CN110951620A

CN110951620A - Compound for resisting activity of helicobacter pylori, preparation method and application thereof

Info

Publication number: CN110951620A
Application number: CN201911102303.3A
Authority: CN
Inventors: 刘玲; 刘高然; 郭龙芳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2020-04-03
Anticipated expiration: 2039-11-12
Also published as: CN110951620B

Abstract

The invention discloses an Aspergillus (Aspergillus sp) ZJU5-1 and a method for preparing a compound thereof, and the compound has good activity on a helicobacter pylori drug-resistant strain (H.pylori BHKS 159).

Description

Compound for resisting activity of helicobacter pylori, preparation method and application thereof

Technical Field

The present invention relates to compounds having anti-helicobacter pylori activity.

Background

Helicobacter pylori is a gram-negative bacterium, is specifically planted on human gastric mucosa, is a main cause of human chronic gastritis, peptic ulcer, gastric cancer and gastric lymphoma, and the number of people who have died of gastric cancer is gradually increased every year in the world. The key point of treating stomach diseases such as gastric cancer is to kill helicobacter pylori, while drug therapy plays an important role, and an anti-tumor drug taking a natural product as a source has the advantages of obvious activity, unique structure and action mechanism different from that of a common micromolecule chemotherapy drug. Therefore, there is an urgent need to find drugs for inhibiting helicobacter pylori to treat gastric diseases such as gastric cancer.

Disclosure of Invention

The invention aims to provide a strain, which is named ZJU5-1, belongs to aspergillus (Aspergillus sp.), is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Hospital No.1 Kyoh West Chen-Lu-1 of the sunny region in Beijing city) in 11 months and 06 days in 2019, and is registered with the registration number of CGMCC No. 18829.

The second purpose of the invention is to provide two compounds with anti-helicobacter pylori activity, the structural formula of which is shown in formula I:

the third object of the present invention is to provide a process for the preparation of a compound of formula i, comprising the steps of:

fermenting Aspergillus (Aspergillus sp.) ZJU5-1 in a solid medium to obtain a fermentation product, and extracting, separating and purifying the fermentation product to obtain the compound of formula I.

The compound provided by the invention has better activity of resisting helicobacter pylori (H.pylori BHKS159), and is suitable for preparing the helicobacter pylori-resisting medicine.

Drawings

FIG. 1 shows a scheme for preparing a compound (1)¹H nuclear magnetic resonance spectrum.

FIG. 2 shows a scheme for preparing a compound (1)¹³C nuclear magnetic resonance spectrum.

FIG. 3 shows a scheme for preparing a compound (2)¹H nuclear magnetic resonance spectrum.

FIG. 4 shows a scheme for preparing a compound (2)¹³C nuclear magnetic resonance spectrum.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were obtained from a conventional biochemical reagent store. The quantitative tests in the following examples were all set up for 3 replicates and the results were averaged.

Example 1

Isolation and characterization of Strain ZJU5-1

First, collection and separation of strain ZJU5-1

A strain is separated from the sediment of the southern Atlantic in 7 months of 2012 and is named as a strain ZJU 5-1.

The strain ZJU5-1 is a marine fungus.

II, identification of strain ZJU5-1

1. Molecular identification

The partial sequence of 18S ribosomal RNA of the strain ZJU5-1 is shown in sequence 1 of the sequence table, for example, GENBANKACCESSION NO. KR611594.1 has the highest sequence similarity, and the similarity is 99.81%.

According to the above identification results, the strain ZJU5-1 belongs to Aspergillus (Aspergillus sp.).

Third, preservation of Strain ZJU5-1

The strain ZJU5-1 belongs to Aspergillus (Aspergillus sp.), and has been preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 of Xilu No.1 of Beijing, Chaoyang district) in 11.06.2019, with the preservation number of CGMCC No. 18829.

Example 2

Preparation of the Compounds

PDA culture medium: mixing potato 200g, glucose 20g, agar 15g and water 1000mL, and sterilizing with high pressure steam at 121 deg.C for 30 min.

Rice culture medium: 200g of rice was soaked in 200mL of water overnight and autoclaved at 121 ℃ for 30 min.

1 solid fermentation of Strain ZJU5-1

1.1 activation of Strain ZJU5-1, 2 cells of 3cm²The size of the clump was inoculated into a 1000mL Erlenmeyer flask containing rice medium together with the medium, and aseptically cultured at 25 ℃ for 30 days.

The strain ZJU5-1 was cultured on PDA plates for 5d, and colonies were waited for the plates to grow.

1.2 solid fermentation of Strain ZJU5-1

When colonies grew on the plate, 5 colonies of 3cm were placed²The size of the clump was inoculated into a 1000mL Erlenmeyer flask containing rice medium together with the medium, and aseptically cultured at 25 ℃ for 30 days.

2 extraction of the Compound

2.1 to the flask (containing the solid fermentation) obtained in step 1.2, 2.0L of an organic reagent (ethyl acetate: methanol ═ 4:1) was added, and the mixture was extracted at room temperature for 24 hours, and the first supernatant was collected.

2.2 to the flask of step 2.1 was added 2.0L of organic reagent (ethyl acetate: methanol ═ 4:1) again, extracted at room temperature for 24h and the second supernatant collected.

2.3 in the triangular flask of step 2.2, 2.0L of organic reagent (ethyl acetate: methanol ═ 4:1) was added again, extracted at room temperature for 24h, and the third supernatant was collected.

2.4 combine the first supernatant of step 2.1, the second supernatant of step 2.2, and the third supernatant of step 2.3, and distill to dryness under reduced pressure to obtain 11g of crude extract.

3. Separation and purification of compounds

3.1 vacuum silica gel column chromatography to obtain the eluent

And (3) carrying out reduced pressure silica gel column chromatography on the crude extract obtained in the step 2.4.

The types of the columns are as follows: 6 x 35 cm;

the fillers are: thin layer chromatography silica gel H;

the elution process is as follows: 500mL of an eluate (composed of 9.8 parts by volume of n-hexane and 0.2 part by volume of methylene chloride) was collected, and the eluate after passing through the column was collected at a flow rate of 50 mL/min.

3.2 subjecting the chromatographic eluate to gel chromatography to obtain a gel eluate

The parameters of the gel chromatographic separation are as follows:

the types of the columns are as follows: 3 × 50 cm;

the fillers are: sephadex LH-20;

the mobile phase is a mixture of 2 parts by volume of dichloromethane and 1 part by volume of methanol, and the flow rate is 0.5 mL/min;

collecting the gel eluent with the retention volume of 40-80mL after passing through the column.

3.3 reverse phase HPLC separation is carried out on the gel eluent to obtain HPLC eluent

The parameters of the reverse phase HPLC separation are as follows:

the types of the columns are as follows: eclipse XDB-C18(5 μm; 9.4X 250 mm);

the elution process is as follows: isocratic elution is carried out for 15min by using 35% acetonitrile water solution by volume percentage, gradient elution is carried out for 5min by using 35-45% acetonitrile water solution by volume percentage, gradient elution is carried out for 15min by using 45-60% acetonitrile water solution by volume percentage, and the flow rate is 3 mL/min.

The collection retention time is 5.8-6.2min (retention time of peak value t)_R6.0min) and 28.4-29.5min (retention time of peak value t)_R29.0min) of the eluate after passing through the column.

3.4 distillation under reduced pressure of the HPLC eluent collected in step 3.3 to dryness gives 3.3mg and 4.4mg of product, respectively.

4 characterization of the Compounds

And (3) carrying out organic mass spectrometry and nuclear magnetic resonance spectrum analysis on the product of the step 3.4.

The characterization data of the product are as follows:

compound (1)

A yellow solid;

the molecular formula is as follows: c₁₆H₁₂O₆(ii) a Molecular weight: 300, respectively;

¹the H nuclear magnetic resonance spectrum is shown in figure 1,¹³c nuclear magnetic resonance spectrum is shown inFIG. 2;

compound (2)

A yellow solid;

the molecular formula is as follows: c₁₆H₁₂O₇(ii) a Molecular weight: 316;

¹the H-NMR spectrum is shown in figure 3,¹³the C NMR spectrum is shown in FIG. 4;

based on the above characterization data and the physicochemical data and profile of the above compound, the product of step 3.4 is a compound of formula (I).

Example 3

A compound represented by the formula (I) has an inhibitory effect on H.pylori BHKS 159.

The minimum inhibitory concentration (MIC, 100 μ L system) of the compound represented by formula (I) against h.pylori BHKS159 was examined by microdilution.

Parallel experiments were performed with metronidazole (aladin) as a positive control for the compounds.

The bacteria used in this example were: helicobacter pylori (h. pylori BHKS 159).

The experiment comprises the following specific steps:

1. preparation of MIC plates

Adding a brain heart extract culture medium (BHI) (specific components comprise peptone, dehydrated calf brain extract powder, dehydrated calf heart extract powder, sodium chloride, glucose and disodium hydrogen phosphate, and the pH value is 7.4) to 173.6 mu L of the BHI in the 1 st hole, adding 6.4 mu L of a compound to be detected, and diluting to the 7 th hole in a multiple ratio; no addition was made in

well

8, and 90. mu.L of the medium was retained as a control with no addition of test compound.

2. Preparation of bacterial liquid

Taking helicobacter pylori (H.pyrori BHKS159) growing on a solid plate in a logarithmic phase, preparing a bacterial suspension by using a BHI culture medium, and adjusting the concentration OD₆₀₀Is 0.3 (1X 10)⁸CFU/mL), 10-fold dilution at 1X 10⁷CFU/mL, spare.

3. Inoculated bacterial liquid

Adding 10 μ L into 1-8 th well (the concentration of bacteria liquid in each well is about 1.0 × 10)⁶CFU/mL). And culturing for 72h to judge the result. The compound concentrations in wells 1 to 7 were 64, 32, 16, 8, 4, 2, 1. mu.g/mL, respectively.

4. Result judgment

The MIC was taken as the lowest test compound concentration that completely inhibited bacterial growth in the wells.

The test is meaningful when the bacteria grow significantly in the 8 th well (i.e., no antibiotic) of the positive control well.

When a single jump hole occurs in the microdilution method, the highest concentration of compound that inhibits bacterial growth should be recorded. If a plurality of jump holes appear, the result should not be reported, and the test needs to be repeated.

The bacteriostatic action of the compound shown in the formula (I) on helicobacter pylori (H.pylori BHKS159) needs to be repeated for 3 times.

MIC values of the compounds (1) and (2) represented by the formula (I) against helicobacter pylori (H.pylori BHKS159) were each 16. mu.g/mL.

The MIC value of metronidazole to helicobacter pylori (h. pylori BHKS159) was greater than 16 μ g/mL.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> compound with anti-helicobacter pylori activity, preparation method and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>532

<212>DNA

<213> Aspergillus (Aspergillus sp.)

<400>1

tccgtaggtg aacctgcgga aggatcatta ctgagtgcgg gctgcctccg ggcgcccaac 60

ctcccacccg tgaataccta acactgttgc ttcggcgggg aaccccctcg ggggcgagcc 120

gccggggact actgaacttc atgcctgaga gtgatgcagt ctgagtctga atataaaatc 180

agtcaaaact ttcaacaatg gatctcttgg ttccggcatc gatgaagaac gcagcgaact 240

gcgataagta atgtgaattg cagaattcag tgaatcatcg agtctttgaa cgcacattgc 300

gccccctggc attccggggg gcatgcctgt ccgagcgtca ttgctgccca tcaagcccgg 360

cttgtgtgtt gggtcgtcgt cccccccggg ggacgggccc gaaaggcagc ggcggcaccg 420

tgtccggtcc tcgagcgtat ggggctttgt cacccgctcg actagggccg gccgggcgcc 480

agccgacgtc tccaaccatt tcttcaggtg acctcggatc agtagatgcc ac 532

Claims

1. Aspergillus (Aspergillus sp.) ZJU5-1 CGMCC No. 18829.

2. A compound with anti-helicobacter pylori activity has a structural formula shown as the following formula:

3. a process for the preparation of a compound according to claim 2, comprising the steps of:

an activating strain ZJU 5-1;

solid fermentation strain ZJU 5-1;

leaching the solid fermentation product with ethyl acetate and methanol to obtain crude extract;

performing reduced pressure silica gel column chromatography to obtain eluate;

separating with gel chromatography to obtain gel eluate;

separating by reverse phase HPLC to obtain HPLC eluent.

4. The use of a compound of claim 2 for inhibiting helicobacter pylori.

5. The use according to claim 4, wherein the minimum concentration of the compound to inhibit bacterial growth is 16 μ g/mL.