CN115944658A - Application of paecilomyces variotii extract - Google Patents

Abstract

The invention belongs to the field of natural product development, and provides an application of a paecilomyces variotii extract in preparing an antioxidant and anti-aging reagent, food or medicine, wherein the paecilomyces variotii extract is an ethanol extract of mycelium; the preservation number of the paecilomyces variotii is CGMCC No.10114. The invention provides a basis for developing PVE non-agricultural uses of the paecilomyces variotii extract, such as the utilization in the aspects of oxidation resistance, aging resistance and the like.

Description

Application of paecilomyces variotii extract

Technical Field

The invention belongs to the field of natural product development, and relates to an application of a paecilomyces variotii extract in the aspect of oxidation resistance.

Background

The plant endophyte can live in different parts of host plants and has various types, thus being capable of generating various metabolites and having various biological activities such as antibiosis, antioxidation, anticancer, anti-inflammation and the like. The endophytic fungus DZY5 strain obtained by screening eucommia ulmoides has potential antioxidant application value. The endophyte isolated from Cajanus cajan has antioxidant effect and can scavenge superoxide radical and hydroxyl radical. The extract of the zygophyllum endophytic fungus JR0203 has antioxidant activity and has an inhibiting effect on staphylococcus aureus and the like. The endophyte isolated from rhizome of Curcuma longa has antioxidant activity, and can prolong the life of caenorhabditis elegans (caenorhabditis elegans) and survival rate under oxidative stress. 13 endophytic bacteria were isolated from alfalfa seeds, 4 of which had antioxidant activity and were able to significantly prolong the life of caenorhabditis elegans.

Paecilomyces variotii extract (A)Paecilomyces variotii extract, PVE) is wild sea buckthorn endophytic fungus paecilomyces variotii (A. Varioti)P. variotii) The extract of (1). The PVE active ingredients mainly comprise pyrimidine nucleosides, amino acids, oligosaccharides, cyclic peptides and the like, are environment-friendly and have wide action spectrum, and the active ingredients have obvious effects on various crops, but at present, research mainly focuses on various functions of PVE, such as growth promotion, drought resistance, low temperature resistance, saline and alkaline resistance, disease resistance and the like. Therefore, it is necessary to evaluate whether the PVE has antioxidant activity, so as to provide a basis for further development and utilization of the PVE.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a new application of a paecilomyces variotii extract, which can improve the activity of antioxidant related enzymes, reduce the content of active oxygen in a body and promote the effect of antioxidant action.

In order to achieve the purpose, the invention adopts the following technical scheme.

Application of paecilomyces variotii extract in preparation of antioxidant and anti-aging reagents, foods or medicinesPaecilomyces variotii) Ethanol extract of SJ1 mycelium;

the preservation number of the paecilomyces variotii SJ1 is CGMCC No.10114.

Preferably, the preparation method of the paecilomyces variotii extract comprises the following steps:

(1) Inoculating paecilomyces variotii to a seed culture medium for fermentation to obtain a seed solution;

(2) Inoculating the seed liquid to a fermentation culture medium for fermentation, and performing solid-liquid separation to obtain mycelia;

(3) Washing mycelium, drying, pulverizing, extracting with ethanol, and separating solid and liquid to obtain solution containing Paecilomyces variotii extract.

Preferably, the seed culture medium is a PDB culture medium; the formula of the fermentation medium is as follows: potato extract 1.0L, yeast extract 1.0 g, peptone 3.0 g, glucose 15.0 g.

Preferably, step (3) further comprises a step of removing the solvent in the solution.

Preferably, the subject of the above application is an animal or a human. More preferably, the animal is caenorhabditis elegans.

The invention has the following advantages:

the invention evaluates the antioxidant activity of PVE, finds that the PVE can prolong the life of the caenorhabditis elegans, promote the growth of the caenorhabditis elegans and the resistance to heat stress, can obviously reduce the ROS level of the caenorhabditis elegans, obviously increase the activity of antioxidant enzymes SOD, CAT and GST and the content of GSH, and has no toxic effect on other physiological functions, such as feeding, exercise and reproductive capacity. The plant endophytes are various in variety and have great development and utilization potential, and the invention provides a basis for developing PVE non-agricultural application of the paecilomyces variotii extract, such as the application in the aspects of oxidation resistance, aging resistance and the like.

Biological preservation information

Paecilomyces variotii (A. Varioti)Paecilomyces variotii) SJ1, which is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 08 days in 2014, the preservation address is China Beijing, and the microorganism research institute of China academy of sciences No. 3, xilu No.1 Hospital, north Cheng, the south China area, in Beijing, and the preservation number is CGMCC No.10114.

Drawings

FIG. 1 is a plot of ROS fluorescence in C.elegans after treatment with varying concentrations of Paecilomyces variotii extract;

FIG. 2 shows the ROS levels in C.elegans treated with varying concentrations of Paecilomyces variotii extract;

FIG. 3 is a graph showing the change in the levels of antioxidant-related enzymes in C.elegans treated with various concentrations of Paecilomyces variotii extract;

FIG. 4 is a graph showing the survival curves of C.elegans treated with various concentrations of the Paecilomyces variotii extract;

FIG. 5 is a graph showing the survival curves of C.elegans under heat stress conditions after treatment with various concentrations of the Paecilomyces variotii extract;

FIG. 6 shows the number of offspring of C.elegans treated with varying concentrations of the Paecilomyces variotii extract.

Detailed Description

The present invention will be further described with reference to the following examples and the accompanying drawings, but the present invention is not limited by the following examples.

Example 1 preparation of Paecilomyces Variotii Extract (PVE)

Paecilomyces variotii (A. Varioti)Paecilomyces variotii) SJ1, isolated from wild Hippophae rhamnoides (Hippophae rhamnoidesLinn.) root, as described in CN 104745483A, and the preservation number is CGMCC No.10114.

A Paecilomyces variotii strain SJ1 is inoculated on a panel PDA culture medium, 6 d is cultured at 25 ℃, an agar block of a puncher is inoculated on a 250 mL triangular flask, 50 mL seed culture solution (PDB culture medium) is contained in the bottle, 3d is cultured on a rotary shaking table at 28 ℃ and 120 r/min to be used as seeds, and 10% of the strain is inoculated on a fermentation culture medium containing 150 mL (the fermentation culture medium is potato extract, yeast extract 1.0 g, peptone 3.0 g, glucose 15.0 g. Preparation of the potato extract: peeling potato 200 g, cutting into small pieces, adding 1000 mL water, boiling for 30 min, filtering out potato pieces, adding filtrate to a 500 mL triangular flask of 1000 mL), culturing under the same conditions for 5 d, terminating fermentation, filtering the fermentation liquid, washing the cultured mycelium, drying at 60 ℃, weighing, pulverizing, extracting with fermentation liquid ethanol (24 3535 for 3 times, each time 24 zxft 3535, mixing uniformly by using a magnetic stirrer, oscillating with ultrasonic wave 1 3584, vacuum filtering, wherein the filtrate is light yellow clear liquid, the pH is 6.1-6.2, and the main components are small molecular oligosaccharides, amino acids, pyrimidine nucleosides, glycoproteins, polypeptides and the like with the molecular weight of 181-10000.

Example 2 antioxidant Activity of Paecilomyces Variotii Extract (PVE)

1. To the active oxygen (ROS) content in the body of caenorhabditis elegans

The influence of the paecilomyces variotii extract on the content of active oxygen (ROS) in the body is measured by taking the caenorhabditis elegans as a target, and the specific method is as follows:

caenorhabditis elegans is cultured by 3d until eggs exist in vivo, then the caenorhabditis elegans on a Nematode Growth Medium (NGM) is washed down by using an M9 buffer solution, the caenorhabditis elegans is placed into a 2 mL centrifuge tube, the supernatant is discarded after being centrifuged at 4000 rpm for 2 min, 100 muL of 5 NaClO and 50 muL of 5M NaOH are added, the solution is shaken for 3-5 min, the solution is centrifuged at 4000 rpm for 2 min, the supernatant is discarded, M9 buffer solution is added for washing for 2-3 times, the supernatant is centrifuged and discarded, then the eggs are transferred to a new bacterial plate containing escherichia coli OP50, and 48 h is cultured at the constant temperature of 20 ℃ and is 4-year-old (L4) nematodes. Transferring L4 nematodes to 24-well plates containing PVEs at various concentrations to culture 24 h, then respectively sucking 50 nematodes, transferring the nematodes to 2 mL centrifuge tubes containing 190. Mu.L of PBS buffer, and adding 10. Mu.L of 5. Mu. M H ₂ DCFDA solution is kept at the constant temperature of 20 ℃ in the dark for 2 h, washed for 3 times by PBS buffer solution, added with 5 mM for anesthesia, the anesthetized contrast and test group nematodes are placed on a 2% agarose pad, and the slide glass is placed on a stage of a fluorescence inverted microscope for observation and photographed for storage. The captured images were processed using Image J software. Not less than 20 nematodes were observed for each treatment and the experiment was repeated 3 times.

The effect of different concentrations of PVE treatment on the ROS levels of C.elegans is shown in FIG. 1 and FIG. 2. After treatment of 24 h with 1.0, 10 and 100 ng/mL PVE, the c.elegans L4 stage ROS levels exhibited a trend of significantly decreasing, with relative fluorescence intensities ranging from 64.71% to 80.08%, compared to the control group. After 10 and 100 ng/mL PVE treatment, ROS levels were significantly reduced by 35.29% and 29.30% (r) ((r))p <0.001 No significant difference between the two treatment groups; 1.0 ng/mL treatment also significantly reduced ROS levels: (p <0.05 19.92% lower.

Influence on antioxidant-related enzymes and antioxidant substances of caenorhabditis elegans

Antioxidant enzymes (including SOD, CAT, GST, etc.) can reduce biological oxidative damage and prevent the generation of excessive active oxygen, and are important components of defense systems. SOD is one of the most abundant antioxidant enzymes in animals,SOD plays an important role in maintaining oxidative stress, and is known as the first line of defense against ROS under oxidative stress. Glutathione (GSH) is an important biological free radical scavenger and can effectively remove O ²⁻ Hydrogen peroxide and lipid peroxide (LOOH), etc., GSH levels are often used to assess the body's level of antioxidant. PVEs prepared in example 1 were configured at different concentrations, and synchronized L4 stage nematodes (6000 per treatment) 24 h were treated, and the activity of the enzyme was determined:

centrifuging the treated nematodes at 4000 rpm for 2 min, discarding the supernatant, washing with M9 buffer solution for 3 times, repeatedly centrifuging, collecting in a new centrifuge tube, and homogenizing in a glass homogenizer on ice. 4000 Centrifuging at rpm for 5 min, and collecting supernatant to determine protein concentration and enzyme activity. The protein concentration is measured by adopting a Coomassie brilliant blue method; the determination of the activity of the antioxidant enzyme and the GSH is carried out by adopting a Nanjing constructed kit, the specific operation is carried out according to the relevant kit instructions, and the test is repeated for 3 times.

(1) Effect of PVE on the superoxide dismutase (SOD) of C.elegans

The effect of different concentrations of PVE treatment on the SOD activity of C.elegans is shown in FIG. 3 a. After 1.0, 10 and 100 ng/mL PVE treatment, the SOD activity in the caenorhabditis elegans is remarkably increased and is 79.78, 256.31 and 195.01U/mg prot respectively, and is respectively improved by 21.04%, 288.41% and 195.86% (3262 zxft 3232%) compared with a control group (65.91U/mg prot)p <0.001 ); there were also significant differences between the 1.0, 10 and 100 ng/mL PVE treatment groups ((s))p < 0.05）。

(2) Effect of PVE on Catalase (CAT) of C.elegans

The effect of different concentrations of PVE treatment on CAT activity of C.elegans is shown in FIG. 3 b. The CAT activity of the caenorhabditis elegans after the PVE treatment is obviously improved compared with that of a control group. After 1.0, 10 and 100 ng/mL PVE treatment, the CAT activity in C.elegans is 32.71, 35.90 and 34.11U/mg prot respectively, and is remarkably improved compared with that in a control group (16.35U/mg prot) ((p < 0.05）。

(3) Effect of PVE on C.elegans glutathione mercaptotransferase (GST)

Treatment of caenorhabditis elegans with different concentrations of PVEThe GST activity effect is shown in FIG. 3 c. After the PVE treatment, the GST activity is obviously improved compared with the control group. After being treated by 1.0 and 100 ng/mL PVE, the GST activity in the caenorhabditis elegans is obviously increased by 19.99 percent and 11.97 percent respectively, and the two treatment groups have no obvious difference. 10 GST activity in C.elegans of ng/mL treatment group increased 35.97%, the most significant increase compared to control group: (p < 0.001）。

(4) Effect of PVE on glutathione enzyme (GSH) of C.elegans

The effect of 1.0, 10 and 100 ng/mL PVE treatment on the GSH content of C.elegans is shown in FIG. 3 d. According to statistical data analysis, after the PVE treatment, the content of GSH is changed significantly compared with the control group, after the treatment of 1.0, 10 and 100 ng/mL PVE, the GSH activity in the caenorhabditis elegans is 102.33, 230.53 and 147.32 mg/g prot respectively, and is significantly improved compared with the control group (53.98 mg/g prot) ((S))p < 0.05）。

Example 3 Effect of Paecilomyces Variotii Extract (PVE) on the longevity of caenorhabditis elegans

Uniformly mixing PVE with the concentration and Escherichia coli OP50 which is subjected to overnight shaking with 1:1, coating a proper amount of the mixture on an NGM plate, and culturing at the constant temperature of 37 ℃ overnight to obtain a drug-carrying bacterium plate. 60 C.elegans were picked synchronously on each plate and the C.elegans were transferred every other day to plates containing fresh extract. The number of living C.elegans was counted daily until the last C.elegans died. The head of the caenorhabditis elegans was touched with a needle, and if the caenorhabditis elegans did not respond, the caenorhabditis elegans was considered dead and the test was repeated 3 times.

TABLE 1 Effect of PVE treatment on the longevity of caenorhabditis elegans

As shown in FIG. 4, the longevity of C.elegans was significantly extended over time compared to the control group after 1.0, 10, 100, 1000, and 10000 ng/mL PVE treatment. From 5 d, the control group showed a shorter lifespan and a significantly increased number of aged and dead insects compared to the PVE-treated group at each concentration. As shown in Table 1, exceptThe average life span of the caenorhabditis elegans after the treatment of PVEs with the same concentration is obviously prolonged, wherein the average life span of the caenorhabditis elegans is 15.98 d and 15.33 d after the treatment of 10 and 100 ng/mL PVEs, the life span of the caenorhabditis elegans is most obviously prolonged compared with that of a control group (the life span of the caenorhabditis elegans is prolonged by 10 ng/mL treatment groupp <0.001 Results show that PVE treatment can extend caenorhabditis elegans longevity.

Example 4 Effect of Paecilomyces Variotii Extract (PVE) on the Heat stress ability of C.elegans

The aging process is often accompanied with the balance state of the body being broken and the physiological function of the body being reduced, and the stress capability under the specific environmental stimulation can better reflect the physiological condition of the body and is often used for representing the health state in the aging process. Heat stress is one of the most common stress tests, and the survival of the caenorhabditis elegans in a high-temperature environment at 37 ℃ is generally determined. To explore the effect of PVE on the heat stress ability of C.elegans, the viability of C.elegans was tested at a stress temperature of 37 ℃: l4 nematodes were transferred to NGM plates containing PVEs at various concentrations and incubated at 20 ℃ for 24 h. Then, they were observed to survive at 37 ℃ every 2 h, dead nematodes were picked and recorded until all of the nematodes died. Not less than 50 treatments were performed per treatment and the experiment was repeated 3 times.

The results are shown in FIG. 5, where the longest survival time for the control group nematodes was 16 h;1.0, 10 and 100 ng/mL PVE treatment all extended the survival time of C.elegans under heat stress conditions, with 10 ng/mL treatment having a maximum survival time of 22 h,1.0 ng/mL treatment having a maximum survival time of 20 h, and 100 ng/mL treatment having a maximum survival time of 18 h. The results show that PVE can enhance the heat stress resistance of the caenorhabditis elegans.

Example 5 toxicity of Paecilomyces Variotii Extract (PVE) against caenorhabditis elegans

The PVEs of example 1 were configured to treat L4 nematodes with solutions of 1.0, 10 and 100 ng/mL, and their toxicity was assessed from acute toxicity, effects on feeding, locomotion and reproduction.

The results show that by measuring the toxic effect of PVE on caenorhabditis elegans, 1.0, 10, 100, 1000 and 10000 ng/mL PVE treated groups had no significant difference in nematode survival rates at 24, 48 and 72 h compared to control group(s) ((p > 0.05）It was shown that the chosen concentration of PVE is not toxic to C.elegans.

At least 20 caenorhabditis elegans groups treated by 24 h through PVE with each concentration are picked to a new Escherichia coli OP50 bacterial plate, the frequency of pharyngeal pump jumping during feeding of the nematode in 30 s is observed and recorded under a stereoscopic microscope, and the test is repeated for 3 times. The results showed that there was no significant difference in the pharyngeal pumping frequency of caenorhabditis elegans between the PVE-treated groups and the control group, nor between the PVE-treated groups ((C))p > 0.05）。

The head of the caenorhabditis elegans swings 1 time after swinging from one side to the other side. The caenorhabditis elegans after being post-treated by 24 h with various concentrations of PVEs are picked to an NGM plate without Escherichia coli OP50, the NGM plate is placed under a stereoscopic microscope to observe and record the swinging times of each caenorhabditis elegans in 30 s, at least 20 caenorhabditis elegans in each group are measured, and the test is repeated for 3 times. The results showed no significant difference in the number of head oscillations of caenorhabditis elegans in the PVE-treated group and between the PVE-treated groups, compared to the control group (p > 0.05）。

The daily egg production and the total number of offspring of caenorhabditis elegans are key indicators for the health status of the nematode. The synchronized L4-stage nematodes are picked to NGM drug plates (1 caenorhabditis elegans per plate) containing PVE with various concentrations, the temperature is kept at 20 ℃, new drug plates are replaced every day, the old plates are cultured in a constant temperature incubator at 20 ℃ for 24 h, and then the insects on the plates are counted until the nematodes do not lay eggs any more. At least 20 per treatment group and the experiment was repeated 3 times.

TABLE 2 influence of PVE treatment on the reproduction of caenorhabditis elegans

As shown in Table 2, the number of eggs laid by C.elegans 1 d was increased in the PVE-treated group and 17.88% in the 10 ng/mL-treated group, as compared with the control group (see Table 2)p <0.05 ); the oviposition amount of the 2 d treated group is not obviously different from that of the control group; the oviposition amount of the 3d treated group is obviously reduced compared with the control group, and the oviposition amount of the 10 ng/mL treated group is reduced by 14.47%. No longer oviposition at 4 d; as shown in FIG. 6, the total number of offspring treated groups versus the controlNo significant difference between the treatment groups of PVEs compared with the treatment groups (p >0.05). This indicates that PVE had no significant effect on L4 stage nematode reproduction.

The results show that the paecilomyces variotii extract has no obvious toxicity to the caenorhabditis elegans, does not influence the normal physiological activities of feeding, exercise and the like of the caenorhabditis elegans, and has no obvious reproductive toxicity.

Claims (6)

1. Application of paecilomyces variotii extract in preparation of antioxidant and anti-aging reagents, foods or medicinesPaecilomyces variotii) Ethanol extract of SJ1 mycelium;

the preservation number of the paecilomyces variotii SJ1 is CGMCC No.10114.

2. The use according to claim 1, wherein the preparation method of the paecilomyces variotii extract comprises the following steps:

(1) Inoculating paecilomyces variotii to a seed culture medium for fermentation to obtain a seed solution;

(2) Inoculating the seed liquid to a fermentation culture medium for fermentation, and performing solid-liquid separation to obtain mycelia;

(3) Washing mycelium, drying, pulverizing, extracting with ethanol, and separating solid and liquid to obtain solution containing Paecilomyces variotii extract.

3. Use according to claim 2, wherein the seed medium is PDB medium; the formula of the fermentation medium is as follows: potato extract 1.0L, yeast extract 1.0 g, peptone 3.0 g, glucose 15.0 g.

4. The use of claim 2, wherein step (3) further comprises the step of removing the solvent from the solution.

5. Use according to claim 1, characterized in that the object of action of the use is an animal or a human.

6. The use according to claim 1, wherein the subject of said use is caenorhabditis elegans.

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