Use of a purple mold DQWM-G4 and a microbial agent thereof in preparation of 1-(beta-D-ribofuranosyl)-1H-1,2,4-triazole
The fermentation and purification method of Porphyromonas DQWM-G4 and its inoculant has filled the technological gap in the preparation of 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole by Porphyromonas species, realizing an efficient and environmentally friendly preparation process with high yield and easy industrialization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QUJING NORMAL UNIV
- Filing Date
- 2023-03-31
- Publication Date
- 2026-08-04
AI Technical Summary
There are currently no reports on the use of *Porphyra* species for the preparation of triazole compounds, especially for 1-(β-D-ribofurano)-1H-1,2,4-triazole, which lacks an effective method.
1-(β-D-ribofuranosyl)-1H-1,2,4-triazole was prepared by culturing *Porphyromonas purpureus* DQWM-G4 and its inoculum in PDB medium, followed by fermentation, ultrasonic extraction, and column chromatography purification. The specific steps included inoculation into rice medium for fermentation, soaking in methanol solution, ultrasonic extraction, and column chromatography separation.
A simple, green, pollution-free, and easily industrialized preparation method is provided, which yields high output under mild conditions and successfully prepares 1-(β-D-ribofuranoside)-1H-1,2,4-triazole.
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Figure CN116751684B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and particularly relates to the application of a purple mold DQWM-G4 and its inoculum in the preparation of 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole. Background Technology
[0002] Nucleoside compounds generally refer to substances composed of a base and a sugar. After phosphorylation, nucleosides participate in various cellular processes such as DNA and RNA synthesis, cell signaling, enzyme regulation, and metabolism. They can inhibit cell division and viral transmission, thus often exhibiting antitumor and antiviral effects. Triazoles are an important class of five-membered heterocyclic compounds containing three nitrogen atoms, existing in two isomers: 1,2,3-triazole and 1,2,4-triazole. 1,2,4-triazole derivatives generally possess pharmacological activities such as anticancer, antiviral, antihypertensive, antidiabetic, and antituberculosis effects, and are widely used in biomedicine and materials science. 1,2,4-triazole nucleosides typically have antiviral activity. Ribavirin (1-β-D-ribofurano-1,2,4-triazole-3-hydroxyamide) was the first reported small molecule drug of the 1,2,4-triazole nucleoside class, showing good inhibitory effects against more than ten viruses, including hepatitis C virus and respiratory syncytial virus. 1-(β-D-ribofuranoside)-1H-1,2,4-triazole is a 1,2,4-triazole nucleoside compound that is mainly found in peppermint, seaweed, and sponge Callyspongia sp.
[0003] Microbial metabolism produces a wealth of metabolites, including primary and secondary metabolites. Secondary metabolites often have complex structures, including a large number of bioactive compounds. The technology of obtaining bioactive substances through microbial fermentation plays a vital role in food, medicine, fine chemicals, bioenergy, biomaterials, and biomass resource utilization, making a remarkable contribution to the national economy.
[0004] *Purpureocillium* sp. was established as a new genus in 2011, separating from *Paecilomyces*. Its colonies are pale purple. For example, *Purpureocillium lilacinum*, also known as *Paecilomyces lilacinus*, is an important nematode-eating fungus. Its metabolites have good control effects on most parasitic nematodes and can also promote plant growth. Currently, more than ten pesticide products formulated with *Purpureocillium lilacinum* have been approved and registered in my country. However, there are currently no reports of using *Purpureocillium* species for the preparation of triazole compounds. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide the application of *Porphyra yezoensis* DQWM-G4 and its inoculant in the preparation of 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a *Purpureocillium* sp. DQWM-G4, with accession number CCTCCNO:M 2023331.
[0008] The present invention also provides a fungal agent prepared from the above-mentioned *Porphyra yezoensis* DQWM-G4.
[0009] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising: inoculating the *Porphyra yezoensis* DQWM-G4 into PDB medium and culturing it to obtain the microbial agent.
[0010] Preferably, the culture conditions are: temperature 26-30℃, shaking speed 110-160 rpm, and time 3-7 days.
[0011] The present invention also provides the application of the above-mentioned *Porphyra yezoensis* DQWM-G4 or the above-mentioned fungal agent in the preparation of 1-(β-D-ribofuranoside)-1H-1,2,4-triazole.
[0012] The present invention also provides a method for preparing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole, comprising: inoculating the above-mentioned bacterial agent into rice culture medium for fermentation culture to obtain fermentation product, extracting it by ultrasonication to obtain crude extract, and separating and purifying it by chromatography column to obtain the 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0013] Preferably, the inoculum amount of the microbial agent is 0.1-0.3 mL / g.
[0014] Preferably, the fermentation conditions are: temperature of 25-32℃ and time of 30-60 days.
[0015] Preferably, before ultrasonic extraction, the fermentation product is soaked in a methanol solution; the volume percentage of the methanol solution is 90%-100%, and the soaking time is 10-15 hours.
[0016] Preferably, the conditions for ultrasonic extraction are: power of 230-250W, frequency of 30-50KHz, temperature of 35-45℃, extraction 2-5 times, and extraction time of 30min / time.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] This invention provides a *Porphyra yezoensis* strain DQWM-G4, which can be used to prepare 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole. The method for preparing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole according to this invention is simple, green, pollution-free, and operates under mild conditions, making it easy to industrialize. Furthermore, the yield of 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole prepared by this invention is high.
[0019] Biological Preservation Instructions
[0020] The *Purpureocillium* sp. DQWM-G4 described in this invention, classified as a fungus of the genus *Purpureocillium*, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2023331, deposited on March 15, 2023, at the Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Attached Figure Description
[0021] Figure 1 Compound I of Example 3 13 C NMR and DEPT spectra;
[0022] Figure 2 Compound I of Example 3 1 H NMR spectrum. Detailed Implementation
[0023] This invention provides a *Purpureocillium* sp. DQWM-G4, with accession number CCTCCNO:M 2023331.
[0024] The *Purpureocillium* sp. described in this invention was isolated from the tuberous root of *Euonymus japonicus* and identified as a *Purpureocillium* sp. by ITS sequence analysis. *Purpureocillium* sp. DQWM-G4 was deposited at the China Center for Type Culture Collection on March 15, 2023.
[0025] The present invention also provides a fungal agent prepared from the above-mentioned *Porphyra yezoensis* DQWM-G4.
[0026] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising: inoculating the *Porphyra yezoensis* DQWM-G4 into PDB medium and culturing it to obtain the microbial agent.
[0027] In this invention, the preferred culture conditions are: temperature 26-30℃, shaking speed 110-160 rpm, and time 3-7 days; more preferably, temperature 28℃, shaking speed 140 rpm, and time 5 days. Before obtaining the inoculum, the process preferably includes an activation step of *Porphyra yezoensis* DQWM-G4: inoculating *Porphyra yezoensis* DQWM-G4 onto PDA medium and culturing at a constant temperature of 28℃ for 3-7 days. Preferably, after activation, the present invention immediately performs a seed culture step, or the activated *Porphyra yezoensis* DQWM-G4 is stored at 4℃ for later use.
[0028] The present invention also provides the application of the above-mentioned *Porphyra yezoensis* DQWM-G4 or the above-mentioned fungal agent in the preparation of 1-(β-D-ribofuranoside)-1H-1,2,4-triazole.
[0029] The structure of 1-(β-D-ribofurano)-1H-1,2,4-triazole described in this invention is as follows:
[0030]
[0031] The present invention also provides a method for preparing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole, comprising: inoculating the above-mentioned bacterial agent into rice culture medium for fermentation culture to obtain fermentation product, extracting it by ultrasonication to obtain crude extract, and separating and purifying it by chromatography column to obtain the 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0032] In this invention, the method for preparing the rice culture medium preferably includes: mixing rice with water and then sterilizing it with high-pressure steam; the weight ratio of rice to water is 1:(2-4), more preferably 1:3; the sterilization temperature is preferably 118-125℃, more preferably 121℃; and the sterilization time is preferably 15-25 min, more preferably 20 min.
[0033] In this invention, the inoculum amount of the microbial agent is preferably 0.1-0.3 mL / g, more preferably 0.2 mL / g; the fermentation conditions are preferably: temperature 25-32℃, time 30-60 days, more preferably: temperature 28℃, time 40 days. The fermentation described in this invention is solid-state fermentation.
[0034] In this invention, the process of ultrasonic extraction preferably includes soaking the fermentation product in a methanol solution; the volume percentage of the methanol solution is preferably 90%-100%, more preferably 100%, and the soaking time is preferably 10-15 hours, more preferably 12 hours.
[0035] The preferred conditions for ultrasonic extraction according to this invention are: power of 230-250W, frequency of 30-50KHz, temperature of 35-45℃, extraction 2-5 times, time 30min / extraction; more preferably, power of 240W, frequency of 40KHz, temperature of 40℃, extraction 3 times, time 30min / extraction. After ultrasonication, the fermentation product is preferably filtered, and the filtrate is evaporated to dryness using a rotary evaporator; the evaporation temperature is preferably 40-60℃, more preferably 50℃.
[0036] After evaporation, the crude extract is preferably further purified by column chromatography. The preferred purification step is as follows: the crude extract is dissolved and spotted onto a GF254 TLC plate, developed with dichloromethane-methanol-acetic acid (5:1:0.1), and examined under a 254 nm UV lamp. A dark spot is observed at Rf0.7. The crude extract is then separated using an open-tube flash chromatography column (200-300 mesh silica gel), with a gradient elution of dichloromethane-methanol-acetic acid (10:1:0.2 → 10:2:0.2). TLC monitoring is used to detect the Rf0.7 spot in the dichloromethane-methanol-acetic acid (10:2:0.2) eluent. This spot is combined and evaporated to obtain fraction I. Fraction I is dissolved in methanol and separated by Sephadex LH-20 gel column chromatography to obtain compound I. The compound is then purified by 13C chromatography. NMR and 1H NMR identified compound I as 1-(β-D-ribofurano)-1H-1,2,4-triazole.
[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0038] Example 1
[0039] Obtaining strains
[0040] Screening: Endophytic fungi (including DQWM-G4 and other strains) isolated from the tuberous roots of Euonymus japonicus were fermented in rice culture medium, extracted with methanol, and analyzed by TLC (dichloromethane-methanol (3:1) development). It was found that DQWM-G4 had a clear chromatographic spot at Rf0.8, while other fungi did not have corresponding chromatographic spots at the same position.
[0041] DQWM-G4 was obtained through screening and identified as a *Purpureocillium* sp. by ITS sequence analysis.
[0042] Example 2
[0043] Preparation of *Porphyra yezoensis* DQWM-G4 inoculant
[0044] (1) The purple mold DQWM-G4 was inoculated onto potato dextrose agar (PDA) medium for activation. After being cultured in a constant temperature incubator at 28℃ for 5 days, the activated strain was obtained and stored in a refrigerator at 4℃ for later use.
[0045] (2) The activated strain was inoculated into potato dextrose (PDB) seed culture medium and cultured in a constant temperature shaker at 28℃ for 5 days with a shaking speed of 140 rpm to obtain the purple mold DQWM-G4 inoculum.
[0046] Example 3
[0047] Preparation of 1-(β-D-ribofurano)-1H-1,2,4-triazole
[0048] (1) Weigh 15g of rice, add 45mL of water, sterilize at 121℃ for 20min, and cool to use as fermentation medium.
[0049] (2) The obtained 3 mL of the bacterial agent from Example 2 was inoculated into the above fermentation medium and fermented for 45 days at 28°C to obtain a fermentation product containing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0050] (3) The fermentation product was soaked in 50 mL of 100% methanol solution for 12 h. Under the conditions of ultrasonic power of 240 W, ultrasonic frequency of 40 K Hz and ultrasonic temperature of 40 ° C, it was ultrasonically extracted 3 times for 30 min each time. The product was filtered under normal pressure, the filtrates were combined, and the crude extract was obtained by evaporation at 50 ° C using a rotary evaporator.
[0051] (4) The crude extract was dissolved and spotted onto a GF254 TLC plate. It was developed with dichloromethane-methanol-acetic acid (5:1:0.1). After development, it was examined under a 254 nm UV lamp, and a dark spot was observed at Rf0.7. The substances in the crude extract were separated using an open-tube flash chromatography column (200-300 mesh silica gel). Gradient elution was performed with dichloromethane-methanol-acetic acid (10:1:0.2→10:2:0.2). TLC was used for monitoring, and a spot at Rf0.7 was detected in the dichloromethane-methanol-acetic acid (10:2:0.2) eluent. This spot was combined and evaporated to dryness to obtain fraction I. Fraction I was dissolved in methanol and separated by Sephadex LH-20 gel column chromatography to obtain compound I.
[0052] 25 mg of the compound obtained by weighing was analyzed by 13C NMR (see [reference]). Figure 1 ) and 1H NMR (see Figure 2It was identified as 1-(β-D-ribofurano)-1H-1,2,4-triazole.
[0053] Example 4
[0054] (1) Weigh 15g of rice, add 45mL of water, sterilize at 121℃ for 20min, and cool to use as fermentation medium.
[0055] (2) The obtained 3 mL seed culture solution was inoculated into the above fermentation medium and fermented for 30 days at 28℃ to obtain the fermentation product containing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0056] (3) The fermentation product was soaked in 50 mL of 100% methanol solution for 12 h. Under the conditions of ultrasonic power of 240 W, ultrasonic frequency of 40 K Hz and ultrasonic temperature of 40 ° C, it was ultrasonically extracted 3 times for 30 min each time. The product was filtered under normal pressure, the filtrates were combined, and the crude extract was obtained by evaporation at 50 ° C using a rotary evaporator.
[0057] (4) The crude extract was dissolved and spotted onto a GF254 TLC plate. It was developed with dichloromethane-methanol-acetic acid (5:1:0.1). After development, it was examined under a 254 nm UV lamp, and a dark spot was observed at Rf0.7. The substances in the crude extract were separated using an open-tube flash chromatography column (200-300 mesh silica gel). Gradient elution was performed with dichloromethane-methanol-acetic acid (10:1:0.2→10:2:0.2). TLC was used for monitoring, and a spot at Rf0.7 was detected in the dichloromethane-methanol-acetic acid (10:2:0.2) eluent. This spot was combined and evaporated to dryness to obtain fraction I. Fraction I was dissolved in methanol and separated by Sephadex LH-20 gel column chromatography to obtain compound I.
[0058] The compound obtained by weighing 18 mg was analyzed by TLC thin-layer chromatography with dichloromethane-methanol-acetic acid (5:1:0.1) and examined under a 254 nm UV lamp. The spot position and Rf value were the same as those of the compound obtained in Example 3, and it was identified as 1-(β-D-rifuranose)-1H-1,2,4-triazole.
[0059] Example 5
[0060] (1) Weigh 15g of rice, add 45mL of water, sterilize at 121℃ for 20min, and cool to use as fermentation medium.
[0061] (2) The obtained 2.5 mL seed culture solution was inoculated into the above fermentation medium and fermented for 50 days at 25 °C to obtain a fermentation product containing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
[0062] (3) The fermentation product was soaked in 50 mL of 100% methanol solution for 12 h. Under the conditions of ultrasonic power of 240 W, ultrasonic frequency of 40 K Hz and ultrasonic temperature of 40 ° C, it was ultrasonically extracted 3 times for 30 min each time. The product was filtered under normal pressure, the filtrates were combined, and the crude extract was obtained by evaporation at 50 ° C using a rotary evaporator.
[0063] (4) The crude extract was dissolved and spotted onto a GF254 TLC plate. It was developed with dichloromethane-methanol-acetic acid (5:1:0.1). After development, it was examined under a 254 nm UV lamp, and a dark spot was observed at Rf0.7. The substances in the crude extract were separated using an open-tube flash chromatography column (200-300 mesh silica gel). Gradient elution was performed with dichloromethane-methanol-acetic acid (10:1:0.2→10:2:0.2). TLC was used for monitoring, and a spot at Rf0.7 was detected in the dichloromethane-methanol-acetic acid (10:2:0.2) eluent. This spot was combined and evaporated to dryness to obtain fraction I. Fraction I was dissolved in methanol and separated by Sephadex LH-20 gel column chromatography to obtain compound I.
[0064] The compound obtained by weighing 20 mg was analyzed by TLC thin-layer chromatography with dichloromethane-methanol-acetic acid (5:1:0.1) and examined under a 254 nm UV lamp. The spot position and Rf value were the same as those of the compound obtained in Example 3, and it was identified as 1-(β-D-rifuranose)-1H-1,2,4-triazole.
[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A *Purpureocillium* sp. DQWM-G4, characterized in that, The preservation number of the purple mold DQWM-G4 is CCTCCNO:M2023331.
2. A fungal agent prepared using the *Porphyra yezoensis* DQWM-G4 as described in claim 1.
3. The method for preparing the microbial agent according to claim 2, characterized in that, include: The *Porphyra yezoensis* DQWM-G4 was inoculated into PDB medium and cultured to obtain the inoculum.
4. The preparation method according to claim 3, characterized in that, The culture conditions are: temperature 26-30℃, shaking speed 110-160 rpm, and time 3-7 days.
5. The use of the *Porphyra yezoensis* DQWM-G4 of claim 1 or the fungal agent of claim 2 in the preparation of 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole.
6. A method for preparing 1-(β-D-ribofuranoside)-1H-1,2,4-triazole, characterized in that, include: The bacterial agent described in claim 2 was inoculated into rice culture medium for fermentation to obtain fermentation products. After ultrasonic extraction, a crude extract was obtained, which was then purified by chromatography to obtain 1-(β-D-rifuranose)-1H-1,2,4-triazole.
7. The method according to claim 6, characterized in that, The inoculum concentration of the microbial agent is 0.1-0.3 mL / g.
8. The method according to claim 6, characterized in that, The fermentation conditions are: temperature 25-32℃, time 30-60 days.
9. The method according to claim 6, characterized in that, Before ultrasonic extraction, the fermentation product is soaked in a methanol solution; the methanol solution has a volume percentage of 90%-100% and the soaking time is 10-15 hours.
10. The method according to claim 6, characterized in that, The conditions for ultrasonic extraction are: power of 230-250W, frequency of 30-50KHz, temperature of 35-45℃, extraction 2-5 times, and extraction time of 30min / time.