Glycosylated ganoderic acid and anti-aging application thereof

Abstract

The invention discloses glycosylated ganoderic acid with a chemical structural formula shown in the specification, the compound can effectively improve H2O2-induced cellular morphological change, remarkably reduce generation of reactive oxygen species (ROS) and reduce accumulation of beta-galactosidase in senescent cells, and in addition, cell immunoblotting analysis shows that the compound can be used for preparing glycosylated ganoderic acid. The glycosylated ganoderic acid can significantly down-regulate the expression of H2O2-induced senescence marker proteins p16 and p21, and experimental results prove that the glycosylated ganoderic acid has potential in the aspect of reversing cell senescence, provides a new candidate compound for the development of anti-senescence drugs, and shows a wide application prospect; # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicinal chemistry, and specifically relates to a glycosylated ganoderic acid and its anti-aging application. Background Art

[0002] Ganoderma lucidum is an important medicinal fungus, and its main active ingredient, ganoderic acid, has a wide range of pharmacological activities. However, the hydrophobicity of ganoderic acid leads to its limited water solubility and bioavailability, thus limiting its potential in therapeutic applications. Glycosylation modification can significantly improve the water solubility of ganoderic acid, thereby enhancing its bioavailability. Therefore, obtaining glycosylated ganoderic acid is crucial to improving its medicinal value. At present, there is no report on the direct production of glycosylated ganoderic acid in Ganoderma lucidum. The present invention realizes the in vivo glycosylation synthesis of ganoderic acid for the first time by heterologously expressing a glycosyltransferase gene (Bacillus sp.FQZ25-16345-GA-UGT) in Ganoderma lucidum (Ganoderma lingzhi CGMCC 5.616), and successfully obtains a new glycosylated ganoderic acid GAC2-15-O-β-glucoside with anti-aging activity, which not only opens up a new way for the discovery and preparation of anti-aging drugs, but also provides new candidate compounds for the development of anti-aging drugs, showing broad application prospects. Summary of the invention

[0003] The present invention provides a glycosylated ganoderic acid, the chemical structure of which is as follows:

[0004]

[0005] Another purpose of the present invention is to use glycosylated ganoderic acid in the preparation of anti-aging drugs.

[0006] The present invention applies the above-mentioned glycosylated ganoderic acid to the preparation of anti-aging drugs. The ingredients (or active ingredients) of the drugs are the above-mentioned glycosylated ganoderic acid. One or more excipients acceptable in product production may also be added to improve the absorption effect of the active ingredients or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] 1. The glycosyltransferase gene from Bacillus subtilis (nucleotide sequence as shown in SEQ ID NO: 1) was transferred into the Ganoderma lingzhi engineering strain (CGMCC 5.616) to obtain the recombinant Ganoderma lingzhi engineering strain OE-In-opGTs capable of producing glycosylated ganoderic acid;

[0009] 2. The recombinant Ganoderma lucidum engineered bacteria OE-In-opGTs were cultured to obtain fruiting bodies, and a crude extract containing glycosylated ganoderic acid was extracted from the fruiting bodies using a 70% ethanol solution. The single compound in the crude extract was then separated and purified using chromatography, and after nuclear magnetic resonance identification, glycosylated ganoderic acid with the structural formula shown above was obtained;

[0010] 3. Identification of the anti-aging activity of glycosylated ganoderic acid. The experimental results showed that the compound can effectively improve H2O2-induced cell morphological changes, significantly reduce the generation of reactive oxygen species (ROS), and reduce the accumulation of β-galactosidase in senescent cells. In addition, cell immunoblot analysis showed that glycosylated ganoderic acid can significantly downregulate the expression of H2O2-induced aging marker proteins p16 and p21. The experimental results prove that glycosylated ganoderic acid has the potential to reverse cell aging.

[0011] The present invention provides for the first time a glycosylated ganoderic acid produced by recombinant ganoderma lucidum engineering bacteria and reveals its anti-aging activity, thus providing a new approach for the discovery and preparation of anti-aging drugs and providing new candidate compounds for the development of anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the pJW-EXP-intron-opGTs vector structure;

[0013] Figure 2 This is the screening and verification process of the OE-In-opGTs positive transformants in the present invention, wherein Figure a is a transformant primary screening plate map; Figure b is a transformant secondary screening plate map; Figure c is a schematic diagram of electrophoresis of positive transformants, wherein: M is DL5000 Maker (Takara), P is a positive control, N is a negative control, 1-3 are positive transformants of pJW-EXP-intron-opGTs, and WT is a wild-type strain;

[0014] Figure 3 Schematic diagram of the OE-In-opGTs fruiting body culture process; Figure a is a fungus bag full of hyphae; Figure b is the primordium of the recombinant strain OE-In-opGTs; Figure c is the recombinant strain OE-In-opGTs fruiting body;

[0015] Figure 4 is a 16-minute liquid chromatogram of the crude extract separated in semi-preparative liquid chromatography, wherein WT represents the crude extract of the WT strain, OE-In-opGTs represents the crude extract of the OE-In-opGTs engineered strain, GAC2 represents the unglycosylated ganoderic acid GAC2, and I represents compound I;

[0016] Figure 5 is the COSY and key HMBC correlation of compound I;

[0017] Figure 6 is the result of the CCK8 test on the toxicity of compound I to hAMSCs cells, wherein I is compound I;

[0018] Figure 7 is a schematic diagram of the morphology of hAMSCs cells after treatment with compound I, wherein Control is a blank control group; H2O2 is a positive control group; 5, 1, and 0.2 are hAMSCs cells treated with H2O2 after being cultured with different concentrations of compound I;

[0019] Figure 8 is the ROS detection result after hAMSCs cells were treated with compound I; Control is the blank control group; H2O2 is the positive control group; 5, 1, and 0.2 are hAMSCs cells treated with H2O2 after being cultured with different concentrations of compound I;

[0020] Fig. 9 is the β-galactosidase staining result of hAMSCs cells after treatment with compound I; Control is the blank control group; H2O2 is the positive control group; 5, 1, and 0.2 are hAMSCs cells treated with H2O2 cultured with different concentrations of compound I;

[0021] Fig.10 It is the result of immunoblotting detection of the expression of cell senescence marker proteins (p16 and p21) in hAMSCs cells after treatment with compound I; wherein GAC2 is unglycosylated ganoderic acid, and I is compound I. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the contents described above. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified;

[0023] Seed culture medium (g / L) in the following examples: glucose 35, peptone 5, yeast extract 2.5, potassium dihydrogen phosphate 1, magnesium sulfate 0.5, vitamin B1 0.05;

[0024] CYM medium (g / L): glucose 20, maltose 10, yeast powder 2, peptone 2, MgSO4 0.5, KH2PO44.6, agar 10;

[0025] Formula of mushroom bag: 58% cottonseed hull, 20% oak sawdust, 20% wheat bran, 1% calcium sulfate, 1% sucrose (mass percentage). Soak the cottonseed hull and oak sawdust overnight, add wheat bran, calcium sulfate and sucrose, adjust the humidity until a small amount of water seeps out when squeezed tightly; pack into mushroom bags, and sterilize in a high-pressure steam sterilizer at 121°C for 60 minutes.

[0026] Example 1: Obtaining glycosylated ganoderic acid

[0027] 1. Obtain the Bacillus subtilis glycosyltransferase gene sequence from the NCBI public database, and add the first exon, the first intron, and the first three bases of the second exon of the Ganoderma lucidum glycerol triphosphate dehydrogenase gene to the 5' end of the sequence, and entrust Sangon Biotech (Shanghai) Co., Ltd. to optimize and synthesize according to the codon preference of Ganoderma lucidum to obtain the gene sequence shown in SEQ ID NO: 1 (the 1st to 6th bases are the first exon of Ganoderma lucidum glycerol triphosphate dehydrogenase; the 7th to 73rd bases are the first intron of Ganoderma lucidum glycerol triphosphate dehydrogenase; the 74th to 76th bases are the first three bases of the second exon of Ganoderma lucidum glycerol triphosphate dehydrogenase).

[0028] 2. Expression vector construction

[0029] The gene fragment synthesized in step 1 was digested with Sma I and Nhe I by double restriction enzyme digestion, and ligated to the linearized pJW-EXP vector using T4 DNA ligase (Takara) at 16°C for 30 min. The construction of the linearized pJW-EXP vector was prepared according to the method of YU X, JI SL, HE YL, et al. Development of an Expression Plasmid and its Use in Genetic Manipulation of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes) [J / OL]. International Journal of Medicinal Mushrooms, 2014, 16 (2). Afterwards, the ligation product was transformed into E. coli DH5α competent cells, and the transformants were verified using primers F: 5'-CGAGTGACGCAGGTGGTGAC-3' and R: 5'-TTACTTGATCTTGACGTAGGGCTTC-3'. The plasmid was extracted and sequenced, and finally the pJW-EXP-intron-opGTs vector ( Figure 1 ).

[0030] 3. Construction of engineered strains

[0031] According to the method in Zhang Dehuai. High expression of oxidized squalene synthase gene and co-expression of 3-hydroxy-3-methylglutaryl-CoA reductase gene in Ganoderma lucidum to improve the production of ganoderic acid [D / OL]. Kunming University of Science and Technology, 2018, the pJW-EXP-intron-opGTs plasmid was transferred into Ganoderma lucidum cells (CGMCC 5.616), and positive transformants were selected from CYM selective medium containing 2 mg / L Carboxin; and primers F: 5'-CGAGTGACGCAGGTGGTGAC-3' and R: 5'-TTACTTGATCTTGACGTAGGGCTTC-3' were used to verify the transformants. The results are as follows Figure 2 , and obtained the recombinant Ganoderma lucidum engineering strain OE-In-opGTs.

[0032] 4. Fruiting body cultivation

[0033] The recombinant Ganoderma lucidum engineered strain OE-In-opGTs was cultured on a PDA slant medium at 30°C for 7 days, the mycelium was scraped into a 250mL conical flask containing 50mL seed culture medium, and cultured at 30°C and 120rpm for 5 days to obtain a primary seed culture solution; 45mL seed culture medium and 5mL primary seed culture solution (about 500mg DW / L, the primary culture was inoculated after being broken with glass beads) were added to a 250mL shake flask, and cultured at 30°C and 120rpm for 2 days to obtain a secondary culture;

[0034] Add 30 mL of secondary culture (about 500 mg DW / L, the secondary culture is broken with glass beads and then inoculated) to each bacterial bag and culture it in a constant temperature incubator at 30°C until the entire bacterial bag is full of hyphae about 15 days after inoculation.

[0035] Remove the plastic film from the upper part of the fungus bag covered with mycelium, then place the entire fungus bag in the soil, cover it with a soil layer about 1 cm thick, place it in a warm and humid environment (temperature 28-30℃, humidity 85%-90%), and cover it with a transparent lid or plastic bag; during the cultivation period, ventilate and water it every day, and provide light (300-1000lx). After about 60 days, the mycelium in the fungus bag will grow into mature fruiting bodies, and the results are as follows: Figure 3 .

[0036] 5. Preparation of crude extract

[0037] The fruiting bodies were collected, dried at 37°C and fully ground, and a 70% ethanol solution was added to the fruiting body powder at a solid-liquid ratio of 1:10 (g:mL). The mixture was allowed to stand overnight, and then ultrasonically treated for 3 times, each for 30 minutes, and centrifuged at 10,000 rpm for 5 minutes. The supernatant was dried in an oven at 50°C to obtain a crude extract.

[0038] 6. Separation and purification of compounds

[0039] The crude extract was dissolved in methanol and sent to semi-preparative liquid chromatography for separation. The liquid chromatogram was as follows Figure 4 As shown, the eluate corresponding to the target product peak in the chromatogram is collected and concentrated under reduced pressure to obtain compound I;

[0040] Chromatographic conditions: Agilent Eclipse Plus C18 column (1.8 μm, 2.1×150 mm); phase A was 0.01% acetic acid water, phase B was acetonitrile; flow rate was 0.4 mL / min; detection wavelength was 254 nm, and the chromatographic detection procedure is shown in Table 1;

[0041] Table 1 Separation and purification chromatographic detection procedures for compounds

[0042]

[0043]

[0044] The isolated compound I was subjected to mass spectrometry analysis and nuclear magnetic resonance detection, ESI-MS m / z: 679.2 [MH] - Its molecular formula is determined to be C 36 H 56 O 12 ; Compound I 1H NMR (500MHz, Methanol-d4) δ values ​​are 4.75 (m, 1H), 4.58 (dd, J = 8.0, 9.5Hz, 1H), 4.26 (d, J = 8.0Hz, 1H), 3.76 (d, J = 11.5Hz, 1H), 3.56 (m, 1H), 3.24 (t, J = 9.0Hz ,1H),3.17(m,1H),3.16(m,1H),3.11(t,J=8.5Hz,1H),3.03(dd,J=4.5,11. 5Hz,1H),2.76(d,J=16.0Hz,1H),2.70(m,1H),2.65(m,1H),2.62(m,1H),2. 45(d,J=16.5Hz,1H),2.35(m,1H),2.30(d,J=16.0Hz,1H),2.16(m,1H),2.1 0(m,1H),2.00(m,1H),1.87(m,1H),1.76(m,1H),1.71(m,1H),1.50(m,1H), 1.49(m,1H),1.17(s,3H),1.16(s,3H),1.03(d,J=6.5Hz,3H),0.90(s,3H), 0.88(s,3H),0.84(m,1H),0.79(m,1H),0.76(d,J=6.5Hz,3H),0.73(s,3H); 13 CNMR (125MHz, Methanol-d4)δ values ​​are 211.0, 200.7, 181.8, 160.4, 142.3, 103.8, 82.1, 77.5, 77.1, 76.5, 74.2, 70.1, 67.3, 61.3, 53.6, 51.5, 49.2, 48.9, 48.6, 47.0, 46.3, 38.2, 38.0, 36.8, 35.3, 34.6, 32.4, 28.2, 27.2, 26.9, 20.1, 18.6, 17.9, 16.8, 16.2, 14.7.

[0045] COSY and key HMBC correlations of compound I are shown in Figure 2. Figure 5As shown, the following key HMBC correlations are included: H-1 (C-2,3,5,10,19), H-2 (C-3), H-3 (C-4,28,29), H-5 (C-4,10,19,29), H-6 (C-5,7,8,10), H-7 (C-8,9), H-12 (C-9,11,13,14,18), H-15 (C-1',8,14,30), H-16 (C-13,14,15,17,20), H-17 (C-13,16,18,20), H-18 (C-12,13,14,17 ), H-19(C-1,5,9,10), H-21(C-17,20,22), H-22(C-20,21,23), H-24(C-23), H-27(C-24,25,26), H-28(C-3,4,5,29), H-29( C-3,4,5,28), H-30(C-8,13,14,15), H-1'(C-15,2',3',5'), H-2'(C-1',3'), H-3'(C-2',4'), H-4'(C-5'), H-6'(C-4',5').

[0046] Example 2: Activity detection experiment of compound I

[0047] 1. Weigh the compound I prepared in Example 1, prepare it into a 25 μM stock solution with DMSO, and then dilute it into solutions of compound I with concentrations of 5 μM, 1 μM, and 0.2 μM by a 5-fold dilution method;

[0048] Test cells: human amniotic mesenchymal stem cells (hAMSCs);

[0049] Culture medium: DMEM culture medium and F12 culture medium were mixed at a volume ratio of 1:1, and then a final concentration of 10% fetal bovine serum, 1% double antibody (a mixture of penicillin and streptomycin) and 10 ng / mL bFGF (basic fibroblast growth factor) were added to prepare the culture medium;

[0050] 2. CCK8 detection of compound I cytotoxicity to hAMSCs

[0051] hAMSCs were cultured at 5×10 3 / well were inoculated in a 96-well cell culture plate, and after culturing for 24 hours at 37°C and 5% CO2, solutions of compound I with final concentrations of 25 μM, 5 μM, 1 μM, and 0.2 μM were added, respectively, and the culture was continued for 48 hours, followed by adding 10 μL of CCK8 reagent, and after incubation for 2 hours, the absorbance was measured at 450 nm, and the cell survival rate was calculated by the absorbance value, thereby evaluating the cytotoxicity of compound I; a blank control group without adding compound I was also set up;

[0052] Results Figure 6 As can be seen from the figure, compared with the blank control group, compound I had no toxicity to hAMSC cells in the concentration range of 0.2-25 μM.

[0053] 3. Test of compound I on reversing aging of hAMSCs cells

[0054] A. Cell morphology observation

[0055] hAMSCs were cultured at 5×10 4 / well were inoculated in a 12-well cell culture plate, cultured for 2 hours at 37°C, 5% CO2, and then treated with 200 μM H2O2 for 2 hours, and then replaced with a culture medium containing 5 μM, 1 μM, and 0.2 μM compound I. After 48 hours of culture, the cell morphology was observed and photographed under a microscope, and a blank control group (no compound, H2O2) and a positive control (no compound, H2O2) were set up at the same time;

[0056] Results Figure 7 From the figure, it can be seen that the normal hAMSCs cells in the blank control group are long spindle-shaped and grow rapidly; in the positive control group, the cells grow slowly after H2O2 treatment, and some cells grow flat and wide. After treatment with compound I, the cell morphology induced by H2O2 is restored, the cells gradually show a long spindle shape, and the growth rate is accelerated. Compound I has a certain reversal effect on H2O2-induced cell senescence.

[0057] B. ROS staining experiment

[0058] hAMSCs were cultured at 5×10 4 The cells were seeded at a density of 100 μg / well in a 12-well cell culture plate and cultured at 37°C, 5% CO2 for 24 h. After treatment with 200 μM H2O2 for 2 h, the culture medium was replaced with the medium containing 5 μM, 1 μM, and 0.2 μM compound I. The culture was continued for 48 h. At the same time, a blank control group (no compound, H2O2) and a positive control (no compound, H2O2) were set up.

[0059] Before staining, the cell culture medium was removed, the cells were washed once with PBS, and then 1 mL of 10 μM DCFH-DA working solution (Solarbio, China, CA1410) was added and incubated at room temperature in the dark for 30 minutes, and then washed three times with PBS to remove the probe that did not enter the cells. Then, the green fluorescence intensity of each group of cells was observed and recorded under a fluorescence microscope (FITC channel);

[0060] Results Figure 8From the figure, it can be seen that the green fluorescence of normal young hAMSCs cells in the blank control group is darker. After H2O2 treatment in the positive control group, some senescent cells are stained with strong green fluorescence; after compound I treats H2O2-induced cells, the green fluorescence weakens, indicating that compound I can effectively reduce the generation of reactive oxygen species (ROS) and alleviate H2O2-induced cellular oxidative stress.

[0061] C. β-galactosidase staining experiment

[0062] hAMSCs were cultured at 5×10 4 / well were inoculated in a 12-well cell culture plate, and cultured at 37°C, 5% CO2 for 24 hours, then 200 μM H2O2 was added for 2 hours, and then replaced with a culture medium containing 5 μM, 1 μM, and 0.2 μM compound I, and cultured for 48 hours. At the same time, a blank control group (no compound, H2O2) and a positive control (no compound, H2O2) were set up. After the culture was completed, the cell culture medium was removed, the cells were washed once with PBS, 0.5 mL of β-galactosidase staining fixative (Beyotime, China, C0602) was added, and fixed at room temperature for 15 minutes; the cells were washed 3 times with PBS buffer, 1 mL of β-galactosidase staining working solution (Beyotime, China, C0602) was added to each well to ensure that the cells were completely covered, and the culture plate was incubated at 37°C in a CO2-free environment for 12 hours. After the incubation was completed, the staining results were observed and recorded using a microscope.

[0063] Results Fig. 9 From the figure, it can be seen that the normal young hAMSCs cells in the blank control group are not blue, and some senescent flat and wide cells in the positive control group are blue after H2O2 treatment; after compound I treats H2O2-induced cells, the blue color weakens, indicating that compound I can effectively reduce the accumulation of β-galactosidase in senescent cells and has a certain anti-aging effect.

[0064] D. Immunoblotting of cell senescence marker proteins (p16 and p21)

[0065] hAMSCs were cultured at 5×10 4 / well were inoculated in a 12-well cell culture plate, cultured at 37°C, 5% CO2 for 24 hours, then treated with 200 μM H2O2 for 2 hours, and then replaced with a culture medium containing compound I or unglycosylated ganoderic acid GAC2 (all 5 μM, 1 μM and 0.2 μM), and cultured for 48 hours. At the same time, a blank control group (no compound, H2O2) and a positive control (no compound, H2O2) were set up. After the culture was completed, the culture medium was aspirated, the cells were washed twice with cold PBS, ice-cold RIPA lysis buffer was added, and the cells were lysed on ice for 20 minutes. The lysate was collected and centrifuged at 4°C, 12000 rpm for 15 minutes to obtain the supernatant, and the protein concentration was quantified using the BCA method. An equal amount of protein sample was mixed with SDS loading buffer, boiled for 5 minutes, and then loaded onto SDS-PAGE gel. Electrophoresis was performed at 80 V until the sample entered the separation gel, and then the protein was separated at 120 V. After the electrophoresis, the protein was transferred to a PVDF membrane, transferred at 100 V at 4°C for 2 hours, and then blocked with 5% skimmed milk powder for 1 hour. Subsequently, the membrane was incubated with primary antibodies p16 (Abcam ab108349 1:2000 dilution), p21 (Proteintech ab109349 1:1000 dilution) and GAPDH internal control antibody (Proteintech 10494-1-AP 1:10000 dilution) at 4°C overnight. The next day, the membrane was washed three times with TBST, and HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing again with TBST three times, ECL chemiluminescent substrate was used for color development, and the bands of p16, p21 and GAPDH were detected and recorded under a chemiluminescent imaging system.

[0066] Results Fig.10 Compared with the blank control group, H2O2 treatment in the positive control group upregulated the expression of p16 and p21; while after treatment with unglycosylated ganoderic acid GAC2 (5μM) and compound I (5, 1, 0.2μM), the expression levels of p16 and p21 in senescent hAMSCs cells were downregulated. In contrast, the inhibitory effect of compound I was more significant and the effect was dose-dependent, indicating that compound I could more effectively inhibit the expression of senescence marker proteins induced by H2O2 than unglycosylated GAC2, and had a better anti-aging effect.

[0067] The above results indicate that compound I has a significant effect in resisting H2O2-induced hAMSCs cell senescence, showing its potential for development as an anti-aging drug.

Claims

1. A glycosylated ganoderic acid, the chemical structure of which is as follows:

2. Use of the glycosylated ganoderic acid according to claim 1 in the preparation of anti-aging drugs.

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