Therefore, the present invention uses the above-mentioned fermented wolfberry juice freeze-dried powder to alleviate the high sugar-induced metabolism-related aging of Caenorhabditis elegans, which has the following beneficial effects: The reducing sugar content in the fermented wolfberry juice freeze-dried powder prepared by the Kluyveromyces marxianus fermentation process was significantly reduced to ≤1.5%, while the polysaccharide retention rate was ≥97%, the zeaxanthin dipalmitate retention rate was ≥98%, and the betaine retention rate was ≥87%.

[0012] The lifespan of nematodes was measured. In the aqueous solution of fermented wolfberry juice freeze-dried powder with a dosage of 7.65 mg / mL, 3.83 mg / mL and 1.53 mg / mL, the high sugar-induced lifespan of nematodes was significantly prolonged, with the maximum lifespan extended by 28.00%, 25.00% and 14.20%, respectively, and the average lifespan extended by 24.20%, 23.10% and 16.70%, respectively.

[0013] The determination of lipofuscin deposition in nematodes showed that the fluorescence intensity of lipofuscin in nematodes induced by high sugar was significantly reduced in the aqueous solution of lycium barbarum juice freeze-dried powder at the dosage concentrations of 7.65 mg / mL, 3.83 mg / mL and 1.53 mg / mL ( p <0.01), which decreased by 54.39%, 52.88% and 52.24% respectively.

[0014] The ROS level in nematodes was tested. In the aqueous solution of fermented wolfberry juice freeze-dried powder at concentrations of 7.65 mg / mL, 3.83 mg / mL, and 1.53 mg / mL, the fluorescence intensity of ROS in nematodes induced by high sugar was significantly weakened ( p <0.01), which decreased by 57.24%, 50.34% and 38.62% respectively.

[0015] The SOD content in the nematodes was tested. In the aqueous solution of fermented wolfberry juice freeze-dried powder with a dosage concentration of 7.65 mg / mL, the SOD content in the nematodes induced by high sugar was significantly increased ( p <0.01), an increase of 71.77%.

[0016] In nematodes age-1 and sir-2.1 The relative expression levels were tested. In the aqueous solution of fermented wolfberry juice freeze-dried powder with a dosage concentration of 7.65 mg / mL, the high sugar-induced nematodes age-1 and sir-2.1 The relative expression level was significantly downregulated ( p <0.01), down by 40.13% and 57.74% respectively.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The study of the present invention reveals the application of fermented wolfberry juice freeze-dried powder to nematodes under glucose-induced metabolic-related aging conditions after eliminating the interference of the sugar content of wolfberry itself: compared with the high sugar group and the non-fermented wolfberry group, the fermented wolfberry juice freeze-dried powder prolonged the maximum lifespan and average survival rate of nematodes under high sugar-induced metabolic-related aging conditions; reduced the accumulation of lipofuscin water in the intestine of nematodes under high sugar-induced metabolic-related aging conditions; reduced the ROS level in nematodes under high sugar-induced metabolic-related aging conditions; increased the SOD enzyme activity in nematodes under high sugar-induced metabolic-related aging conditions; inhibited the abnormal activation of the IIS pathway in nematodes under high sugar-induced metabolic-related aging conditions and downregulated the lifespan-related age-1 and sir-2.1 Gene expression.

[0018] 2. The present invention has discovered new uses for the fermented wolfberry juice freeze-dried powder, laying a foundation for the future development of new food and daily chemical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The invention discloses the effects of different dosages of the freeze-dried powder of fermented wolfberry juice in aqueous solution on the life span of nematodes.

[0020] Figure 2The invention discloses the effects of different dosages of the fermented wolfberry juice freeze-dried powder aqueous solution on the accumulation of lipofuscin in nematodes.

[0021] Figure 3 The present invention shows the effects of different doses of the fermented wolfberry juice freeze-dried powder aqueous solution on the ROS level in nematodes.

[0022] Figure 4 This invention discloses the effects of different dosages of fermented wolfberry juice freeze-dried powder aqueous solution on the SOD content in nematodes.

[0023] Figure 5 The effects of different doses of the fermented wolfberry juice freeze-dried powder aqueous solution on the nematodes age-1 and sir-2.1 Effect of relative expression Specific implementation methods

[0024] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings.

[0025] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0026] Example 1 Effects of different concentrations of fermented wolfberry juice freeze-dried powder on the life span of high-sugar-induced nematodes 1 Materials and Instruments 1.1 Materials and reagents The nematode is N2 wild-type Caenorhabditis elegans.

[0027] The nematode food is uracil-deficient Escherichia coli ( Escherichia coli OP 50 ).

[0028] The reagents include fresh wolfberry puree, agar, glucose, magnesium sulfate, sodium chloride, dipotassium hydrogen phosphate, disodium hydrogen phosphate, trypsin, cholesterol, paraformaldehyde, a total superoxide dismutase (SOD) detection kit, and 2',7'-dichlorodihydrofluoresceindiacetate (DCFH-DA).

[0029] Preparation of Lycium barbarum juice freeze-dried powder: A single colony of Kluyveromyces marxianus was inoculated into YPD liquid medium for activation, and then inoculated into fresh Lycium barbarum puree at an inoculation rate of 10 11 ~1012 CFU, 30±2 ℃ shaking fermentation for 22-26 h. Remove ethanol by vacuum rotary evaporation, pasteurize, centrifuge and vacuum freeze-dry the supernatant to obtain the fermented wolfberry juice freeze-dried powder, the polysaccharide retention rate in the freeze-dried powder is ≥97%, the zeaxanthin dipalmitate retention rate is ≥98%, the betaine retention rate is ≥87%, and the reducing sugar content is ≤1.5%.

[0030] Prepare NGM medium: NGM ( C.elegans Growth Media (NGM) contains peptone, NaCl, agar, deionized water, CaCl2, MgSO4, cholesterol solution, and phosphate buffer (KPI). Mix well and dispense into culture dishes. After cooling to room temperature, apply fresh E. coli OP50 was cultured in a 37°C incubator overnight and then placed in a 4°C refrigerator for later use.

[0031] 1.2 Instruments The instruments are a biochemical incubator; a clean workbench; a high-speed refrigerated centrifuge; an Olympus microscope; an ETC811 PCR instrument; and a KZ-III-F high-speed tissue grinder.

[0032] 2 Methods 2.1 Synchronization of nematodes After the transfer plate is taken, synchronize the nematodes in the egg-laying stage, wash the plate several times with M9 buffer, collect the washing liquid and mix it with the lysis buffer in a certain proportion, vortex to lyse the worm body, centrifuge to remove the supernatant, wash the lysis buffer with M9 again, and re-inoculate the purified eggs on the plate coated with E. coli OP 50 on plates and cultured at 20±1 ℃ for 46-50 h to obtain worms in the L3-L4 stage for subsequent experiments.

[0033] 2.2 Nematode exposure and grouping The synchronized nematodes were divided into control group (CON), high sugar group (HG), non-fermented group (GQ) and high, medium and low dosing groups (HGQ, MGQ, LGQ) for treatment. The control group was treated with M9 buffer; the high sugar group was exposed to 50 mmol / L glucose solution; the non-fermented group was exposed to 50 mmol / L glucose solution and 7.65 mg / mL non-fermented fresh wolfberry juice freeze-dried powder solution; the high, medium and low dosing groups were exposed to 50 mmol / L glucose solution and low-sugar fermented wolfberry juice freeze-dried powder solutions of high, medium and low dosing concentrations. Among them, the non-fermented group and the three high, medium and low dosing concentration solutions (7.65 mg / mL, 3.83 mg / mL and 1.53 mg / mL) were prepared with M9 buffer. All groups were placed in a 20 ℃ incubator for exposure for 24 hours before subsequent experiments.

[0034] 2.3 C. elegans lifespan experiment After synchronization and exposure for 24 h, the nematodes were placed on fresh E. coli OP 50 Every day, the surviving nematodes were transferred to fresh NGM medium using a platinum pick. E. coli OP 50 The survival of nematodes was observed and recorded on NGM medium until all nematodes died. The survival rate was calculated as the percentage of live nematodes to the total number of nematodes. Each group had no less than 105 nematodes and 3 parallels were set.

[0035] 2.4 Data Processing The experimental data were statistically analyzed using SPSS statistics 27.0, and the results were expressed as mean ± standard deviation (SD). ANOVA was used for significance analysis. p <0.05 indicates significant difference. p <0.01 indicates a very significant difference. Image J was used to process the experimental photos, and Origin 2019 was used for drawing.

[0036] 3 Results Analysis Figure 1 The effects of different doses of fermented wolfberry juice freeze-dried powder aqueous solution on the life span of nematodes. Figure 1 As shown in the results, the high sugar environment in the HG group significantly shortened the lifespan of Caenorhabditis elegans, with the maximum lifespan and average lifespan reduced by 25.00% and 21.90%, respectively. Moreover, the unfermented fresh wolfberry juice in the GQ group could not prolong the lifespan of nematodes induced by high sugar. The maximum lifespan and average lifespan of the GQ group were reduced by 8.02% and 11.11%, respectively, compared with the HG group. However, after being treated with different concentrations of low-sugar fermented wolfberry juice, the lifespan of nematodes was restored to varying degrees. The maximum lifespan of the HGQ group, MGQ group, and LGQ group was extended by 28.00%, 25.00%, and 14.20%, respectively, and the average lifespan was extended by 24.20%, 23.10%, and 16.70%, respectively. In particular, the effects of the HGQ and MGQ treatment groups were statistically significant ( p <0.05).

[0037] Example 2 Effects of different concentrations of fermented wolfberry juice freeze-dried powder on high sugar-induced lipofuscin accumulation in nematodes Take the synchronized nematodes and transfer them to fresh E. coli OP 50 The cells were cultured on NGM medium for 10 days. An appropriate number of nematodes were selected and fixed with paraformaldehyde, and then transferred to a 2% agarose pad. The cells were photographed under the DAPI channel. Each group had no less than 20 nematodes and three parallels were set.

[0038] Lipofuscin is a lipid and protein complex deposited during cell aging. It often appears in aging cells and various degenerative diseases and is associated with cell dysfunction and tissue damage. Figure 2 The results show that the effects of different doses of fermented wolfberry juice freeze-dried powder aqueous solution on lipofuscin deposition in nematodes. The high sugar environment accelerates the aging of nematodes. Compared with the CON group, the lipofuscin deposition in the HG group increased significantly ( p <0.01), the fluorescence intensity of lipofuscin increased by 33.2%; the aqueous solution of fresh wolfberry juice freeze-dried powder in the GQ group increased the lipofuscin content in the nematodes induced by high sugar in the HG group, and the fluorescence intensity of lipofuscin in the GQ group increased by 3.45% compared with the HG group; compared with the HG group, the fluorescence intensity of lipofuscin in the nematodes in the HGQ group, MGQ group and LGQ group was significantly reduced ( p <0.01), which decreased by 54.39%, 52.88% and 52.24% respectively.

[0039] Example 3 Effects of different concentrations of fermented wolfberry juice freeze-dried powder on the level of ROS in high-sugar-induced nematodes After synchronization and exposure for 24 h, add 2 μL DCFH-DA working solution, place in a shaker at 100 rpm, 37 °C, and stain for 5 h in the dark, then remove the supernatant. After staining, add 2 μL anesthetic, transfer an appropriate amount of nematodes to a 2% agarose pad, cover with a coverslip, and take pictures under the FITC channel. Each group should have no less than 20 nematodes, and set up 3 parallels.

[0040] As an aerobic organism that relies on oxygen for energy, nematodes will produce ROS free radicals when subjected to high sugar stress, thus being in a state of oxidative stress, which will accelerate the aging of the body. Figure 3 The effects of different doses of fermented wolfberry juice freeze-dried powder aqueous solution on ROS levels in nematodes. Figure 3 As shown in Figure 2, the average optical density intensity of ROS in the HG group was significantly higher than that in the CON group ( p <0.01), which was 38.62% higher. However, the unfermented fresh wolfberry juice freeze-dried powder aqueous solution in the GQ group could not significantly reduce the ROS level in the nematodes induced by high sugar. The average optical density intensity of ROS in the GQ group was only reduced by 6.16% compared with the HG group. After being treated with different doses of fermented wolfberry juice, the growth of ROS in the nematodes was significantly inhibited, among which the HGQ group and MGQ group had the best effect, and the average optical density intensity was extremely significantly weakened ( p <0.01), and decreased by 57.24% and 50.34% respectively. This shows that the fermented wolfberry juice freeze-dried powder can reduce the ROS level in high-fat nematodes, alleviate the oxidative stress caused by high sugar, and improve the survival ability of nematodes in a high-sugar environment.

[0041] Example 4 Effects of different concentrations of fermented wolfberry juice freeze-dried powder on SOD activity in high-sugar-induced nematodes The high, medium and low dosage groups (HGQ, MGQ and LGQ) had no toxic side effects on nematodes and the HGQ group had the best effect on improving the aging indicators of high-sugar-induced nematodes. Therefore, the HGQ group was determined as the dosage group to investigate its evaluation of SOD enzyme activity in high-fat nematodes. Synchronized nematodes were obtained after exposure for 24 h. The nematodes were mechanically homogenized in an ice water bath and the supernatant was collected. The SOD enzyme activity in nematodes was determined using a kit.

[0042] from Figure 4 It can be clearly seen that compared with the CON group, the SOD content in the HG group decreased by 51.92%. Compared with the HG group, the SOD content in the nematodes of the GQ group increased by only 3.44%, while the SOD content in the nematodes of the HGQ group increased by 71.77%.

[0043] Example 5 Effects of different concentrations of fermented wolfberry juice freeze-dried powder on the growth of nematodes induced by high sugar age-1 and sir-2.1 Effect of relative expression The nematodes were synchronized and exposed for 24 h. The total RNA of the tissues was extracted by chloroform extraction method, and the concentration of RNA was measured. The RNA with too high concentration was diluted in an appropriate proportion to make the final concentration of 200 ng / μL. The RNA was used as a template for reverse transcription to synthesize cDNA, and the fluorescence quantitative PCR method was used for age-1 and sir-2.1 Gene expression was detected. -△△CT The relative gene expression was calculated by the method. The primer sequences of each gene are shown in Table 1.

[0044]

[0045] The IIS pathway is crucial in regulating nematode metabolism, growth, development and aging. age-1 Belongs to the upstream of the IIS pathway, age-1 Downregulation can extend the lifespan of nematodes. sir-2.1 Regulated by the IIS signaling pathway, it affects the metabolism and antioxidant capacity of nematodes, and its reduced expression can extend lifespan. age-1 and sir-2.1 The relative expression of Figure 5 Compared with the CON group, the HG group age-1 and sir-2.1 The relative expression levels of GQ group and HG group were significantly increased by 14.54% and 33.99%, respectively. age-1 The relative expression level increased by 35.38%. sir-2.1The relative expression level was only down-regulated by 3.93%, indicating that the unfermented fresh wolfberry juice freeze-dried powder aqueous solution could not regulate the IIS pathway of nematodes. age-1 and sir-2.1 Compared with the HG group, they were significantly downregulated, down by 40.13% and 57.74%, respectively, indicating that fermented wolfberry juice freeze-dried powder may alleviate the metabolic-related aging of nematodes caused by high glucose by inhibiting the abnormal activation of the IIS pathway.

Claims

1. An application of fermented wolfberry juice freeze-dried powder in alleviating high sugar-induced metabolism-related aging in Caenorhabditis elegans, characterized in that: The freeze-dried powder is mixed with a high sugar solution at a concentration of 1.53-7.65 mg / mL and then applied to nematodes, thereby achieving an anti-aging effect by inhibiting abnormal activation of the nematode IIS pathway, extending the lifespan of the nematodes, reducing lipofuscin deposition, lowering ROS levels, and increasing SOD enzyme activity.

2. The use according to claim 1, characterized in that: The concentration of the high sugar solution is 50-100 mM, the nematodes are N2-type Caenorhabditis elegans at the L3-L4 stage, the exposure time is 22-26 hours, and the ambient temperature is 20±1°C.

3. The use according to claim 1, characterized in that: The preparation method of the fermented wolfberry juice freeze-dried powder comprises the following steps: (1) Inoculate Kluyveromyces marxianus into fresh wolfberry puree at a rate of 10 11 ~10 12 CFU, shaking fermentation at 30±2℃ for 22-26 hours; (2) After fermentation is completed, the fermentation liquid is subjected to vacuum rotary evaporation to remove ethanol, and the supernatant is centrifuged after pasteurization and freeze-dried to obtain freeze-dried powder; wherein the polysaccharide retention rate in the freeze-dried powder is ≥97%, the zeaxanthin dipalmitate retention rate is ≥98%, the betaine retention rate is ≥87%, and the reducing sugar content is ≤1.5%.

4. The use according to claim 1, characterized in that: The anti-aging effect is achieved by inhibiting the abnormal activation of the IIS pathway of nematodes induced by high sugar, which is specifically manifested as age-1 and sir-2.1 Gene expression was significantly downregulated.

5. The use according to claim 1, characterized in that: When the dosage concentration was 7.65 mg / mL, the life span of nematodes induced by high sugar was extended by 28.00%, lipofuscin deposition was reduced by 54.39%, ROS level was reduced by 57.24%, SOD enzyme activity was increased by 71.77%, age-1 and sir-2.1 The gene expressions were down-regulated by 40.13% and 57.74%, respectively.

6. The use according to claim 1, characterized in that: The fermented wolfberry juice freeze-dried powder is used for preparing food and daily chemical products for alleviating high-sugar-induced metabolism-related aging.

7. The use according to claim 1, characterized in that: The fermented wolfberry juice freeze-dried powder is used to prepare anti-aging creams, eye creams, essences, facial masks, and oral beauty products for alleviating high-sugar-induced metabolism-related aging.

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