Beta-eudesmol synthase CcTPS6 and its encoding gene and application thereof

Abstract

The application discloses a kind of for synthesizing β - terpene synthase CcTPS6 of eucalyptol and its encoding gene and its product β - the application of eucalyptol as anti-aging drug belongs to synthetic biology and natural medicine chemistry technical field. The application starts from Labiatae plant torch flower ( Colquhounia coccinea var. mollis ) Cloning and functional identification of a synthetic β - eucalyptol single-function terpene synthase CcTPS6 encoding gene, its nucleotide sequence is as shown in Seq ID No.2, after codon optimization and site-directed mutagenesis, connect with expression vector pCold-TF, construct into the recombinant plasmid capable of expressing in escherichia coli, again the recombinant plasmid is transformed into escherichia coli and is constructed into engineering cell, realizes the heterologous high-efficiency synthesis compound β - eucalyptol in escherichia coli. The gene engineering cell constructed by the application is safe and stable, and the production cycle is short, which shows its great value in application development. The CcTPS6 product provided by the application β - eucalyptol has anti-aging activity and can be applied in the preparation of anti-aging drugs.

Description

Technical fields:

[0001] This invention relates to the fields of synthetic biology and natural product chemistry, specifically to β-eucalyptol sesquiterpene synthase and its encoding gene, its applications, and the use of its products in the preparation of anti-aging drugs. Background technology:

[0002] Terpenes are the most diverse and chemically varied class of natural products, widely found in higher plants and microorganisms, and possess significant economic and medicinal value. The sesquiterpene β-cineole is typically found in the volatile oils of plants and exhibits a wide range of pharmacological activities, including anti-inflammatory, antitumor, angiogenesis-inhibiting, gastric emptying-promoting, and acetylcholine-coenzyme-inhibiting activities; however, its anti-aging activity has not been reported in the literature. Summary of the Invention:

[0003] The purpose of this invention is to provide the cDNA sequence of the gene encoding β-eucalyptol synthase CcTPS6 and its applications.

[0004] To achieve the above-mentioned objectives of this invention, the present invention provides the following technical solution:

[0005] β-Cineole CcTPS6, which is:

[0006] (1) The protein composed of the amino acid sequence shown in Seq ID No.1;

[0007] (2) Derivative proteins with the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues according to the amino acid sequence shown in Seq ID No.1.

[0008] The gene encoding the β-eucalyptol synthase CcTPS6 is as follows:

[0009] (a) The nucleotide sequence shown in Seq ID No. 2;

[0010] (b) Nucleotide sequences of the nucleotide sequence shown in Seq ID No.2 that have been substituted and / or deleted and / or added with one or more nucleotides and express the same functional protein.

[0011] A recombinant vector containing the CcTPS6 encoding gene.

[0012] An expression cassette containing the gene encoding the β-eucalyptol synthase CcTPS6.

[0013] Transgenic cell lines containing the gene encoding the β-eucalyptol synthase CcTPS6.

[0014] Recombinant bacteria containing the β-eucalyptol synthase CcTPS6 encoding gene.

[0015] The application of the β-cineole synthase CcTPS6 or its encoding gene in the preparation of recombinant vectors, expression cassettes, transgenic cell lines, and recombinant bacteria containing the β-cineole synthase CcTPS6 encoding gene.

[0016] The application of the β-cineole synthase CcTPS6 or its encoding gene in the preparation of fermentation broth containing the compound β-cineole is described in that: an expression vector containing the gene encoding the gene is constructed, the recombinant vector is transformed into Escherichia coli, and the obtained genetically engineered bacteria are fermented to obtain a fermentation broth containing β-cineole.

[0017] A method for preparing compound β-eucalyptol (c1) includes the following steps: constructing an expression vector containing a gene encoding β-eucalyptol synthase CcTPS6; transforming the recombinant vector into Escherichia coli; fermenting the obtained genetically engineered bacteria to obtain a fermentation broth containing β-eucalyptol; extracting the broth with petroleum ether or ethyl acetate to obtain an extract containing β-eucalyptol; and separating and purifying the extract by silica gel column chromatography to finally obtain compound β-eucalyptol (c1).

[0018]

[0019] The application of the β-eucalyptol synthase CcTPS6 or its encoding gene in the preparation of the compound β-eucalyptol.

[0020] Application of β-Cineole synthase CcTPS6 or its encoding gene in the preparation of fragrance oils, daily cosmetics, and anti-aging drugs.

[0021] The application of β-eucalyptol, a sesquiterpene compound obtained by any of the above preparation methods, in the preparation of fragrance oils, daily cosmetics, and anti-aging drugs.

[0022] The anti-aging drugs include the strains of Escherichia coli and Nematode elegans used.

[0023] A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound β-cineole. The pharmaceutical composition may be in the form of tablets, capsules, granules, oral liquid formulations, intravenous injections, or intramuscular injections.

[0024] This invention also provides specific primer pairs for obtaining the cDNA sequence of the β-eucalyptol synthase CcTPS6 encoding gene, including:

[0025] Forward primer (CcTPS6-F): 5'-ATGGCTGCACTTATCGCCG-3'

[0026] Reverse primer (CcTPS6-R): 5'-TTAAATCTCTATTTGATCGACGAGC-3'

[0027] The primers mentioned above were synthesized by Beijing Qingke Xinyue Biotechnology Co., Ltd.

[0028] The open reading frame (ORF) of the β-cineole synthase CcTPS6 gene provided by this invention is 1650 bp (Seq ID No. 2), encoding 549 amino acids (Seq ID No. 1). BLASTN analysis performed on it in NCBI showed that it has 80% homology with LcTPS2 of the Lamiaceae plant Colquhounia coccinea var. mollis.

[0029] The β-cineole synthase CcTPS6 gene provided by this invention is a terpene synthase gene cloned from *Colquhounia coccinea var. mollis*. It can specifically catalyze the synthesis of the single product sesquiterpene β-cineole from the direct precursor of sesquiterpenes, farnesyl pyrophosphate (FPP). This gene is the first to be cloned from a plant, and the discovery of this enzyme enriches the diversity of terpene synthases.

[0030] This invention clones and functionally identifies the sesquiterpene synthase CcTPS6 from the medicinal plant *Torchella esculenta* (Lamiaceae family), and utilizes it to generate β-eucalyptol from *Escherichia coli*.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] This invention starts with the plant *Colquhounia coccinea var. mollis* (Lamiaceae family), clones and functionally identifies a monofunctional terpene synthase gene, CcTPS6, encoding β-cineole. Its nucleotide sequence is shown in Seq ID No. 2. After codon optimization and site-directed mutagenesis, the gene is ligated into the expression vector pCold-TF to construct a recombinant plasmid capable of expression in *E. coli*. The recombinant plasmid is then transformed into *E. coli* to construct engineered cells, achieving heterologous and efficient synthesis of the compound β-cineole in *E. coli*. The genetically engineered cells constructed by this invention are safe and stable, with a short production cycle, demonstrating significant value in application development. The product provided by this invention possesses anti-aging activity and can be used in the preparation of anti-aging drugs. The results of the examples show that the β-cineole provided by this invention can extend the lifespan of the model organism *Caenorhabditis elegans* by approximately 32%, an effect similar to the lifespan extension effect of metformin hydrochloride (35%). Attached Figure Description

[0033] Figure 1 This is a multiple sequence alignment diagram of β-eucalyptol synthase CcTPS6 in Example 1.

[0034] Figure 2 This is a predicted three-dimensional structure of β-eucalyptol synthase CcTPS6 in Example 1.

[0035] Figure 3 The total ion chromatogram of the GC-MS analysis of the extract of the engineered Escherichia coli expressing the β-eucalyptol synthase CcTPS6 gene in Example 3 is shown.

[0036] Figure 4 The total ion chromatogram of GC-MS analysis of the extract of engineered Escherichia coli expressing the β-eucalyptol synthase CcTPS6 gene after codon optimization and site-directed mutagenesis in Example 4.

[0037] Figure 5 This is a graph showing the effect of β-eucalyptol on the lifespan of wild-type Caenorhabditis elegans in Example 5.

[0038] Figure 6 This is a graph showing the effect of β-eucalyptol on the stress resistance of Caenorhabditis elegans in Example 5.

[0039] Figure 7 This is a diagram showing the effect of β-eucalyptol on the reproductive capacity of Caenorhabditis elegans in Example 5.

[0040] Figure 8 This is a diagram showing the effect of β-eucalyptol on lipofuscin accumulation in *C. elegans* in Example 5.

[0041] Figure 9 The figure shows the effect of β-eucalyptol on reactive oxygen species accumulation in Caenorhabditis elegans in Example 5.

[0042] Figure 10 This is a graph showing the effect of β-eucalyptol on lipid accumulation in *C. elegans* in Example 5.

[0043] Figure 11 The graph shows the effects of β-cineole on body length development, pharyngeal pump frequency, and head twitching frequency in Example 5.

[0044] Figure 12 This is a diagram showing the effect of β-eucalyptol on the metabolism of *C. elegans* in Example 5.

[0045] Figure 13 This is a graph showing the effect of β-eucalyptol on the growth curve of E. coli OP50 in Example 5.

[0046] Figure 14 This is a graph showing the effect of heat-inactivated E. coli OP50 on the lifespan of Caenorhabditis elegans in Example 5.

[0047] Figure 15 This is a graph showing the effect of β-eucalyptol on the lifespan of the loss-of-function mutant *C. elegans* in Example 5.

[0048] Figure 16 This is a diagram showing the effect of β-eucalyptol on the nuclear translocation of DAF-16 in Example 5.

[0049] Figure 17 The diagram shows the effects of β-eucalyptol on the skn-1, hsp-16.2 and gst-4 genes in Example 5.

[0050] Figure 18 This is a diagram showing the effect of β-eucalyptol on aging-related genes in Example 5.

[0051] Figure 19 This is a schematic diagram of the expression plasmid structure of E. coli expressing the β-eucalyptol synthase CcTPS6 gene in Examples 2 and 3. Detailed Implementation

[0052] The following description, in conjunction with the accompanying drawings, illustrates the essential aspects of the present invention through embodiments, but is not intended to limit the scope of the invention. Unless otherwise specified, all experimental examples were conducted under standard experimental conditions, such as those recommended in the Molecular Cloning Handbook, relevant references, or manufacturer's instructions.

[0053] Example 1

[0054] Acquisition and bioinformatics analysis of the cDNA sequence encoding the monofunctional sesquiterpene synthase CcTPS6 gene:

[0055] RNA was extracted from *C. tectorum* (a type of flower). The procedure was described in the Molecular Cloning Handbook. RNA was obtained and reverse transcribed into cDNA using the One-Step gDNA Removal and cDNA Synthesis SuperMix kit. PCR amplification was then performed using specific primer pairs (CcTPS6-F: 5'-ATGGCTGCACTTATCGCCG-3', CcTPS6-R: 5'-TTAAATCTCTATTTGATCGACGAGC-3') to obtain the full-length cDNA sequence of the CcTPS6 gene (e.g., Seq ID No. 2).

[0056] The open reading frame (ORF) of the gene encoding the monofunctional sesquiterpene synthase CcTPS6 is 1650 bp (Seq ID No. 2), encoding 549 amino acids (Seq ID No. 1), with a molecular weight of 63 kDa. The amino acid sequence encoded by this monofunctional sesquiterpene synthase CcTPS6 gene contains typical motifs [RxR], [DDxx(D,E)], and [Rxx(N,D)Dxx(S,T,G)xxxE]. Homology searches of the gene encoding the monofunctional sesquiterpene synthase CcTPS6 were performed using BLASTN in NCBI. Nucleotide-level analysis and alignment showed 80% homology with the sesquiterpene / diterpene synthase LcTPS2 from the Lamiaceae plant *Leucosceptrum canum*.

[0057] Example 2

[0058] Construction of the expression vector for the β-eucalyptol synthase CcTPS6 encoding gene:

[0059] RNA was extracted from *Torchbearer* using the Trizol method. The procedure was described in the Invitrogen Trizol kit instructions. RNA was obtained and analyzed using SMART... TMUsing the 5'-CDS primer in the RACE cDNA Amplification Kit as a template for reverse transcription to synthesize cDNA, and the aforementioned CcTPS6-F and CcTPS6-R primers, PCR amplification was performed using the high-fidelity enzyme PrimeSTAR HSDNA Polymerase. The PCR system consisted of 50 μL, and the reaction program was as follows: 10 μL 5×PrimeSTAR HS Buffer, 4 μL dNTP Mixture (2.5 mM each), 1 μL Primer F, 1 μL Primer R, 0.5 μL Template cDNA, 0.5 μL PrimeSTAR HS DNA Polymerase, and deionized water to a final volume of 50 μL. The PCR program was: 98℃ for 10 sec, 60℃ for 15 sec, 72℃ for 2 min, for 35 cycles. After the program, the band size was detected by 1% agarose gel electrophoresis, and the product was recovered and purified. Both the purified product and the pCold-TF vector were digested with restriction endonucleases Kpn I and Pst I, reacted at 37°C for 3 h, and the band size was detected by 1% agarose gel electrophoresis. The product was then recovered and purified. The digested PCR product was ligated into the pCold-TF vector, i.e., the cDNA encoding the β-eucalyptol synthase CcTPS6 gene was cloned into the pCold-TF expression vector containing an N-terminus HIS tag. The vector was transformed into E. coli DH5α, plated on LB agar plates supplemented with ampicillin (100 μg / mL) for screening, and incubated overnight at 37°C until single colonies grew. Single colonies were picked for PCR and restriction enzyme digestion verification, and positive clones were selected for DNA sequencing verification.

[0060] Example 3

[0061] Heterologous expression of β-eucalyptol synthase CcTPS6 in Escherichia coli:

[0062] The recombinant pCold-TF / CcTPS6 plasmid and the pBbA5c-MevT-MBIS plasmid were co-transformed into Escherichia coli BL21(DE3). Transformed colonies were screened using LB agar plates containing ampicillin (100 μg / mL) and chloramphenicol (34 μg / mL). Single colonies were picked for verification. Verified single colonies were inoculated into 500 mL of TB medium containing the same antibiotics and cultured at 37°C with shaking until OD (occurrence limit). 600The value was approximately 0.6. 0.3 mM IPTG was added, and the mixture was induced at 16℃ for 18 h. The temperature was then adjusted to 25℃, and the mixture was cultured for another 3 days. Extraction was performed with petroleum ether, and the organic layer was collected and concentrated to 1 mL under reduced pressure to obtain the in vivo enzyme activity reaction sample. The sample was analyzed using GC-MS. GC-MS chromatographic conditions: HP-5MS quartz capillary (30 m × 250 μm × 0.25 μm); column temperature program: initial temperature 80℃, hold for 2 min; increase to 220℃ at 15℃ / min, hold for 0 min; then increase to 270℃ at 4℃ / min, hold for 2 min; splitless injection mode, injection volume 5 μL, carrier gas helium, helium flow rate 3 mL / min, column inlet pressure 40 kPa. GC-MS mass spectrometry conditions: EI ion source, ion source temperature 250℃, electron energy 70 eV, mass range 35-550 amu.

[0063] The petroleum ether extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate (100:0, 80:1, 70:1, 50:1) to finally obtain the compound β-eucalyptol.

[0064]

[0065] Example 4

[0066] Codon optimization and modification of the β-cineole synthase gene CcTPS6 in Example 1 were performed to increase the yield of β-cineole.

[0067] Specific methods:

[0068] Codon optimization for β-eucalyptol synthase gene CcTPS6:

[0069] The codons of the β-eucalyptol synthase gene CcTPS6 were optimized for the E. coli expression system. The optimized sequence OptCcTPS6 was constructed into the expression vector pCold-TF to obtain the recombinant plasmid pCold-TF / OptCcTPS6. This part of the work was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0070] Construction of mutant expression vectors using site-directed mutagenesis and homologous recombination techniques:

[0071] Table 1 Primers required for constructing mutant expression vectors

[0072]

[0073]

[0074] Table 2 shows the amplification of the target gene using the high-fidelity enzyme Phanta Max DNA Polymerase. The reaction system is as follows:

[0075]

[0076] Table 3 shows the above system after vortexing and centrifugation. The target gene is then amplified according to the procedure in the table below:

[0077]

[0078] After the PCR reaction is complete, the DNA fragments are detected by 1.5% agarose gel electrophoresis and the corresponding bands are excised for recovery.

[0079] Table 4 shows the homologous recombination reaction system as follows:

[0080]

[0081] Mix all components thoroughly, incubate at 37°C for 30 min, then incubate on ice for 3 min. Finally, transform the system into E. coli DH5α competent cells for colony PCR verification and sequencing.

[0082] Heterologous expression of the mutant in E. coli:

[0083] Plasmids pCold-TF / OptCcTPS6, C271S, A292S, F305A, and F513A were co-transformed with pBbA5c-MevT-MBIS into *E. coli* strain BL21(DE3). Transformed colonies were screened using LB agar plates containing ampicillin (100 μg / mL) and chloramphenicol (34 μg / mL). Single colonies were picked for verification. Verified single colonies were inoculated into 500 mL of TB medium containing the same antibiotics and cultured at 37°C with shaking until OD (occurrence limit). 600 The value was approximately 0.6. 0.3 mM IPTG was added, and the mixture was induced at 16°C for 18 h. The temperature was then adjusted to 25°C, and the mixture was cultured for another 3 days. The mixture was extracted with petroleum ether, and the organic layer was collected. The mixture was then concentrated to 1 mL by vacuum evaporation to obtain the in vivo enzyme activity reaction sample. The sample was analyzed and detected by GC-MS. The detection conditions are described in Example 3.

[0084] Example 5

[0085] Specific methods for evaluating the anti-aging activity of compound β-cineole in Example 3 using *C. elegans*: Effect of β-cineole on the lifespan of wild-type *C. elegans*:

[0086] Lifespan experiments were conducted using wild-type *Caenorhabditis elegans*, loss-of-function mutant nematodes daf-2, daf-16, and skn-1, and NGM solid medium. 50 mM Met·HCl was used as a positive control, 0.1% DMSO as a blank control, and 200 μM β-eucalyptol was administered to the treatment group. Synchronized L1-stage nematodes were transferred to culture dishes in each group. Two days later, the nematodes entered the L4 stage and were transferred to new culture dishes using a platinum wire spatula, 20 nematodes / dish, 6 dishes / group, and incubated at a constant temperature of 20℃. This was day 0 of the lifespan experiment, denoted as day 0 (d 0), and the nematodes at this stage were referred to as generation P0 nematodes. After the L4 stage, the adult nematodes entered the reproductive stage. During this period, generation P0 nematodes were transferred to new culture dishes daily. After 8 days, the nematodes were transferred to new dishes every other day. The survival, death, and removal of nematodes were observed and recorded daily until all nematodes died. The criterion for determining nematode mortality was the absence of response when the nematode's head was touched with a platinum wire shovel. Exclusion criteria included: accidental death, escape (climbing and drying to death), cysticercosis, and internal incubation. Each group had three replicates, and data were analyzed using GraphPad Prism 7.

[0087] Effects of β-Cineole on the stress resistance of Caenorhabditis elegans:

[0088] Oxidative stress.

[0089] The experiment consisted of a blank control group (0.1% DMSO) and an experimental group (200 μM β-eucalyptol). L1 stage nematodes were transferred to different culture dishes and grown at a constant temperature of 20℃, denoted as day 0. After 3 days, 30 L4 stage nematodes from each group were selected and transferred to a new plate containing 200 μM juglone and grown at a constant temperature of 20℃. The survival of the nematodes was recorded every 1 hour until all nematodes died. The methods for confirming death and removing nematodes were the same as those used in the lifespan experiment, with 3 replicates per group.

[0090] Heat stress

[0091] The experiment consisted of a blank control group (0.1% DMSO) and an experimental group (200 μM β-eucalyptol). L1 stage nematodes were transferred to different culture dishes and grown at a constant temperature of 20℃, denoted as day 0. After 3 days, 30 L4 stage nematodes from each group were selected and placed into corresponding new plates, which were then placed in an incubator at 35℃. The survival status of the nematodes was recorded every 1 hour until all nematodes died. The methods for confirming death and removing nematodes were the same as those used in the lifespan experiment, with 3 replicates per group.

[0092] Effects of β-Cineole on the reproductive capacity of Caenorhabditis elegans:

[0093] Synchronized L1-stage nematodes were transferred to NGM plates containing 200 μM β-eucalyptol or 0.1% DMSO and incubated at 20°C for 60 h. Then, the nematodes were transferred. During transfer, 10 nematodes were placed in each new plate (6 plates per group) and incubated at 20°C. Parental nematodes were transferred to new plates every 24 h until all nematodes ceased oviposition. The plates containing progeny were then incubated in an incubator until L4 stage nematodes were reached, with three replicates per group.

[0094] Effects of β-Cephalophyllin on lipofuscin accumulation in Caenorhabditis elegans.

[0095] The experiment was divided into a blank control group (0.1% DMSO) and experimental groups (100, 150, 200, 250, and 300 μM β-eucalyptol). Synchronized L1-stage nematodes were transferred to different culture dishes and grown at a constant temperature of 20℃, denoted as day 0. After 10 days, the nematodes in each group were washed with M9 Buffer, anesthetized with levamisole solution (25 mM, 3 min), centrifuged, washed with M9 Buffer, and then transferred to laser confocal culture dishes for observation and imaging using a laser scanning confocal microscope. The excitation wavelength was set to 340 nm and the emission wavelength to 430 nm. The autofluorescence intensity of lipofuscin in the whole nematode in the images was analyzed by LAS X. Each group contained at least 60 nematodes.

[0096] Effects of β-Cineole on reactive oxygen species accumulation in Caenorhabditis elegans:

[0097] The experiment consisted of a blank control group (0.1% DMSO) and an experimental group. The experimental group was treated with five concentrations of β-eucalyptol (100, 150, 200, 250, and 300 μM). L1 stage nematodes were transferred to different culture dishes and grown at a constant temperature of 20℃, denoted as day 0. Five days after administration, the nematodes in each group were washed with M9 Buffer, removing most of the larvae through natural sedimentation. This larval washing process was repeated at least three times to obtain approximately 50 μM adult nematodes. The concentration was then supplemented with M9 Buffer to approximately 250 μM. The probe DCFH-DA was added under light-protected conditions, with a final concentration of approximately 50 μM. The nematodes were incubated at 20℃ in the dark for approximately 50 min. Afterward, the nematodes were washed, anesthetized with levamisole solution, and transferred to laser confocal microscopy culture dishes. Observation and photography were performed using a laser confocal microscope with an excitation wavelength of 488 nm, an emission wavelength of 525 nm, and a FITC fluorescence source. Each group contained at least 90 nematodes.

[0098] Effects of β-Cineole on lipid accumulation in *C. elegans*:

[0099] Synchronized L1-stage nematodes were transferred to NGM containing 200 μM β-eucalyptol or 0.1% DMSO and cultured at 20°C for 60 h. The nematodes were collected and washed with 1×PBS buffer. They were then fixed with 1% paraformaldehyde solution at 4°C for 1 h. The nematodes were subjected to two freeze-thaw cycles in liquid nitrogen-ice bath-water bath sequence, centrifuged to collect the nematodes, and the supernatant was discarded. The nematodes were washed three times with PBS. 60% isopropanol (diluted with distilled water) was added, and the mixture was allowed to stand at room temperature for 15 min before centrifugation, discarding the supernatant. The nematodes were stained with Oil Red O staining solution at room temperature in the dark for 4 h. After staining, the staining solution was washed off with PBS. The nematodes were then placed on a glass slide for observation and photographing using a stereomicroscope. The staining degree of the entire nematode was quantified using ImageJ. Three replicates were performed per group, with at least 60 nematodes randomly selected from each replicate for observation.

[0100] Effects of β-Cineole on body length development, pharyngeal pump frequency, and head twitching frequency in Caenorhabditis elegans:

[0101] Synchronized L1-stage N2 nematodes were transferred to NGM culture dishes in the blank control group and the drug-treated group, respectively, and cultured at 20°C for 60 h. The nematodes in each group were washed with M9 Buffer, transferred to blank NGM culture dishes, and labeled accordingly. The nematodes were observed under a stereomicroscope, and the pharyngeal pump frequency and head twitching frequency of the nematodes within 30 seconds were counted manually using a counter. At least 90 nematodes were observed in each group. Subsequently, the nematodes were anesthetized with levamisole hydrochloride solution (25 mM, 3 min), observed and photographed under a stereomicroscope, with at least 90 nematodes in each group. The body length of the nematodes in each group was counted using ImageJ.

[0102] Effects of β-Cineole on the metabolism of Caenorhabditis elegans:

[0103] Synchronized L1-stage nematodes were transferred to NGM plates containing 200 μM β-eucalyptol or 0.1% DMSO and cultured at 20°C for 60 h. Each group of nematodes was washed with M9 buffer, and approximately 400 μL of N2 nematode bodies were retained. 1 mL of methanol was added to each replicate, followed by sonication, centrifugation (12000 rpm, 10 min), and collection of the supernatant. The precipitates were extracted by sonication with 500 μL of acetone and petroleum ether for 30 min, respectively. The supernatants were combined, concentrated under nitrogen, and then reconstituted with 1 mL of methanol. Metabolites were analyzed by LC-MS, with three replicates per group.

[0104] Effects of β-Cineole on the growth curve of E. coli OP50:

[0105] Take E. coli OP50 glycerol bacillus, streak it onto antibiotic-free LB solid agar medium, invert it and incubate overnight at 37°C. The next day, pick a single colony and inoculate it into 100 mL of LB liquid medium, then incubate at 37°C on a shaker until OD reaches 100%.595 After reaching a concentration close to 0.55, 10 mL was taken out and diluted 1000 times for later use. Using the diluted bacterial solution, E. coli OP50 suspensions containing different components were prepared. In this experiment, a suspension containing 0.1% DMSO was used as a blank control, a suspension containing 50 mM metformin hydrochloride (Met·HCl) was used as a positive control, and a suspension containing β-eucalyptol was used as the experimental group. The concentrations of β-eucalyptol were set at 100, 150, 200, 250, and 300 μM, respectively. The suspensions were cultured in 96-well plates, with 5 replicates per group. OD was measured every hour. 595 And plot the growth curve.

[0106] Effects of heat-inactivated E. coli OP50 on the lifespan of Caenorhabditis elegans.

[0107] At a drug concentration of 200 μM, with 0.1% DMSO as a blank control, wild-type *C. elegans* were fed with normal and heat-inactivated (121℃, 15 min) *E. coli* OP50, respectively. Synchronized L1-stage nematodes were transferred to culture dishes in each group. Two days later, the nematodes entered the L4 stage and were transferred to new culture dishes using a platinum wire spatula, 20 nematodes / dish, 6 dishes / group, and cultured at a constant temperature of 20℃. This was day 0 of the lifespan experiment, denoted as day 0 (d 0). The nematodes at this stage are referred to as P0 generation nematodes. After the L4 stage, the adult nematodes enter the reproductive stage. During this period, P0 generation nematodes were transferred to new culture dishes daily. After 8 days, the nematodes were transferred to new dishes every other day. The survival, death, and removal of nematodes were observed and recorded daily until all nematodes died. The criterion for judging nematode death was the absence of reaction when the head of the nematode was touched by the platinum wire spatula. Exclusion criteria included: accidental death, escape (climbing and drying to death), cysticercosis, and internal incubation. Each group had three replicates, and data were analyzed using GraphPad Prism 7.

[0108] Effects of β-Cineole on the genes daf-16, skn-1, hsp-16.2 and gst-4

[0109] The effects of β-eucalyptol on the genes daf-16, skn-1, hsp-16.2, and gst-4 were detected using green fluorescent protein (GFP) fusion strains of nematodes TG356 (daf-16::GFP), LD1008 (skn-1::GFP), TG375 (hsp-16.2::GFP), and CL2166 (gst-4::GFP). The experiment included a blank control group and a drug-treated group. L1-stage synchronized nematodes were transferred to culture dishes containing 100 μM 5-FUDR, denoted as d0. After 5 days of incubation at a constant temperature, the nematodes were washed with M9 buffer and collected. They were then transferred to clean glass slides, anesthetized with levamisole hydrochloride, covered with coverslips, and observed and photographed using a laser confocal scanning microscope. The excitation wavelength was 488 nm, and the emission wavelength was 500-550 nm. Each group contained at least 60 nematodes, and the experiment was repeated three times. daf-16 has three distribution states: cytoplasm, intercytoplasm / nucleus, and nucleus. If a nematode has more than 20 nuclear fluorescent spots, it is in the nuclear state; fewer than 20 nuclear fluorescent spots indicate the intercytoplasmic state; and no nuclear fluorescent spots indicate the cytoplasmic state. Except for TG356, which uses nuclear fluorescent spots to determine daf-16 expression, other nematode species use fluorescence intensity as the criterion. The fluorescence intensity of each group of nematodes was statistically analyzed using LAS X software, and the fluorescence intensity of the treated group relative to the blank control group was calculated.

[0110] Effects of β-Cineole on the expression of aging-related genes:

[0111] Synchronized L1-stage nematodes were transferred to NGM (100 μM, 5-FUDR) plates containing 200 μM β-eucalyptol or 0.1% DMSO, denoted as day 0. After 6 days, the nematodes were washed with M9 buffer, collected from each group, and transferred to RNase-free centrifuge tubes. Total RNA was extracted from *C. elegans* according to the instructions of the Trizol Total RNA Extraction Reagent from Acori. The One-Step gDNARemoval and cDNASynthesis SuperMix kit was used for reverse transcription. The total system volume was 20 μL. The required components and volumes are shown in the table below.

[0112] Table 5 shows the components and volumes required for reverse transcription.

[0113]

[0114] Add the corresponding components to a 200 μL Nase-free centrifuge tube according to the table above (operate on ice), mix and centrifuge, and then perform the PCR reaction. The reaction procedure is as follows:

[0115] Table 6 shows the PCR reaction procedure as follows:

[0116]

[0117] Real-time quantitative PCR:

[0118] According to 2× Follow the instructions for the Master qPCR Mix (SYBR Green I) kit. The 20 μL qRT-PCR reaction mixture is shown in Table 7 below.

[0119] Table 7 20 μL qRT-PCR reaction system

[0120]

[0121] Add each component to a 96-well PCR plate according to the table above. All operations should be performed on ice. The ABI7500 Real-Time PCR System qRT-PCR program settings are shown in Table 8 below (two-step reaction program).

[0122] Table 8 Two-step reaction procedure

[0123]

[0124] Actin-1 was used as an internal reference gene. The primers for the target gene are shown in the table below. (Using 2...) -ΔΔCt The relative expression levels of each gene's mRNA were calculated using this method.

[0125] Table 9 Primers required for qRT-PCR

[0126]

[0127] Pharmaceutical Formulation Examples 1-8:

[0128] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. This application example only demonstrates that the compound β-eudesminol described in this invention can be prepared into different formulations, and does not specifically limit the specific reagents and operations:

[0129] 1. The compound β-eucalyptol is dissolved in anhydrous ethanol, and then water for injection is added according to conventional methods. After fine filtration, the solution is filled, sealed, and sterilized to prepare an injection solution with a concentration of 0.5-5 mg / mL.

[0130] 2. The compound β-eucalyptol was dissolved in dimethyl sulfoxide, then dissolved in sterile water for injection. The solution was stirred until dissolved, filtered through a sterile vacuum funnel, then sterilely filtered again, dispensed into ampoules, freeze-dried at low temperature, and then sterilely sealed to obtain a powder for injection.

[0131] 3. Add compound β-eucalyptol to the excipient at a mass ratio of 9:1 to prepare a powder.

[0132] 4. Add compound β-eucalyptol to the excipient at a mass ratio of 5:1, then granulate and compress into tablets.

[0133] 5. The compound β-eucalyptol was prepared into an oral liquid using conventional oral liquid preparation methods.

[0134] 6. Add compound β-eucalyptol to the excipient at a mass ratio of 5:1 to make capsules.

[0135] 7. Add compound β-eucalyptol to the excipient at a mass ratio of 5:1 to prepare granules.

[0136] 8. Capsules: β-Cephalophyllin 20mg, lactose 180mg, magnesium stearate 5mg.

[0137] Preparation method: Mix the compound with a co-solvent until homogeneous, sieve, and fill the resulting mixture into gelatin capsules. Each capsule weighs 205 mg and contains 20 mg of active ingredient.

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] This invention is not limited to the specific textual description above. It should be understood that after reading the above content of this invention, any modifications or alterations made by those skilled in the art based on this invention are also equivalent forms that fall within the scope defined by the appended claims.

Claims

1. β-Cineole synthase CcTPS6, characterized in that... It is: Proteins consisting of the amino acid sequence shown in Seq ID No. 1, and recombinant proteins that retain the same function after a substitution of one amino acid residue in their amino acid sequence; the amino acid residue substitutions include: Phe (F) at position 305 being replaced by Ala (A) and Ala (A) at position 292 being replaced by Ser (S).

2. The gene encoding β-eucalyptol synthase CcTPS6 according to claim 1, characterized in that... It is: The nucleotide sequence shown in Seq ID No. 2, and the nucleotide sequence in which the encoded protein undergoes amino acid substitution due to the substitution of nucleotides in a specific codon, but still maintains the same expression function; the amino acid substitution is selected from one of F305A and A292S.

3. A recombinant vector containing the encoding gene of β-eucalyptol synthase CcTPS6 as described in claim 2.

4. An expression cassette containing the β-eucalyptol synthase CcTPS6 encoding gene as described in claim 2.

5. A transgenic cell line containing the β-eucalyptol synthase CcTPS6 encoding gene as described in claim 2.

6. A recombinant bacterium containing the β-eucalyptol synthase CcTPS6 encoding gene as described in claim 2.

7. The application of the encoding gene of β-cineole synthase CcTPS6 as described in claim 1 or β-cineole synthase CcTPS6 as described in claim 2 in the preparation of recombinant vectors, expression cassettes, transgenic cell lines, and recombinant bacteria containing the encoding gene of β-cineole synthase CcTPS6.

8. The application of the gene encoding β-cineole synthase CcTPS6 according to claim 1 or β-cineole synthase CcTPS6 according to claim 2 in the preparation of fermentation broth containing the compound β-cineole, characterized in that... The application involves: constructing an expression vector containing the gene encoded therein, transforming the recombinant vector into Escherichia coli, and fermenting the obtained genetically engineered bacteria to obtain a fermentation broth containing β-eucalyptol.

9. A method for preparing compound β-eucalyptol, characterized in that... The method includes the following steps: An expression vector containing the gene encoding β-cineole synthase CcTPS6 was constructed. β-cineole synthase CcTPS6 is a protein with the amino acid sequence shown in Seq ID No. 1, and the encoding gene is the nucleotide sequence shown in Seq ID No.

2. The recombinant vector was transformed into *E. coli*, and the resulting genetically engineered bacteria were fermented to obtain a fermentation broth containing β-cineole. The broth was extracted with petroleum ether or ethyl acetate to obtain an extract containing β-cineole. The extract was then purified by silica gel column chromatography to finally obtain the compound β-cineole, as shown in the following structural formula. 。 10. The use of the gene encoding β-eucalyptol synthase CcTPS6 as described in claim 1 or β-eucalyptol synthase CcTPS6 as described in claim 2 in the preparation of the compound β-eucalyptol.

11. The use of the gene encoding β-eucalyptol synthase CcTPS6 as described in claim 1 or β-eucalyptol synthase CcTPS6 as described in claim 2 in the preparation of anti-aging products.

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