Cordyceps sinensis cyclic peptide compound separated from cordyceps militaris and application thereof

Abstract

The invention discloses a cordyceps sinensis cyclic peptide compound separated from cordyceps militaris and application of the cordyceps sinensis cyclic peptide compound. The structural formula of the cordyceps sinensis cyclic peptide B is as shown in formula I. The preparation method of the cordyceps sinensis cyclic peptide B comprises the following steps: crushing sporocarp of cordyceps militaris, wheat grass, silkworm chrysalis infected by the cordyceps militaris or mycelium of the cordyceps militaris, and extracting by adopting a formic acid aqueous solution; carrying out centrifugal separation on the obtained leaching solution, adding resin into a concentrated extracting solution of supernate for adsorption, putting the resin for adsorbing the cordyceps sinensis cyclic peptide compound into a chromatographic column, and sequentially eluting with water and a methanol aqueous solution; the preparation method comprises the following steps: treating an eluent of methanol water to obtain a crude extract, carrying out chromatographic separation on the crude extract, eluting with methanol water, collecting effluent containing the cordyceps sinensis cyclic peptide A, carrying out separation preparation on concentrate by virtue of an HPLC (High Performance Liquid Chromatography) semi-preparative column, and carrying out isocratic elution by taking a mixed solution of methanol and a trifluoroacetic acid water solution as a mobile phase, thereby obtaining the cordyceps sinensis cyclic peptide A. The compound extracted by the method has the activity of inhibiting proliferation of cancer cells MCF-7 and SW-480, and is expected to be developed into drugs for treating tumors. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the field of edible fungi extraction and separation, and in particular to extraction and separation of cordyceps cyclic peptide compounds from cordyceps militaris, a method and application thereof. Background Art

[0002] Cordyceps militaris (L.) Fr. belongs to the phylum Ascomycota, order Hypocreales, family Cordycipitaceae, genus Cordyceps (Sung et al., 2007). It is an important edible and medicinal fungus and one of the few species in the broad sense of Cordyceps fungi that can be mass-produced. Cordyceps militaris is rich in a variety of active ingredients such as cordycepin and pentostatin, cordyceps polysaccharides, cordycepic acid and carotenoids, and has a variety of pharmacological activities such as antibacterial, antioxidant, anti-fatigue (Lee et al., 2011), anti-tumor, and immunomodulatory, and has important edible and medicinal value.

[0003] The Cordyceps militaris genome contains at least thirty secondary metabolite synthesis gene clusters, and its ability to produce natural products far exceeds people's expectations. Most natural products are in urgent need of exploration. Therefore, it is urgent to develop and prepare natural compounds synthesized by Cordyceps militaris, reveal the material basis of the effective functions of the edible and medicinal fungus Cordyceps militaris, and provide a theoretical basis for its market development and efficient utilization. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting a cyclopeptide Cordyceps militaris cyclopeptide compound from Cordyceps militaris fruiting bodies, and at the same time provide a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present invention, and the separated compound has an inhibitory effect on tumor cells.

[0005] The molecular formula of the novel Cordyceps cyclopeptide compound provided by the present invention is: 27 H 41 N5O 12 S, denoted as Cordyceps cyclopeptide B, includes structures such as N-methyl-β-hydroxytyrosine, β-hydroxyisoleucine, valine, alanine and 2-amino-4-hydroxypentanoic acid-5-sulfinyl, and its chemical structure is as follows:

[0006]

[0007]

[0008] The preparation method of Cordyceps cyclopeptide B provided by the present invention comprises the following steps:

[0009] S1. crushing the fruiting bodies of Cordyceps militaris, wheatgrass, silkworm pupae infected by Cordyceps militaris or mycelium of Cordyceps militaris, and extracting them with a formic acid aqueous solution;

[0010] S2, the extract obtained in step S1 is centrifuged to obtain a supernatant, and the supernatant is concentrated to obtain a concentrated extract;

[0011] S3, adding resin to the concentrated extract for adsorption to obtain a resin for adsorbing Cordyceps cyclic peptide compounds;

[0012] S4, placing the resin adsorbing the Cordyceps cyclic peptide compound in a chromatography column, eluting with water and methanol aqueous solution in sequence, and collecting the eluate of the methanol aqueous solution;

[0013] S5, the eluate of the methanol aqueous solution is treated to obtain a crude extract, the crude extract is subjected to chromatography separation, eluted with a methanol aqueous solution, the effluent containing Cordyceps cyclopeptide A is collected, and concentrated to obtain a concentrate;

[0014] S6. The concentrate is separated and prepared by HPLC semi-preparative column, and a mixture of methanol and trifluoroacetic acid aqueous solution is used as the mobile phase for isocratic elution to obtain the Cordyceps cyclic peptide B.

[0015] In the above-mentioned preparation method, in step S1, the powder is crushed after drying;

[0016] The particle size of the powder obtained after the pulverization is 40 to 60 meshes;

[0017] The ratio of the powder to the formic acid aqueous solution is 1 g: 5-10 mL;

[0018] The volume content of formic acid in the formic acid aqueous solution is 1 to 10%;

[0019] The extraction is performed under ultrasonic conditions, such as ultrasonic extraction in a KQ-500DE numerically controlled ultrasonic cleaner;

[0020] The extraction time is 30 to 60 minutes.

[0021] In the above preparation method, in step S2, centrifugation is performed at 6000-12000 rpm for 10-30 min, and the centrifugation can be performed in Eppendorf's Centrifuge 5810R large-capacity desktop high-speed centrifuge.

[0022] In the above preparation method, in step S3, the resin is XAD4 resin produced by Dow Chemical Company;

[0023] After adsorption, the sample was placed in a HCY-12B combined full-temperature oscillator for overnight adsorption at room temperature.

[0024] The ratio of the concentrated extract to the resin is 10-20:1.

[0025] In the above-mentioned preparation method, in step S4, the resin adsorbing the Cordyceps cyclic peptide compound is placed in the chromatography column after being sieved (such as through a 60-mesh sieve);

[0026] Use 2 to 5 column volumes of water for elution, using a natural flow rate;

[0027] The aqueous methanol solution with a volume content of 0-5% is used for elution in sequence for 5 column volumes, and then the aqueous methanol solution with a volume content of 5-15% is used for elution.

[0028] In the above preparation method, in step S5, the stationary phase used in the chromatographic separation is Sephadex LH-20;

[0029] The volume content of the methanol aqueous solution is 5-20%.

[0030] HPLC was used to monitor whether the effluent contained Cordyceps cyclopeptide A, and the detection conditions were: 0.1% trifluoroacetic acid in methanol: water, with a mobile phase ratio of 10:90.

[0031] In the above preparation method, in step S6, the volume content of the trifluoroacetic acid aqueous solution is 0.1%;

[0032] The volume ratio of the methanol to the trifluoroacetic acid aqueous solution is 5-30:70-95;

[0033] The eluent contains cordyceps cyclic peptide A and the cordyceps cyclic peptide B.

[0034] The cordyceps cyclic peptide B provided by the present invention can inhibit the proliferation of cancer cells. The present invention proves through experiments that the compound has the activity of inhibiting the proliferation of cancer cells MCF-7 and SW-480, and is expected to be used for developing drugs for treating tumors.

[0035] The present invention has the following beneficial technical effects:

[0036] (1) By adopting the method of the present invention, only four purification steps (formic acid water extraction, XAD concentration, Sephadex LH-20 separation and semi-preparative column preparation) are required to obtain Cordyceps cyclic peptide with a purity higher than 90%, and a new compound Cordyceps cyclic peptide B is obtained.

[0037] (2) The compound extracted by the method of the present invention has the activity of inhibiting the proliferation of cancer cells MCF-7 and SW-480, and is expected to be used to develop drugs for treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a high-resolution mass spectrum of the Cordyceps cyclopeptide compound Cordyceps cyclopeptide B of the present invention.

[0039] Figure 2 It is a high-resolution secondary mass spectrum (positive mode) of the cordyceps cyclic peptide compound Cordyceps cyclic peptide B of the present invention.

[0040] Figure 3 It is a high-resolution secondary mass spectrum (negative mode) of the cordyceps cyclic peptide compound Cordyceps cyclic peptide B of the present invention.

[0041] Figure 4 It is a fragment ion of the cordyceps cyclic peptide compound Cordyceps cyclic peptide B of the present invention.

[0042] Figure 5 The cordyceps cyclic peptide compound cordyceps cyclic peptide B of the present invention 1 H-NMR spectrum.

[0043] Figure 6 Cordyceps cyclopeptide compound Cordyceps cyclopeptide A 1 H-NMR spectrum.

[0044] Figure 7 The cordyceps cyclic peptide compound cordyceps cyclic peptide B of the present invention 13 C-NMR spectrum.

[0045] Figure 8 The cordyceps cyclic peptide compound cordyceps cyclic peptide B of the present invention 13 A partial enlarged view of the C-NMR spectrum.

[0046] Fig. 9 The cordyceps cyclic peptide compound cordyceps cyclic peptide B of the present invention 1 H-1HCOSY spectrum. DETAILED DESCRIPTION

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1: Isolation of Cordyceps militaris cyclic peptide B from silkworm pupae infected with Cordyceps militaris

[0050] Step 1, taking Cordyceps militaris to infect silkworm pupae to obtain dead insects, drying them and setting them aside;

[0051] Step 2: The raw materials in step 1 are ground into powder using an 800S tissue crusher and set aside;

[0052] Step 3, adding 5% formic acid water to the raw material dry powder obtained in step 2 according to a material-liquid ratio of 1:10, and ultrasonically extracting for 30 minutes using a KQ-500DE CNC ultrasonic cleaner;

[0053] Step 4: Pour the extract in step 3 into a centrifuge bucket and centrifuge it in a Centrifuge 5810R large-capacity desktop high-speed centrifuge from Eppendorf at a speed of 8000 rpm for 30 min, and take the supernatant for later use;

[0054] Step 5: Pour the supernatant obtained in step 4 into a rotary evaporator and concentrate using a RE-3000 rotary evaporator;

[0055] Step 6: Add XAD4 resin produced by Dow Chemical Company to the concentrated extract obtained in step 5, and place in HCY-12B modular full-temperature oscillator for overnight adsorption at room temperature; wherein the mass ratio of the concentrated extract to the XAD4 resin is 10:1.

[0056] Step 7: The XAD4 resin adsorbing the Cordyceps cyclic peptide compound obtained in step 6 is filtered and collected using a 60-mesh filter screen;

[0057] Step 8: The XAD4 resin adsorbing the cordyceps cyclic peptide compound obtained in step 7 was added to a normal pressure glass chromatography column (16 mm×30 cm, Henghuibio), and firstly rinsed with 5 column volumes of water at a natural flow rate;

[0058] Step 9: Add 5 column volumes of 5% formic acid water to the XAD4 resin in step 8 to elute the compound, and collect the effluent;

[0059] Step 10: Continue to elute the compound from the XAD4 resin in step 9 with 10% methanol, and collect the effluent;

[0060] The eluates from step 11, step 9 and step 10 are used to detect cordyceps cyclic peptide compounds by high performance liquid chromatography (HPLC), and the effluents containing cordyceps cyclic peptides are mixed together and evaporated to dryness by rotary evaporation to obtain a crude extract for standby use;

[0061] Step 12: The crude extract obtained in step 11 is separated by pretreated Sephadex LH-20 chromatography, eluted with 10% methanol, and detected by HPLC. The effluent containing Cordyceps cyclopeptide A is mixed together and evaporated to dryness by rotary evaporation to obtain a concentrate for use; wherein, the mobile phase used for HPLC detection is: methanol with 0.1% trifluoroacetic acid: water, the ratio is 10:90.

[0062] Step 13: The concentrate obtained in step 12 is separated and prepared by HPLC semi-preparative column, and isocratic elution is performed with methanol: 0.1% trifluoroacetic acid water in a ratio of 10:90 as the mobile phase to prepare Cordyceps cyclic peptide compounds, and finally the known Cordyceps cyclic peptide A and the new compound Cordyceps cyclic peptide B are obtained.

[0063] In the above step 3, 5% methanol water was used for extraction at the same time, and other conditions remained unchanged. The results showed that the extraction efficiency was 10% lower than that of 5% formic acid water.

[0064] In the above step 6, XAD16 resin produced by Dow Chemical Company was used for adsorption at the same time, and other conditions remained unchanged. The results showed that the adsorption rate was 30% of the adsorption rate of XAD4 resin.

[0065] The molecular formula of Cordyceps cyclopeptide B is: 27 H 41 N5O 12 S, whose chemical structure is as follows and its H NMR data are shown in Table 1.

[0066]

[0067] Table 1 H NMR data of Cordyceps cyclopeptide B

[0068]

[0069] The cordyceps cyclic peptide B separated by the present invention is a white compound insoluble in acetonitrile, and its m / z is 658.2392 ([MH] - ) and 660.2550([MH] + ,like Figure 1 shown.

[0070] In the process of preparing the new compound, the known Cordyceps cyclic peptide A was prepared. The structure of the new compound was confirmed by MS, secondary mass spectrometry, and nuclear magnetic resonance data. The identification process is as follows:

[0071] The molecular weight of cordyceps cyclic peptide B is 14 greater than that of the reported compound cordyceps cyclic peptide A, which suggests that the compound has one more methyl group than cordyceps cyclic peptide A. To confirm this speculation, the secondary mass spectrometry of the new compound, namely UPLC-MS / MS, was further measured. Figure 2 The results of the secondary mass spectrum shown in the figure show that the compound has fragment ions with m / z ratios of 88, 180, 459, 479 and 465 (negative mode) and Figure 3 The fragment ions with charge-to-mass ratios of 330, 571, and 640 in the positive mode are shown. Figure 4As shown, the fragment ion with a mass-to-charge ratio of 88 is presumed to be alanine, indicating that the glycine in Cordyceps cyclic peptide A is replaced by alanine; while the fragment ions with a mass-to-charge ratio of 330, 465, 479 and 640 all contain a methyl substitution at the C-19 position. The chemical shifts of H at the C19 position are 3.67 (d, J = 17.2, 1H) and 3.73 (d, J = 17.2, 1H), while the chemical shift of H at the C19 position of Cordyceps cyclic peptide A is δH 4.16 (q, J = 7.3Hz, 1H); in addition, Figure 5 The cordyceps cyclic peptide B 1 The H-NMR spectrum has a doublet with a chemical shift of 1.35 (d, J = 7.3 Hz, 3H) compared to cordyceps cyclic peptide A. Based on this information, it is speculated that the new compound is a methyl substitution reaction at the C19 position of cordyceps cyclic peptide A. The methyl substitution reaction at the C19 position is further confirmed by 13 C-NMR and 1 H- 1 HCOSY confirmed. Figure 7 and Figure 8 shown 13 The C-NMR spectrum results showed that the δC 44.2 signal at C19 disappeared, and was replaced by signals at chemical shifts δC 49and 15 similar to those at C6 and C24; Fig. 9 shown 1 H- 1 The HCOSY spectrum showed that δH 4.16 was coupled with δH1.35, and δH 0.97 was coupled with δH 4.44, indicating that the new compound had two CH-CH3 groups, proving the structure of Cordyceps cyclopeptide B.

[0072] Example 2: CCK-8 method to determine the inhibitory activity of compounds on tumor cell proliferation

[0073] 1. After the cells have been activated and subcultured for 2 to 3 generations, pour out the culture medium in the culture flask and add 2 mL of phosphate buffered saline (PBS) to wash twice.

[0074] 2. Use 1 mL of trypsin to enzymatically dissociate the adherent cells. Add 2 mL of complete culture medium to terminate the enzymatic dissociation and pipette evenly.

[0075] 3. Take 1 mL of cell fluid and add it into centrifuge tubes containing 20 mL of culture medium, mix well by pipetting.

[0076] 4. Take 100 μL of cell solution and add it to a 96-well plate, keeping 1×10 3 cells and cultured for 18 h.

[0077] 5. When the cell adhesion rate is determined to be about 80% under an inverted microscope, the cell fluid is aspirated and 100 μL of culture medium containing 1 μM, 10 μM, 20 μM and 40 μM drug gradients is added again, with three parallels for each gradient.

[0078] 6. Place in an incubator and incubate at 37°C and 5% CO2 for 24h and 48h.

[0079] 7. Add CCK-8 solution 24h and 48h after drug action (be careful not to generate bubbles to avoid affecting the readings), and incubate for another 3h.

[0080] 8. The absorbance at 450 nm was measured using an ELISA instrument, and statistical analysis was performed to compare the inhibitory activity of Cordyceps cyclopeptide B on the proliferation of different cell lines.

[0081] 9. The experimental results show that when the concentration of Cordyceps cyclopeptide B is 40μM, it has an inhibitory effect on the proliferation of both breast cancer cell MCF-7 and colon cancer cell SW-480 cell lines. The inhibition rates after 24 hours are 10.2% and 20.7%, respectively, and the inhibition rates after 48 hours are 13.6% and 21.3%, respectively.

Claims

1. Cordyceps cyclic peptide B shown in formula Ⅰ; 2. The method for preparing the Cordyceps cyclic peptide B according to claim 1, comprising the following steps: S1. crushing the fruiting bodies of Cordyceps militaris, wheatgrass, silkworm pupae infected by Cordyceps militaris or Cordyceps militaris mycelium, and extracting them with a formic acid aqueous solution; S2, the extract obtained in step S1 is centrifuged to obtain a supernatant, and the supernatant is concentrated to obtain a concentrated extract; S3, adding resin to the concentrated extract for adsorption to obtain a resin for adsorbing Cordyceps cyclic peptide compounds; S4, placing the resin adsorbing the Cordyceps cyclic peptide compound in a chromatography column, eluting with water and methanol aqueous solution in sequence, and collecting the eluate of the methanol aqueous solution; S5, the eluate of the methanol aqueous solution is treated to obtain a crude extract, the crude extract is subjected to chromatography separation, eluted with a methanol aqueous solution, the effluent containing Cordyceps cyclopeptide A is collected, and concentrated to obtain a concentrate; S6. The concentrate is separated and prepared by HPLC semi-preparative column, and a mixture of methanol and trifluoroacetic acid aqueous solution is used as the mobile phase for isocratic elution to obtain the Cordyceps cyclic peptide B.

3. The preparation method according to claim 3, characterized in that: In step S1, the particle size of the powder obtained after the pulverization is 40 to 60 mesh; The volume content of formic acid in the formic acid aqueous solution is 1 to 10%; Carrying out the leaching under ultrasonic conditions; The extraction time is 30 to 60 minutes.

4. The preparation method according to claim 2 or 3, characterized in that: In step S2, centrifugal separation is performed at 6000-12000 rpm for 10-30 min.

5. The preparation method according to any one of claims 2 to 4, characterized in that: In step S3, the resin is XAD4 resin; After adsorption, place the mixture in HCY-12B modular full-temperature oscillator for overnight adsorption at room temperature; The mass ratio of the concentrated extract to the resin is 10-20:

1.

6. The preparation method according to any one of claims 2 to 5, characterized in that: In step S4, the resin adsorbing the cordyceps cyclic peptide compound is placed in the chromatography column after being sieved; Use 2 to 5 column volumes of water for elution; The aqueous methanol solution with a volume content of 0-5% is used for elution in sequence for 5 column volumes, and then the aqueous methanol solution with a volume content of 5-15% is used for elution.

7. The preparation method according to any one of claims 2 to 6, characterized in that: In step S5, the stationary phase used for the chromatographic separation is Sephadex LH-20; The volume content of the methanol aqueous solution is 5-20%.

8. The preparation method according to any one of claims 2 to 7, characterized in that: In step S6, the volume content of the trifluoroacetic acid aqueous solution is 0.1%; The volume ratio of the methanol to the trifluoroacetic acid aqueous solution is 5-30:70-95.

9. The Cordyceps cyclic peptide B according to claim 1 is used in any of the following: 1) Preparation of drugs for treating cancer; 2) Preparation of drugs for treating breast cancer or colon cancer; 3) Drugs that inhibit cancer cell proliferation; 4) Drugs that inhibit the proliferation of breast cancer cells or colon cancer cells.

10. A drug for treating cancer, wherein the active ingredient is the Cordyceps cyclic peptide B as claimed in claim 1; The cancer is breast cancer or colon cancer.

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