AU2021207155A1

AU2021207155A1 - High-yield eurotium cristatum strain containing diketopiperazine dimer, and method for separating and purifying diketopiperazine dimer therefrom

Info

Publication number: AU2021207155A1
Application number: AU2021207155A
Authority: AU
Inventors: Daidi Fan; Junfeng HUI; Gang Liu; Hui Zhang; Chenhui ZHU
Original assignee: North West University
Current assignee: Northwest University
Priority date: 2020-01-15
Filing date: 2021-01-15
Publication date: 2022-09-08
Also published as: CN111235036A; WO2021143833A1; CN111235036B

Abstract

Provided are a high-yield eurotium cristatum strain containing a diketopiperazine dimer, a method for separating and purifying the diketopiperazine dimer therefrom, and a use of a diketopiperazine dimer in the preparation of a hypoglycemic drug.

Description

FE00325AU

HIGH-YIELD EUROTIUM CRISTATUM STRAIN CONTAINING DIKETOPIPERAZINE DIMER, AND METHOD FOR SEPARATING AND PURIFYING DIKETOPIPERAZINE DIMER THEREFROM

This patent application claims the priority of Chinese patent application 202010039961.9 (application date: January 15, 2020, title of invention: an Eurotium cristatum and a method for separating and purifying a diketopiperazine dimer therefrom).

FIELD OF THE INVENTION The invention belongs to the technical field of biomedicine, and in particular relates to an Eurotium cristatum strain with high-level diketopiperazine dimers, a method for separating and purifying the diketopiperazine dimer therefrom, and the use of the diketopiperazine dimer in the preparation of a hypoglycemic medicament.

STATE OF THE ART Fu brick tea has a long history in China, from the original hand-made tea to the modem machine-made tea, from the "official tea" that was only provided to special groups in the Qing Dynasty to the current side-sale tea, from the special product that can only be produced in Shaanxi to the tea that can now be produced in the whole country, but the fact that Fu brick tea is made by the process of "flowering" remains unchanged. Therefore, Fu brick tea is unique among all kinds of tea drinks. Fu brick tea can not only supplement some special substances needed by the human body, but also can reduce the blood lipids and blood sugar in the human body , and have the effect of losing weight. Studies have shown that the dominant microorganism in Fu brick tea is Eurotium cristatum, and the"Golden Flower" is actually the yellow closed capsule produced by this fungus. Literature (Phytochemistry Letters, 2012, 5, 717-720) reported that a new compound named diketopiperazine dimer was isolated, which was derived from Eurotium cristatum KUFC7356 on marine sponges. The colonies were light orange as a whole, and were irregularly radially circular (as shown in Figure 1). However, the diketopiperazine dimer has not been further studied in the literature. The basic structure of diketopiperazines (DKPs) is a cyclic dipeptide (DKP) formed by the condensation of two amino acids, since its framework has a stable six membered ring structure, and possesses two hydrogen bond donors and two hydrogen bond receptors, this makes DKPs an important pharmacophore in medicinal chemistry, and their research also lays the foundation for the research of polypeptide chemistry. At present, a large number of active DKPs have been found in sponge symbiotic microorganisms, but few DKPs have been found in Eurotium cristatum derived from Fu brick tea.

SUMMARY OF INVENTION The inventor's research found that the diketopiperazine dimer can also be produced by Eurotium cristatum derived from Fu brick tea, and it has a significant regulating effect on sugar metabolism, but currently, the preparation method of the diketopiperazines dimer in Eurotium cristatum derived from Fu brick tea has not been reported in the literature. Therefore, it is of great value to prepare diketopiperazine dimer by modem separation and purification methods and apply it to the study of various pharmacological activities of monomer compounds of the diketopiperazine dimer. The technical problem to be solved by the present invention is to provide an Eurotium cristatum in view of the above-mentioned deficiencies of the prior art. The strain is derived from Fu brick tea, which is ecologically safe, has highm biologicaleffects such as hypoglycemic and lipid-lowering activities, and can be used as a strain with high activity and high production of secondary metabolites. In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is: an Eurotium cristatum, characterized in that, the Eurotium cristatum is Eurotium cristatum XD-05, deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 19024, a deposit date of November 22, 2019, a preservation institution of the China General Microbiological Culture Collection Center, address: Beichen West Road 3-1, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. 1. The strain has the following properties: Morphological characteristics: Inoculating it on a PDA solid plate, and after culturing it at 28°C for 24h, white velvety colonies can be seen, the diameter of the colony can reach 15mm in 48h, and the hyphae at the center of the colony begin to turn yellow. At 72h, the whole colony is bright yellow and the diameter can reach 45-50mm. Continue to cultivate until 144h, the central part of the colony has black-brown exudate, the whole colony is brown, and there is also brown pigment on the back of the medium, and the diameter of the colony is 50-55mm. The shape of the colony inoculated into the Czapek medium is substantially the same as that in the PDA plate, but the growth is slower, and the final colony diameter is also small, at 35-40mm. Physiological and biochemical characteristics: This strain has strong aerobic properties and poor shear resistance, when the stirring speed exceeds 300 rpm, the hyphae are easy to break and the growth becomes slow. The pH range for growth is 4.0-7.0 with an optimum pH of 6.0. The highest growth temperature is 38°C, and the optimum growth temperature is 28°C. The strain can utilize a variety of carbon sources, but the optimal carbon source is sucrose; it can utilize a variety of nitrogen sources, the inorganic nitrogen source is not preferred, and yeast powder is the optimal organic nitrogen source.

Further, the present invention also provides a method for separating and purifying a diketopiperazine dimer from the above-mentioned Eurotium cristatum, characterized in that it

comprises the following steps:

Step 1, preparing Eurotium cristatum spores; Step 2, extracting the Eurotium cristatum spores prepared in step 1 to obtain a spore extract;

Step 3, adsorbing the spore extract in step 2 using a macroporous adsorption resin, and then

carrying out gradient elution to collect the eluent of a target fraction; Step 4, separating and purifying the eluent of the target fraction collected in step 3 using

reversed-phase high performance liquid chromatography, collecting the eluent with the largest

chromatographic peak, concentrating and then freeze-drying the collected eluent to obtain the diketopiperazine dimer.

The above-mentioned method is characterized in that in step 1, the preparation method of the

Eurotium cristatum spores is as follows: inoculating the Eurotium cristatum on a PDA plate to culture for 5 to 7 days, picking out the Eurotium cristatum spores, formulating the picked Eurotium

cristatum spores into a spore suspension, inoculating the spore suspension on the PDA plate, and

collecting golden Eurotium cristatum spores after 5 -7 days. The above-mentioned method is characterized in that, in step 2, the solvent used for extraction

is one or more selected from water, ethanol, ethyl acetate, acetone and diethyl ether, and a ratio of

the volume of the solvent to the mass of the Eurotium cristatum spores is (10-100):1, wherein the unit of the volume is mL and the unit of the mass is g.

The above-mentioned method is characterized in that the solvent used in the extraction is a

mixed solution of ethanol and water. The above-mentioned method is characterized in that the volume percentage of ethanol in the

mixed solution of ethanol and water is 50%.

The above-mentioned method is characterized in that the type of the macroporous adsorption resin described in step 3 is S-8, AB-8, D-101, X-5, NRR or D-3520.

The above-mentioned method is characterized in that the eluent used in the gradient elution in step 3 is an aqueous ethanol solution. The above-mentioned method is characterized in that in step 4, an XDB-C18 reversed-phase chromatographic column is used for the reversed-phase high-performance liquid chromatography, and the mobile phase is an aqueous ethanol solution with a volume percent concentration of 25% to %.

The structural formula of the diketopiperazine dimer obtained by the separation and

purification of the present invention is shown in the following formula:

0 H ~H HNN

O -- 12O

8 9w 'N1io 4NH H H 0 19

18 Compared with the prior art, the present invention has the following advantages:

1. The Eurotium cristatum strain of the present invention is derived from Fu brick tea, the

strain is ecologically safe, and the strain has a high yield of diketopiperazine dimer. 2. The Eurotium cristatum strain of the present invention has high biological activity, and has

obvious active effects such as hypoglycemic and lipid-loweringactivities, and can be used as a

strain with high activity and high production of secondary metabolites. 3. The present invention is the first to separate and purify the diketopiperazine dimer from Fu

brick tea-derived Eurotium cristatum, the separation and purification method is simple and efficient,

and the purity of the isolated and purified diketopiperazine dimer is over 98%. 4. The solvent used in the separation and purification process of the present invention is cheap

and easy to obtain, and the purification process is simple, economical and efficient, and has high

industrial application value.

DESCRIPTION OF THE FIGURES

Figure 1 is a morphological diagram of the colony of Eurotium cristatum on marine sponges.

Figure 2 is a morphological diagram of Eurotium cristatum XD-05 of the present invention.

Figure 3 is the ESI/MS spectrum (negative ion) spectrum of the diketopiperazine dimer separated and purified in Example 2 of the present invention.

Figure 4 is the ESI/MS spectrum (positive ion) spectrum of the diketopiperazine dimer

separated and purified in Example 2 of the present invention. Figure 5 is the 1H NMR spectrum of the diketopiperazine dimer separated and purified in

Example 2 of the present invention.

Figure 6 is the 13C NMR spectrum of the diketopiperazine dimer separated and purified in Example 2 of the present invention.

Figure 7 is the high performance liquid chromatogram of the diketopiperazine dimer separated

and purified in Example 2 of the present invention. Figure 8 is a graph showing the effect of the isolated and purified diketopiperazine dimer on

cell activity.

Figure 9 is a graph showing the effect of the isolated and purified diketopiperazine dimer of the present invention on the glucose uptake of Hep-G2 cells induced by high sugar and insulin.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Screening, mutagenesis and identification of the Eurotium cristatum strain

Screening of strains:

Step 1. Enrichment: 1Og of Fu brick tea into powder was ground and made into a suspension using physiological saline. The method of dilution spreading plate method was used to dilute the above

suspension by 10 times in a gradient, and spread the plate. Large and bright yellow colonies were

picked out as Eurotium cristatum spores; Step 2. Primary screening and purification: the picked spores of Eurotium cristatum were made

into a spore suspension with a spore viability of xI107 cfu/mL. 800 L of the suspension was

inoculated on a PDA plate for culture, and incubated in a constant temperature incubator at 28°C for days to collect golden spores;

Step 3; Re-screening: the spores obtained in the previous step were streaked on a plate containing

PDA to continue to screen for 8 generations, cultured on a PDA slant, and stored at 4°C. Mutagenesis of strains:

(1) Preparation of a spore fluid

The strain obtained by re-screening on a PDA plate is inoculated, and cultured to the golden

spore-producing stage (about 120h).The spores are washed with sterile saline to obtain a crude

spore suspension, and the crude spore suspension is filtered with sterile lens-wiping paper to obtain

the spore fluid. Spore Mutagenesis

The prepared spore solution is diluted to three concentrations of 107 CFU/ml, 106 CFU/ml and

105 CFU/ml respectively, placed under ultraviolet light, irradiated for 10 min, 30 min and 60 min, and the 80% lethal irradiation time at each concentration is determined. Continuous UV

mutagenesis is performed for 80% of the lethal irradiation time corresponding to each

concentration,. The irradiated spores are coated on a PDA solid-state plate in the dark, and after culturing in the dark for 96 h, the colony size is used as an index to screen the colonies with a

diameter of more than 140% of the original colonies as mutagens.

The mutagen is inoculated on a PDA plate and cultured to the golden spore stage, the spores are collected, and the ethanol aqueous extract with a percentage content of 50% is added at a solid

liquid ratio of 1:20 (w/v), and ultrasonically treated for 15 minutes. The extraction is repeated 3

times to obtain a spore extract, which is injected into high performance liquid chromatography. The peak time corresponding to the standard sample of diketopiperazine dimer as the screening time,

and the peak area is taken as the index to screen the strain with the largest peak area.

A total of 1456 mutagen strains are obtained through ultraviolet mutagenesis, and the diketopiperazine dimer production of these strains is determined by liquid chromatography, and the

strain with the highest production of diketopiperazine dimer is obtained, numbered as XD-05.

Compared with the Eurotium cristatum before mutagenesis, this strain has a increased yield of diketopiperazine dimer of 1.5 times that of the original strain.

Identification of the XD-05 strain: (1) Morphological identification:

It is inoculated on a PDA solid plate, and after culturing it at 28°C for 24h, white velvety

colonies can be seen. The diameter of the colony can reach 15mm at 48h, and the hyphae at the center of the colony begin to turn yellow. The whole colony is bright yellow at 72h, and the

diameter can reach 45mm. Continue to cultivate until 144h with black-brown exudate in the center

of the colony, as shown in Figure 2, the colony is bright yellow as a whole, the center of the colony is dark-brown, and has a more regular circular structure and is denser. There is also brown pigment

on the back of the medium, and the colony has a diameter of 50mm. The shape of the colony inoculated into the Czapek medium is substantially the same as that in the PDA plate, but the growth is slower, and the final colony diameter is also small of 35mm.

(2) Physiological and biochemical identification:

The strain has strong aerobic properties, and the optimum pH is 6.0. The highest growth temperature was 38°C, and the optimum growth temperature was 28°C. The strain can utilize a

variety of carbon sources, but the optimal carbon source is sucrose; it can utilize a variety of

nitrogen sources, the inorganic nitrogen source is not preferred, and yeast powder is the optimal organic nitrogen source.

(3) Amplification and sequencing of 18SrDNA:

The identification of the strain was completed by the Chinese Perservation and Administration Center for Industrial Microorganism.

The identification process of strains includes strain DNA extraction, PCR amplification, gel

electrophoresis, purification and recovery, ligation, preparation of competent cells, transformation of ligation products, plasmid extraction and sequencing, and the purified 18SrDNA sequences are

compared on the ribosome database; the primer sequences used by PCR amplification are:

Upstream primer: 5'-TCCGTAGGTGAACCTGCGG-3' Downstream primer: 5'-TCCGTAGGTGAACCTGCGG-3'

Reaction system of the PCR amplification

reagent Volume (pL)

Template (genome DNA 20-50ng/pL) 0.5

xbuffer (with Mg2+) 2.5

Dntp (respectively 2.5mM) 1

enzyme 0.2

F (10pL) 0.5

R (10OpL) 0.5

Add double distilled water until 25

18SrDNA sequence analysis and comparison of the strain: the fragment size of the PCR amplified product is 519bp, and the 18SrDNA partial gene sequence (SEQ ID NO. 3) obtained by

sequencing is 100% homologous to the related fragment sequence of Eurotium cristatum published

in GenBank. Based on the results of physiological and biochemical identification and 18S rDNA sequence

analysis and identification, the above-mentioned strain XD-05 is identified as Eurotium cristatum, which is deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 19024, the deposit date is November 22, 2019, and the address of the preservation institution is the Institute of Microbiology, Chinese Academy of Sciences, Beichen

West Road 1-3, Chaoyang District, Beijing, China.

Example 2 Separation and purification of the diketopiperazine dimer from Eurotium

cristatum XD-05 Step 1. Eurotium cristatum is inoculated on a PDA plate to culture for 5 days, and golden Eurotium

cristatum spores are collected; the picked Eurotium cristatum spores are made into a spore

suspension with the spore viability of1x10cfu/mL, 800 L of the suspension is inoculated on a PDA plate, and the golden Eurotium cristatum spores are collected after 6 days;

Step 2,50g of the Eurotium cristatum spores collected in step 1 is placed in a1-liter beaker, 1000

mL of the aqueous ethanol extract is added with a volume percentage of 50% according to a solid to-liquid ratio of 1:20 (w/v), and ultrasonically treated for 15 minutes. The extraction is repeated 3

times to obtain a spore extract;

Step 3: the spore extract in Step 2 is adsorbed on D-101 (also can be replaced by S-8, AB-8, X-5, NRR or D-3520) macroporous adsorption resin, and then 30% aqueous ethanol solution is added to

elute for 5 column volumes, and then 60% aqueous ethanol solution is added to elute for 5 column

volumes. The 60% aqueous ethanol elute is concentrated under reduced pressure at a temperature of °C and a vacuum of 0.8 MPa to obtain a crude diketopiperazine dimer;

Step 4: using an XDB-C18 reversed-phase chromatographic column with a pore size of 10 m and a

size of 20x250 mm to separate and purify the eluent of target fraction collected in step 3, the column temperature is 30°C, and the mobile phase is an aqueous ethanol solution with a volume

percent concentration of 35%, and the flow rate is 25mL/min. The eluent with the largest

chromatographic peak is collected, the collected eluent is concentrated under reduced pressure under the condition that the temperature is 55°C and the vacuum degree is 0.8MPa, and freeze

drying is performed to obtain 250mg diketopiperazine dimer with a purity of 98.8%.

ESI/MS spectral test is carried out on the diketopiperazine dimer separated and purified in this example, and the results are shown in Figure 3 and Figure 4.

(A) M+H: m/z 591.2757, (B) M+Na: m/z 603.1984, as can be seen from the figure, the ESI/MS spectrum (negative ion) of the sample shows that m/z 567. 2249 and the ESI/MS spectrum of the test sample [M+H] m/z 567.2720 are consistent; the ESI/MS spectrum of the sample (positive

ion) shows that m/z 591.2757 and the ESI/MS spectrum of the test sample [M+ Na]m/z 591.2696 are consistent.

The diketopiperazine dimer isolated and purified in this example is subjected to NMR spectral

analysis (DMSO, 600MHz), the results are shown in Figure 5 and Figure 6. Figure 5 is the 1H

NMR spectrum, Figure 6 is the 13C NMR spectrum, and the 1H NMR spectrum graph shows a total of 18 proton signals, the same as the number of protons in the sample to be measured. The 13C

NMR spectrum shows a total of 16 effective monomer peaks, including 8 tertiary carbon peaks, 1

secondary carbon peak, 5 quaternary carbon peaks and 2 primary carbon peaks. The diketopiperazine dimer separated and purified in this example was analyzed by high

performance liquid chromatography. The results are shown in Figure 7, and the liquid phase purity

showed that the purity was 98.8%.

Example 3 Separation and purification of the diketopiperazine dimer from Eurotium

cristatum XD-05 Step 1. Eurotium cristatum is inoculated on a PDA plate to culture for 7 days, and golden Eurotium

suspension with the spore viability of1x10cfu/mL, 800 L of the suspension is inoculated on a PDA plate, and the golden Eurotium cristatum spores are collected after 5 days;

Step 2: 50g of the Eurotium cristatum spores collected in step 1 is placed in a 1-liter beaker, 1000

mL of the aqueous ethanol extract is added with a volume percentage of 60% according to the solid to-liquid ratio of 1:20 (w/v), and ultrasonically treated for 20 minutes. The extraction is repeated 3

times to obtain a spore extract;

Step 3: the spore extract in Step 2 is adsorbed on D-101 (also can be replaced by S-8, AB-8, X-5, NRR or D-3520) macroporous adsorption resin, and then 40% aqueous ethanol solution is added to

elute for 5 column volumes, and then 70% aqueous ethanol solution is added to elute for 5 column

volumes. The 70% aqueous ethanol eluate is concentrated under reduced pressure at a temperature of 60°C and a vacuum of 0.8 MPa to obtain a crude diketopiperazine dimer;

size of 20 x 250 mm to separate and purify the eluent of target fraction collected in step 3, the column temperature is 30°C, and the mobile phase is an aqueous ethanol solution with a volume

percent concentration of 25%, the flow rate is 20mL/min, the eluent with the largest

chromatographic peak is collected, and the collected eluent is concentrated under reduced pressure under the condition that the temperature is 50°C and the vacuum degree is 0.7MPa, and the freeze

drying is performed to obtain 230mg diketopiperazine dimer with a purity of 98.4%.

Example 4 Separation and purification of diketopiperazine dimer from Eurotium cristatum

XD-05

Step 1. Eurotium cristatum is inoculated on a PDA plate to culture for 6 days, and golden Eurotium

suspension with the spore viability of1x10cfu/mL, 800 L of the suspension is inoculated on a PDA plate, and the golden Eurotium cristatum spores are collected after 7 days;

Step 2, the Eurotium cristatum spores collected in step 1 are extracted to obtain a spore extract; the

solvent used in the extraction is one or more selected from water, ethanol, ethyl acetate, acetone and ether, preferably a mixed solution of ethanol and water, wherein the volume percentage of ethanol

in the mixed solution of ethanol and water is 50%, and the ratio of the volume of the solvent to the

mass of Eurotium cristatum spores is 100:1, wherein the unit of volume is mL and the unit of mass is g;

Step 3: the spore extract described in Step 2 is adsorbed on D-101 (also can be replaced by S-8,

AB-8, X-5, NRR or D-3520) macroporous adsorption resin, and then 20% aqueous ethanol solution is added to elute for 5 column volumes, and then 70% aqueous ethanol solution is added to elute 5

column volumes. The 50% aqueous ethanol eluent is concentrated under reduced pressure at a

temperature of 60°C and a vacuum of 0.8 MPa to obtain a crude diketopiperazine dimer; Step 4: using an XDB-C18 reversed-phase chromatographic column with a pore size of 10 m and a

size of 20x250 mm to separate and purify the eluent of target fraction collected in step 3, the

column temperature is 30°C, and the mobile phase is an aqueous ethanol solution with a volume percent concentration of 35%, and the flow rate is 30mL/min. The eluent with the largest

chromatographic peak is collected, the collected eluent is concentrated under reduced pressure

under the condition that the temperature is 60°C and the vacuum degree is 0.8MPa, and the freeze drying is performed to obtain 250mg diketopiperazine dimer with a purity of 98.2%.

Example 5 Separation and purification of diketopiperazine dimer from Eurotium cristatum XD-05

cristatum spores are collected; the picked Eurotium cristatum spores are made into a spore suspension with the spore viability of1x10cfu/mL, 800 L of the suspension is inoculated on a

PDA plate, and the golden Eurotium cristatum spores are collected after 7 days;

Step 2, the Eurotium cristatum spores collected in step 1 are extracted to obtain a spore extraction

solution; the solvent used for extraction is one or more selected from water, ethanol, ethyl acetate,

acetone and ether, preferably a mixed solution of ethanol and water, wherein the volume percentage

of ethanol in the mixed solution of ethanol and water is 50%, the ratio of the volume of the solvent to the mass of Eurotium cristatum spores is 50:1, and the unit of volume is mL, the unit of mass is g;

Step 3: the spore extract described in Step 2 is adsorbed on AB-8 (also S-8, D-101, X-5, NRR or D

3520 can be used instead) macroporous adsorption resin, and then 40% aqueous ethanol solution is added to elute for 5 column volumes, and then 70% aqueous ethanol solution is added to elute for 5

column volumes, the 70% aqueous ethanol eluate is concentrated under reduced pressure at a

size of 20 x 250 mm to separate and purify the eluent of target fraction collected in step 3, the

column temperature is 30°C, and the mobile phase is an aqueous ethanol solution with a volume percent concentration of 45%, and the flow rate is 25mL/min. The eluent with the largest

under the condition that the temperature is 60°C and the vacuum degree is 0.8MPa, and the freeze drying is perforemed to obtain 230mg diketopiperazine dimer with a purity of 98.5%.

Example 6 Separation and purification of diketopiperazine dimer from Eurotium cristatum XD-05

Step 1, Eurotium cristatum is inoculated on a PDA plate to culture for 6 days, and golden Eurotium

PDA plate, and the golden Eurotium cristatum spores are collected after 7 days;

Step 2, the Eurotium cristatum spores collected in step 1 are collected to obtain a spore extraction solution; the solvent used for extraction is one or more selected from water, ethanol, ethyl acetate,

of ethanol in the mixed solution of ethanol and water is 50%, the ratio of the volume of the solvent to the mass of the Eurotium cristatum spores is 10:1, and the unit of volume is mL, the unit of mass

is g;

Step 3: the spore extract described in Step 2 is adsorbed on S-8 (also D-101, AB-8, X-5, NRR or D 3520 can be used instead) macroporous adsorption resin, and then 40% aqueous ethanol solution is

added to elute for 5 column volumes, and then 70% aqueous ethanol solution is added to elute for 5 column volumes. The 70% ethanol water eluate is concentrated under reduced pressure at a temperature of 60°C and a vacuum of 0.8 MPa to obtain a crude diketopiperazine dimer;

percent concentration of 35%, the flow rate is 20mL/min. The eluent with the largest

chromatographic peak is collected, and the collected eluent is concentrated under reduced pressure at a temperature of 60°C and a vacuum of 0.8 MPa, and the freeze-drying is performed to obtain

240 mg diketopiperazine dimer with a purity of 98.7%.

Example 7 In vitro hypoglycemic experiment using diketopiperazine dimer separated and

purified from Eurotium cristatum XD-05

Experimental cells: Hep-G2 human hepatoma cells are purchased from Shanghai Bogu Biotechnology Co., Ltd., and the number of generations is within 10.

Test method and results:

Effects of diketopiperazine dimer on glucose consumption in high-sugar induced Hep-G2 insulin resistant cells

Human hepatoma cells Hep-G2 are seeded at 10,000 /well in a 96-well plate, with 100 L of

culture medium per well. After 24h, the culture medium is changed to serum-free high-sugar DMEM medium containing different concentrations of diketopiperazine dimer, 0.5mM palmitic

acid, 0.5%BSA, and lnM insulin. In the experiment a cell-free blank group (cell-free, serum-free

low-sugar DMEM medium containing 0.5% BSA, and lnM insulin), a control group (serum-free low-sugar DMEM medium containing 0.5% BSA, and lnM insulin), a model group (serum-free

high-sugar DMEM medium containing 0.5mM Palmitic acid, 0.5% BSA, and 1 nM insulin) and a

positive medicament groups (serum-free high-sugar DMEM medium containing 1 mM metformin, 0.5 mM palmitic acid, 0.5% BSA, and 1 nM insulin) are set. After 24 hours of incubation, 2 L of

supernatant is taken from each well, and the glucose consumption in each well is measured by a

glucose assay kit (glucose oxidase-peroxidase method). The culture medium in the 96-well plate is removed, and the absorbance value of each well is measured by MTT colorimetry at a wavelength

of 570 nM.

The glucose consumption in each well is obtained by subtracting the glucose content in the test wells seeded with cells from the glucose content in the cell-free blank group. At the same time, the

cell number is corrected by dividing by the MTT value of cells in each well. The experimental results show that the diketopiperazine dimer has no toxic and side effects on insulin-resistant Hep G2 cells at doses of 15 M and 30 M. As shown in Figure 8 and Figure 9, both 15 M and 30 [M of diketopiperazine dimer significantly promote the glucose consumption of insulin-resistant Hep

G2 cells, and the high dose is more effective than the low dose in view of glucose consumption. This indicates that the diketopiperazine dimer separated and purified from the strain XD-05 of

Eurotium cristatum has obvious hypoglycemic activity, and the strain can be used as a strain with

high activity and high production of secondary metabolites. The above are only preferred embodiments of the present invention, and do not limit the

present invention. Any simple modifications, changes and equivalent structural changes made to the

above embodiments according to the technical essence of the invention fall within the protection scope of the technical solution of the present invention.

Industrial applicability According to the present invention, provided are a strain of Eurotium cristatum with high yield

of diketopiperazine dimer and a method for separating and purifying diketopiperazine dimer

therefrom, and the use of the diketopiperazine dimer in the preparation of hypoglycemic medicaments..

Claims

CLAIMS 1. An Eurotium cristatum, characterized in that, the Eurotium cristatum is Eurotium cristatum

XD-05, deposited in China General Microbiological Culture Collection Center with a deposit

number of CGMCC No.19024.

2. A method for separating and purifying a diketopiperazine dimer from the Eurotium

cristatum according to claim 1, comprising the following steps:

Step 1, preparing Eurotium cristatum spores;

Step 2, extracting the Eurotium cristatum spores prepared in step 1 to obtain a spore extract;

Step 3, adsorbing the spore extract in step 2 using a macroporous adsorption resin, and then

carrying out gradient elution to collect the eluent of a target fraction;

Step 4, separating and purifying the eluent of the target fraction collected in step 3 using a

reversed-phase high performance liquid chromatography, collecting the eluent with the largest

chromatographic peak, concentrating and then freeze-drying the collected eluent to obtain the

diketopiperazine dimer.

3. The method according to claim 2, characterized in that, the preparation method of the

Eurotium cristatum spores in step 1 is:

inoculating the Eurotium cristatum on a PDA plate to culture for 5 to 7 days, picking the

Eurotium cristatum spores, formulating the picked Eurotium cristatum spores into a spore

suspension, inoculating the spore suspension on the PDA plate, and collecting golden Eurotium

cristatum spores after 5 -7 days.

4. The method according to claim 2, characterized in that, in step 2, the solvent used for

extraction is one or more selected from water, ethanol, ethyl acetate, acetone and ether, and a ratio

of the volume of the solvent to the mass of the Eurotium cristatum spores is (10-100):1, wherein the

unit of volume is mL and the unit of mass is g.

5. The method according to claim 3, characterized in that, the solvent used for extraction is a

mixed solution of ethanol and water.

6. The method according to claim 4, characterized in that, the volume percentage of ethanol in

the mixed solution of ethanol and water is 50%.

7. The method according to claim 2, characterized in that, the type of the macroporous

adsorbent resin in step 3 is S-8, AB-8, D-101, X-5, NRR or D-3520.

8. The method according to claim 2, characterized in that, the eluent used in the gradient elution in step 3 is an aqueous ethanol solution.

9. The method according to claim 2, characterized in that, an XDB-C18 reversed-phase

chromatographic column is used for the reversed-phase high-performance liquid chromatography in

step 4, and the mobile phase is an aqueous ethanol solution with a volume percent concentration of

% to 45%.

10. Use of a diketopiperazine dimer in the preparation of a hypoglycemic medicament.

FE00325AU 1/5

Drawings

Figure 1

Figure 2

1005 96.7