Bioactive oligopeptide as well as extraction method and application thereof

Abstract

The invention provides a cyclic oligopeptide. The oligopeptide provided by the invention has an obvious inhibiting effect on lymphoma, leukemia, breast cancer, lung cancer, liver cancer or myeloma, has a certain inhibiting effect on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and salmonella paratyphi B, and also has a certain uric acid reducing effect. Therefore, the compound has great significance in preparation of antitumor drugs, antibacterial drugs and uric acid reducing drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and more specifically, to a method for extracting bioactive oligopeptides and application thereof. Background Art

[0002] Xuefeng Cordyceps is a dried complex of the fruiting body and larval corpse of the fungus Xuefeng Cordyceps (Ophiocordyceps xuefengensis T.C.Wen, RCZhu, JCKang & K.D.Hyde) parasitizing the larvae of the bat moth family insects. Fungal taxonomy and molecular biology studies have shown that Xuefeng Cordyceps is the species most closely related to Cordyceps sinensis in the same genus. Xuefeng Cordyceps has the effects of nourishing the lungs and kidneys, relieving cough and reducing phlegm. It is mainly used to treat kidney deficiency, spermatorrhea, impotence, sore waist and knees, long-term cough and asthma, etc. It is used by the people to enhance immunity, strengthen the body, prevent and treat cancer, and has significant efficacy.

[0003] Modern pharmacological studies have shown that Cordyceps sinensis has anti-tumor and antioxidant activities. The IC50 of its ethyl acetate fraction on NB4 cells and U937 cells is 3.76 and 1.91 μg / mL, and its in vitro anti-tumor activity is better than the positive control cisplatin. Animal experiments have also proved that the ethyl acetate fraction of Cordyceps sinensis has strong in vivo anti-tumor activity. Studies have shown that the ethyl acetate fraction of Cordyceps sinensis contains a large number of oligopeptide compounds, which have become one of the hot spots in the development of anti-tumor and antibacterial drugs, especially in the discovery of new drug lead compounds. It plays an indispensable role.

[0004] Bioactive oligopeptides are a class of short peptides composed of a small number of amino acids. They have attracted much attention due to their unique biological activities and broad application prospects. Bioactive oligopeptides are considered to be an important source for the development of a new generation of drugs due to their significant anti-tumor, antibacterial, anti-inflammatory and immunomodulatory biological activities, simple structure, high bioavailability and good safety. They are also used to develop foods and nutritional supplements with antioxidant, anti-fatigue and immune-enhancing functions. In addition, oligopeptides with antibacterial activity can be used to develop antibacterial coatings and medical devices to reduce the risk of infection and improve medical safety.

[0005] Therefore, targeted separation, purification and structural identification of oligopeptide compounds in Cordyceps sinensis can be carried out to clarify their anti-tumor, antibacterial and other biological activities and action characteristics, which can improve their resource utilization and provide lead compounds for the development of innovative drugs. Summary of the invention

[0006] The first object of the present invention is to provide an oligopeptide having biological activity.

[0007] The second object of the present invention is to provide a method for extracting oligopeptides with biological activity.

[0008] The third object of the present invention is to provide a use of an oligopeptide having biological activity.

[0009] An oligopeptide having the following structure:

[0010]

[0011] An oligopeptide preparation comprising the oligopeptide as claimed in claim 1.

[0012] The oligopeptide preparation includes a lyophilized preparation of the oligopeptide.

[0013] The oligopeptide is used to prepare a drug for treating cancer; or

[0014] For use in the preparation of a medicament for treating bacterial infection; or

[0015] For use in the preparation of drugs for treating cancer and bacterial infections; or

[0016] Used in the preparation of uric acid-lowering drugs.

[0017] The cancers include lymphoma, leukemia, breast cancer, lung cancer, liver cancer and / or myeloma.

[0018] The bacteria include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and / or Salmonella paratyphi B. The oligopeptide is used to prepare a drug for down-regulating the expression of Nrf2 in tumor cells; or

[0019] Used in the preparation of drugs for down-regulating the expression of Bcl-2 in tumor cells; or

[0020] Used in the preparation of drugs for activating the expression of Caspase 3 in tumor cells; or

[0021] It is used to prepare antibacterial drugs that inhibit NorA protein.

[0022] It is used to prepare drugs for inhibiting xanthine oxidase.

[0023] The method for extracting oligopeptides comprises the following steps:

[0024] S1. Crush the Xuefeng Cordyceps herb and add 95% ethanol, mix thoroughly, reflux and extract, cool, filter, obtain a filtrate and a filter residue, and retain the filtrate;

[0025] S2: adding the filter residue into 95% ethanol again, extracting under reflux, cooling, filtering, and obtaining a filtrate;

[0026] S3: combining the filtrates obtained in step S1 and S2, condensing and concentrating under reduced pressure, and then freezing the filtrate and performing vacuum freeze drying to obtain an extract;

[0027] S4: dissolving the extract described in step S3 in water and extracting with ethyl acetate to obtain an ethyl acetate extract;

[0028] S5: concentrate the ethyl acetate extract of step S4 under reduced pressure, and freeze it in vacuum to obtain ethyl acetate extract;

[0029] S6: dissolving the ethyl acetate extract in step S5 in methanol, pouring the sample into an ODS chromatography column, eluting with methanol, collecting 20 fractions, and separating the fractions to obtain the oligopeptide.

[0030] The ratio of the Xuefeng Cordyceps to the 95% ethanol is 150:1200 ml.

[0031] The reflux extraction time is 2 hours

[0032] The temperature of the condensation and reduced pressure concentration is 60°C;

[0033] The vacuum degree of the condensation and decompression concentration is ≤0.095MPa;

[0034] The freeze-drying temperature is -50°C.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The oligopeptide provided by the present invention has a significant inhibitory effect on lymphoma, leukemia, breast cancer, lung cancer, liver cancer or myeloma, and has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella paratyphi B, and also has a certain uric acid lowering effect. Therefore, the compound is of great significance for the preparation of anti-tumor drugs, antibacterial drugs and uric acid lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 1H-1H COSY and HMBC correlation spectra of the bioactive oligopeptide compound provided by the present invention;

[0038] Figure 2 The nuclear magnetic resonance 1H NMR spectrum of the biologically active oligopeptide compound provided by the present invention;

[0039] Figure 3 The nuclear magnetic resonance 13C NMR spectrum of the biologically active oligopeptide compound provided by the present invention;

[0040] Figure 4 The NMR HSQC spectrum of the biologically active oligopeptide compound provided by the present invention;

[0041] Figure 5The nuclear magnetic resonance HMBC spectrum of the biologically active oligopeptide compound provided by the present invention;

[0042] Figure 6 The nuclear magnetic resonance of the biologically active oligopeptide compound provided by the present invention 1 H- 1 H COSY spectrum;

[0043] Figure 7 An immunoblot band diagram of the biologically active oligopeptide compound provided by the present invention;

[0044] Figure 8 The present invention provides a three-dimensional diagram of the docking of the biologically active oligopeptide compound and the NorA molecule.

[0045] Fig. 9 Provides a two-dimensional diagram of the docking of the biologically active oligopeptide compound and the NorA molecule according to the present invention;

[0046] Fig.10 A three-dimensional diagram of the docking of the biologically active oligopeptide compound provided by the present invention and the xanthine oxidase molecule;

[0047] Fig.11 This is a two-dimensional diagram of the docking of the biologically active oligopeptide compound provided by the present invention with the xanthine oxidase molecule. DETAILED DESCRIPTION

[0048] The present invention found that the Xuefeng Cordyceps is rich in oligopeptide components of 6-15 amino acids, and the cell fishing technology can confirm that these compounds have strong anti-tumor activity. Therefore, the separation, purification, identification of oligopeptides in Xuefeng Cordyceps and the study of their anti-cancer, antibacterial and uric acid-lowering mechanisms are conducive to finding new anti-cancer, antibacterial and uric acid-lowering compounds and preparing them into corresponding preparations.

[0049] Specifically, according to the first aspect of the present invention, a separation and purification method for extracting bioactive oligopeptides from Cordyceps sinensis is provided, comprising the following steps:

[0050] 1) Crush the Xuefeng Cordyceps medicinal material into fine powder, weigh 150 g of the powder, put it in a round-bottom flask, add 1200 ml of 95% ethanol, mix thoroughly, reflux and extract for 2 hours, cool to room temperature, filter, and obtain a filtrate and a filter residue, and retain the filtrate;

[0051] 2) adding the filter residue in step 1) into 900 ml of 95% ethanol again, reflux extraction for 2 hours, cooling to room temperature, filtering, and obtaining a filtrate;

[0052] 3) combining the filtrates obtained in steps 1) and 2), condensing and concentrating under reduced pressure at 60° C. (vacuum degree ≤ 0.095 MPa) until the volume of the filtrate is about 100 ml, and then freezing the filtrate and placing it in a -50° C. vacuum freeze dryer to freeze into an extract;

[0053] 4) dissolving the extract described in step 3) in water, and extracting with ethyl acetate to obtain an ethyl acetate extract;

[0054] 5) condensing the ethyl acetate extract of step 4) at 60° C. under reduced pressure (vacuum degree ≤ 0.095 MPa), and freezing in a -50° C. vacuum freeze dryer to form an ethyl acetate extract;

[0055] 6) Dissolve the ethyl acetate extract in methanol in step 5), pour the sample into an ODS chromatography column, and elute with 80% methanol at a flow rate of 1 mL / min. Collect 20 fractions, and use HPLC-Q-TOF-MS to detect and analyze different fractions, and guide the merging. The HPLC-Q-TOF-MS parameters are as follows:

[0056] Chromatographic conditions: Inertsil ODS-2 column (4.6×250mm, 5μm); flow rate 0.5mL / min; column temperature 30℃; injection volume 20μL; detection wavelength 230nm, full wavelength scanning range 190nm~400nm; mobile phase methanol (B)-formic acid water (0.1%) (A); gradient elution: 0–30min, 50~100% B.

[0057] Mass spectrometry conditions: electrospray ionization (ESI); drying gas (N2) volume flow rate: 8.0 L / min; drying gas temperature: 350°C; sheath gas temperature: 320°C; nebulize 35 psig; sheath gas flow rate: 11 L / min; capillary voltage: 3.5 kV; nozzle voltage: 1.0 kV; collision voltage: 130 V; skimmer voltage 65 V; octopole RF 750 V; detection method: positive and negative ion full scan mode; scanning range: m / z 100 to 1000 Da.

[0058] After performing HPLC-Q-TOF-MS detection and analysis on the 20 fractions separated in 6), the inventors found that the 10th to 14th fractions all contained a compound with the same retention time and mass-to-charge ratio. Therefore, the inventors combined the separated 10th to 14th fractions, condensed them under reduced pressure at 60° C. (vacuum degree ≤ 0.095 MPa) to form a solution with a volume of 1 ml, and further separated and purified the concentrated solution by analytical preparative liquid chromatography to obtain compound I. The chromatographic conditions used were as follows:

[0059] Chromatographic column: Agilent Zorbax Eclipse XDB C18 column (9.4mm×250mm, 5μm); mobile phase: methanol (A)-water (B); mobile phase was filtered through a 0.22μm microporous membrane before use and ultrasonically degassed for 15 minutes; elution conditions were isocratic elution, mobile phase ratio: A:B=75:25; detection wavelength was 230nm; column temperature was 30℃; mobile phase flow rate was 2.5mL / min; injection volume was 50μL. The purity of the isolated compound I was 95%.

[0060] The specific process of structural identification of compound Ⅰ is as follows:

[0061] Compound Ⅰ, off-white powder, HPLC-UV (MeOH-H2O) λmax: 228, 281 nm. High-resolution mass spectrometry HPLC-Q-TOF-MS / MS m / z: 909.6157 [M+H]+ (Calcd for C 48 H 84 N4O 12 , 909.6158), indicating that the molecular formula of the compound is C 48 H 84 N4O 12 .

[0062] The 1H NMR spectrum (600MHz, CDCl3-d) of compound Ⅰ shows two d peaks at high field, δH 0.67 (3H, d) and δH 0.87 (3H, d), and the two-dimensional display shows that the two methyl groups are connected to carbon, which is speculated to correspond to leucine. The number of integrated peaks between δH 0.55-1.05ppm is about 16 methyl groups, with a total of 48 protons, and it is speculated that there are 8 residues containing dimethyl groups; the range of δH 4.43-5.65ppm is defined as the α-H region, with an integrated number of 8, and it is speculated that there are 8 α-H; there are 4 main s peaks in the range of δH 2.85-3.25ppm, which are typical methyl signals of methoxy or methylamine groups. Combined with the possibility of residues, it is speculated that there are 4 residues of aminomethyl substitution; there is no signal in the range of δH 6.5-8.0ppm, confirming the existence of no aromatic ring, confirming the absence of residues containing aromatic rings, no active hydrogen (-NH / OH), and no COOH.

[0063] The 13C spectrum (600 MHz, CDCl3-d) showed a total of 48 carbon signals, including 8 quaternary carbon signals [δC171.62, 171.58, 171.14, 170.63, 170.42, 170.32, 169.57, 168.78], and these signals were the carbonyl quaternary carbon signals of the residues; the negative peak of DEPT-135 showed that the compound contained 4 methylene (-CH2-) carbon signals [δC38.6 4, 38.08, 37.78, 36.91], and the methylene groups are densely concentrated in a narrow range, suggesting that the possible group environments are similar; the DEPT-90 spectrum shows that the compound has 16 methine (-CH-) carbon signals [δC76.95, 75.38, 74.45, 74.370, 57.63, 54.41, 54.36, 53.42, 31.10, 30.36, 29.76, 28.38, 25.5 6, 25.36, 25.04, 24.88], and these signals are composed of four groups of signals, each group has four signals, indicating that there are four tertiary methyl -CH- forms in the molecule, among which δC76.95, 75.38, 74.45, 74.370 are ester-O-α-CH-; comparing DEPT-135 with DEPT-90, it is found that there are 20 methyl carbon signals in the molecule [δC31.75, 31.00, 30. 66, 29.74, 23.86, 23.75, 23.69, 23.59, 21.17, 20.99, 20.90, 20.75, 20.31, 18.87, 18.63, 18.58, 18.33, 18.22, 15.83, 14.87], the positions are very close and partially overlapped, among which δC31.75, 31.00, 30.66, 29.74 are nitrogen methyl (N-CH3). According to the above data analysis and inference, there are 8 carbons in the carbonyl region of the oligopeptide, which may be an octapeptide condensation; there is no aromatic carbon, so there is no aromatic ring residue; the carbon peaks in the high-field alkyl region are dense, and the hydrogen spectrum confirms that there are many terminal methyl and methylene groups connected to the alkyl. It is speculated that the oligopeptide contains more amino acid residues, such as valine, proline, leucine or isoleucine.

[0064] Table 1 1H NMR and 13C NMR data of compound I (CDCl3-d)

[0065]

[0066]

[0067] / , not provided

[0068] By analyzing the HSQC, HMBC and 1H-1H COSY spectra of compound Ⅰ, H-2 (δH4.43, 1H, dd) in fragment 1 (N-Me-Leu-1) is the first α-H, HSQC is correlated with C-2 (δC 57.63, CH), in the HMBC spectrum, H-2 is correlated with C-1 (δC 171.58), C-3 (δC 38.08), C-4 (δC 25.04), C-6 (δC 20.75), C-8 (δC 169.57), C-2 is correlated with H-7 (δH2.89, 3H, s), it can be inferred that the adjacent position of the proton carbonyl carbon is 171.58 (C-1), the related amino group is a nitrogen methyl group, and the proton is δH2.89ppm; in the 1H-1H COSY spectrum, H-2 and H-3 (δH The HSQC site is near δC38.08 (C-3), and combined with DEPT, it is seen to be a -CH2-methylene structure; the alkyl carbon related to H-2 in HMBC (δH 4.43, 1H, dd) is δC 38.08 (C-3), δC 25.04 (C-4), δC 20.75 (C-6), DEPT90 confirmed that δC 25.04 is a tertiary methyl structure -CH-, and δC 20.75 is a methyl group, so the fragment is analyzed to be a N-Me-Leu residue. Structural analysis of fragment 2 (α-hydroxyisovaleric acid-1, S-α-HOIva-1). In the HMBC spectrum, the H of the nitrogen methyl group (δH 2.89, 3H, s, H-7; δC 29.74, N-CH3, C-7) is correlated with C-8 (δC 169.57, C), and it can be deduced that the amide carbonyl carbon connected to it is δC 169.57; δC 169.57 is correlated with a CH (δH 4.99, 1H, d, H-9; δC 76.95, CH, C-9), and the δH and δC of the CH are shifted to the low field, confirming the existence of ester -O-α-CH-; H-9 is correlated with C-7 (δC 29.74), C-8 (δC 169.57), C-10 (δC 28.38), C-11 (δC The 1H-1H COSY spectrum showed that H-9 was connected to H-10, and H-10 was connected to H-11, which further confirmed the structure of this fragment.

[0069] Structural analysis of fragment 3 (N-Me-Leu-2). In the HMBC spectrum, H-9 is weakly correlated with the third carbonyl group (δC 170.32, C, C-13), and C-13 is correlated with the second nitrogen methyl group (δH 3.25, 3H, s, H-19; δC 31.75, N-CH3, C-19); at the same time, H-19 is correlated with a CH (δH 5.43, 1H, d, H-14; δC 54.41, CH, C-14); H-14 is correlated with C-15 (δC 36.91), C-16 (δC 25.56), C-17 (δC 18.58), C-18 (δC 21.17), and C-19, so the fragment is analyzed to be another N-Me-Leu residue. In the 1H-1H COSY spectrum, H-14 was connected to H-15, and H-15 was connected to H-16, which further confirmed the existence of this fragment.

[0070] Structural analysis of fragment 4 (S-α-HOIva-2). In the HMBC spectrum, H-19 is associated with C-20 (δC 171.62, C), C-20 is associated with a CH (δH 5.45, 1H, d, H-21; δC 74.75, CH, C-21), and the δH and δC of the CH are shifted to the low field, confirming the existence of ester -O-α-CH-; H-21 is associated with another CH (δH 2.23, 1H, dd, H-22; δC 31.10, CH, C-22); C-21 is associated with two CH3 (δH 0.94, 3H, s, H-23; δC 23.75, CH3, C-23; δH 1.02, 3H, s, H-24; δC18.33, CH3, C-24); confirming that the fragment is another S-α-HOIva residue. In the 1H-1H COSY spectrum, H-21 is connected to H-22, and H-22 is connected to H-23, further confirming the structure of this fragment.

[0071] Structural analysis of fragment 5 (N-Me-Leu-3). In the HMBC spectrum, H-21 is weakly correlated with the fourth carbonyl group (δC 170.42, C, C-25), and C-25 is correlated with the third nitrogen methyl group (δH 3.04, 3H, s, H-31; δC 31.00, N-CH3, C-31). At the same time, H-31 is correlated with a CH (δH 5.62, 1H, dd, H-26; δC 54.36, CH, C-26). In the 1H-1H COSY spectrum, H-26 is connected to H-27 (δH 1.79, 2H, m), while in the HMBC spectrum, H-27 is associated with one CH (δH 1.61, 1H, m, H-28; δC 24.88, CH, C-28), and H-27 is also associated with two CH3 (δH 0.96, 3H, s, H-29; δC 20.90, CH3, C-29; δH 0.91, 3H, s, H-30; δC 20.31, CH3, C-30). Therefore, the fragment was analyzed to be the third N-Me-Leu residue.

[0072] Structural elucidation of fragment 5 (S-α-HOIva-3). In the HMBC spectrum, H-26 and H-31 are both related to C-32 (δC 170.63, C), C-32 is related to a CH (δH 5.51, 1H, d, H-33; δC 74.37, CH, C-33), and the δH and δC of this CH are shifted to the low field, confirming the existence of ester-O-α-CH-; H-33 is related to another CH (δH 2.10, 1H, m, H-34; δC 29.76, CH, C-34) and two CH3 (δH 1.01, 3H, s, H-35; δC 18.63, CH3, C-35; δH 0.97, 3H, s, H-36; δC 15.83, CH3, C-36); H-35 is related to C-36, and H-36 is related to C-34, confirming that the fragment is the third S-α-HOIva residue. In the 1H-1H COSY spectrum, H-33 is connected to H-34, and H-34 is connected to H-35, further confirming the structure of this fragment.

[0073] Structural analysis of fragment 7 (N-Me-Leu-4). In the HMBC spectrum, H-33 is weakly correlated with the fourth carbonyl group (δC 168.78, C, C-37), and C-37 is correlated with the fourth nitrogen methyl group (δH 3.01, 3H, s, H-43; δC 30.66, N-CH3, C-43). Meanwhile, H-43 is correlated with a CH (δH 5.65, 1H, dd, H-38; δC 53.42, CH, C-38), which is correlated with C-43 and a CH2 (δH 1.76, 2H, m, H-39; δC 53.42, CH, C-39). H-39 is correlated with another CH (δH 1.42, 1H, s, H-40; δC 25.36, CH, C-40), and one CH3 (δH 0.91, 3H, s, H-41; δC 20.99, CH3, C-41); H-41 is related to another CH3 (δH 0.92, 3H, s, H-42; δC 23.59, CH3, C-42), so the fragment is analyzed to be the fourth N-Me-Leu residue. In the H-1H COSY spectrum, H-38 is connected to H-39, H39 is connected to H-40, and H-40 is connected to H-42, which further confirms the structure of this fragment.

[0074] Structural analysis of fragment 8 (S-α-HOIva-4). In the HMBC spectrum, H-38 is related to C-44 (δC 171.14, C), C-44 is related to a CH (δH 5.28, 1H, d, H-45; δC 75.38, CH, C-45), and the δH and δC of the CH are shifted to the low field, confirming the existence of ester -O-α-CH-; H-45 is related to C-43, and is also related to another CH (δH 2.18, 1H, dd, H-46; δC 30.36, CH, C-46) and a CH3 (δH 0.95, 3H, s, H-47; δC 23.96, CH3, C-47); H-46 is related to C-43; H-48 (δH 0.93, 3H, s) correlated with C-45, confirming that the fragment was the fourth S-α-HOIva residue. In the 1H-1H COSY spectrum, H-45 was connected to H-46, further confirming the structure of this fragment.

[0075] Through the above inference, it is deduced that the compound is a cyclic octapeptide structure, which is formed by the alternating condensation of four leucines and four oxo-valine / α-hydroxyisovaleric acid. Due to the existence of a certain configuration in space, the peaks in each region of the NMR spectrum are different, but the overall structure is similar. The planar structure of the compound is as follows.

[0076]

[0077] The compound was found to be a new compound not reported in the literature through SciFinder search, and was named Xuefenglastatin A. The present invention uses the following method to study the anticancer, antibacterial and uric acid-lowering activities of compound I.

[0078] Monomer anticancer activity detection:

[0079] 1. Cell lines: U937, MCF-7, Hep G2, A549, H460, and N2A cell lines were selected.

[0080] 2. Experimental Methods

[0081] 1) Cells in the growth phase were cultured at 1.25×10 5 The cells were inoculated into a 96-well culture plate at a density of 10 μL / mL, and 90 μL was added to each well. Physiological saline was set as the negative control group, cisplatin was set as the positive control group, and the amount of each sample added was 10 μL per well.

[0082] 2) Place the culture plate in an environment of 37°C and 5% carbon dioxide for 48 hours. Subsequently, add 10 μL of MTS solution (concentration of 5 mg / mL) to each well and continue to culture for 4 hours. Then, add 100 μL of triple solution (consisting of 10% sodium dodecyl sulfate, 5% isopropanol and 0.012 mol / L hydrochloric acid) to each well. After standing at room temperature for 12 hours, use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 595 nm.

[0083] The calculation formula of inhibition rate is: inhibition rate (%) = (OD value of control group - OD value of test group) / OD value of control group. Samples with inhibition rate greater than 50% will be rescreened and IC 50 The results are shown in Table 2.

[0084] Table 2

[0085]

[0086] Furthermore, since the occurrence of cancer is mostly caused by the blockage of cell apoptosis, inducing apoptosis of cancer cells is helpful for the treatment of cancer. Therefore, the present invention compares the effect of compound I on apoptosis of lung cancer cells.

[0087] Specifically, in order to further explore the effect of compound I on apoptosis of lung cancer cells, in order to reveal the potential mechanism of its anti-tumor effect. The present invention first observed the effect of compound I on cell morphology by phase contrast microscopy, and evaluated its effect on cell nuclei by Hoechst 33258 staining to verify its ability to induce apoptosis of lung cancer cells. The specific experimental steps are as follows:

[0088] (1) Cell culture

[0089] The cleaned cell slides were placed in a six-well plate and cultured at 4.0 × 10 5 The well-growing cells were inoculated with a density of 1:1 (A549 cells) to ensure that the cells were evenly distributed. Subsequently, the six-well plate was placed in an environment of 37° C., 5% CO 2 and saturated humidity for 24 hours.

[0090] (2) Drug treatment

[0091] After the cells adhered to the wall, different concentrations of compound I (0 nM, 40 nM, 60 nM, 80 nM) were added and culture was continued for 24 hours.

[0092] (3) Cell morphology observation

[0093] After drug incubation, a 40x phase contrast microscope was used to observe and record changes in cell morphology.

[0094] The results showed that under high concentration treatment, the cells treated with compound I became rounded and gradually detached from the culture dish wall.

[0095] (4) Hoechst staining observation

[0096] After the drug incubation, the old culture medium was discarded, and 1 mL of cell fixative was added to each well to fix the cells for 10 minutes. After fixation, the fixative was discarded, washed three times with pre-cooled PBS, and then 500 μL of Hoechst 33258 was added for light-proof staining for 5 minutes. After the staining was completed, the staining solution was discarded, washed three times with PBS, and observed and recorded under an inverted fluorescence microscope (20×). The results showed that in the samples treated with compound I, the number of cell nuclei decreased and showed varying degrees of fragmentation, and the staining results became brighter at high concentrations, indicating that the cell nuclei shrank, which is a typical nuclear change feature during cell apoptosis, indicating that compound I can effectively induce apoptosis of lung cancer cells.

[0097] On this basis, the present invention further carries out cell cycle detection and related apoptosis protein detection.

[0098] The specific steps and results are as follows:

[0099] (1) Cell cycle detection

[0100] Collect the cells treated in the same way and make a single cell suspension of 1×10^6 cells / ml. Set up a negative control and a blank control. Wash the cells with PBS, discard the supernatant, keep 100μL PBS to blow the cells apart, and then add pre-cooled 75% ethanol to fix the cells overnight while vortexing. Centrifuge the fixed cells, wash them with PBS once, centrifuge again, discard the supernatant, and add the prepared IP staining working solution to stain for 10 minutes. Finally, place them on a flow cytometer for detection.

[0101] The results showed that compound I could significantly block A549 cells at the G2 / M phase at concentrations of 40 nM and 80 nM, and could block H460 cells at the G2 / M phase at 80 nM.

[0102] (2) Detection of apoptotic proteins

[0103] The supernatant of the treated cells was discarded, and the cells were washed with PBS containing PMSF, and then 100 μL of SDS cell lysis solution was added to lyse the cells, and the cell lysate was collected, and then heated in a 100-degree metal bath for denaturation for 10 minutes. The collected cell proteins were separated by SDS-PAGE of different concentrations (8%-15%) according to the molecular weight of the target protein, and then the proteins in the gel were transferred to NC membrane or PVDF membrane. The membrane was blocked with 5% milk solution for 1 hour, and then the target protein was incubated with primary antibody overnight. After the incubation, it was washed three times with TBST (TBS containing 1‰ Tween 20) for 5 minutes each time, followed by incubation with secondary antibody for 1 hour. After the incubation, it was washed again with TBST three times for 5 minutes each time. Finally, it was treated with an enhanced chemiluminescence detection kit, and the target protein blot was obtained using the Bio-Rad gel imager Gel Docxrs imaging system.

[0104] The results showed that compound I could significantly regulate the expression levels of nuclear factor-E2-related factor 2 (Nrf2) and apoptosis-related proteins B-cell lymphoma-2 (Bcl-2) and cysteine ​​protease 3 (Caspase-3). Compound I enhanced the oxidative stress level of A549 cells by downregulating the expression of Nrf2, and induced cell apoptosis by downregulating the expression of anti-apoptotic protein Bcl-2 and activating the expression of pro-apoptotic protein Caspase 3. Figure 7 As shown. NRF2 is a reliable driver gene and a key therapeutic target for carcinogenesis. Abnormal expression of Nrf2, Bcl-2, and Caspase-3 is correlated with the progression and malignant biological behavior of lung cancer. Combined with the previously observed changes in cell morphology and nucleus, these results confirm that compound I can induce apoptosis of lung cancer cells in a dose-dependent manner.

[0105] Monomer antibacterial activity detection:

[0106] The test tube dilution method was used to detect the MIC values ​​of the corresponding strains, and the antibacterial activity of compound I against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella paratyphi B was observed.

[0107] The specific steps and results are as follows:

[0108] The test tube dilution method was used to dilute the drug with liquid culture medium. Ten sterile test tubes were taken, and 2.0 mL of liquid culture medium was added to each tube. After adding 5 mg / mL of the test drug to the first tube and mixing with the liquid culture medium, 2 mL was added to the second tube, and then 2 mL was added to the third tube... and so on, so that the final concentrations of the experimental drugs in each tube were 0.25, 0.125, 0.0625, 0.0313, 0.0157, 0.0078, 0.0039, and 0.0020 mg / mL (the final total volume of each tube was 2 mL). 0.1 mL of the logarithmic growth phase culture of each strain with a concentration of 1×10^7 CFU / mL was added to each experimental tube. In addition, a control of adding an equal amount of culture medium without any drugs and bacteria and a blank control of culture medium containing bacteria but no drugs were set up. The bacteria were cultured at 37°C for 24 hours, and the growth of different strains was observed. The highest dilution of the drug at which no bacterial growth was observed was the minimum inhibitory concentration (MIC) of the drug. According to the MIC of each bacteria, compound I has a certain inhibitory effect on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella paratyphi B, with MICs of 0.0157 mg / mL, 0.0313 mg / mL, 0.0625 mg / mL, and 0.125 mg / mL, respectively. The results are shown in Table 3.

[0109] Table 3 In vitro antibacterial activity MIC test results of compound I

[0110]

[0111] Note: "+" indicates that the test tube is turbid and bacteria grows when observed with the naked eye; "-" indicates that the test tube is clear and there is no bacteria growth when observed with the naked eye. Similarly, the inventor further used molecular docking technology to explore the antibacterial mechanism and active groups of compound I. NorA was selected as its target protein. The receptor protein was retrieved from the PDB (http: / / www.rcsb.org / pdb) database, and the receptor protein was dehydrated and deliganded using PyMOL 2.3.4 software. The receptor protein was modified by hydrogenation and charge balance using AutoDockTools software. The Grid Box command under the Grid program was used to open the Grid Option tool to process the receptor protein. The docking space site pocket was generated according to the target protein's own ligand, and the receptor protein was converted into pdbqt format. Compound I was used as a ligand and NorA was used as a receptor. AutoDock Vina 1.1.2 was used for molecular docking. The affinity of the receptor-ligand complex was evaluated by calculating the spatial effect, repulsion, hydrogen bond, hydrophobic interaction, and molecular flexibility of the receptor-ligand complex, and finally the affinity energy score was given. The molecular docking results were analyzed using pymol software (https: / / pymol.org / 2 / ). The results showed that compound I had a strong binding interaction with NorA through hydrophobic interaction, with an affinity of -7.6 kcal / mol. Figure 8-9 This indicates that compound I can prevent bacteria from transporting it from inside the bacteria to outside the bacteria, thereby prolonging its antibacterial time.

[0112] In order to evaluate the advantages of compound I in antibacterial effect, the inventors further evaluated its antibacterial duration in combination with its binding results with NorA. The specific operation method is the same as the above antibacterial test, that is, the revived and activated test strain is inoculated into the corresponding culture medium, shaken and cultured at 37°C to the logarithmic growth phase, and then diluted to a certain concentration with sterile saline to make the bacterial solution concentration reach 1×10^6CFU / mL. Different experimental groups and control groups were set up in 96-well plates. The experimental group added different concentrations of compound I and bacterial solution, and the control group added equal amounts of sterile water and bacterial solution. Three replicates were set for each concentration. The 96-well plate was placed in a constant temperature incubator and incubated at 37°C. Within 0h-48h, the 96-well plate was taken out every 1 hour, and the bacterial solution in each well was gradiently diluted by the viable bacteria counting method, applied to the plate culture medium, and cultured at 37°C for 24h, and the number of colonies on the plate was counted. Evaluate the duration of compound I when the drug is maintained at the minimum inhibitory concentration. The results are shown in Table 4.

[0113] Table 4 Antibacterial duration test results of compound I

[0114] Bacteria Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Salmonella Paratyphi B Effective antibacterial time (h) 35 41 40 33

[0115] Monomer uric acid-lowering activity test:

[0116] Cancer patients may experience hyperuricemia, which is mainly due to the massive proliferation of tumor cells, which leads to an increase in metabolites, thus causing an increase in uric acid levels. In addition, cancer patients may also receive chemotherapy, radiotherapy and other treatments, which cause a large number of tumor cells to be destroyed or cause kidney damage, further leading to increased uric acid production and reduced excretion, thus causing an increase in uric acid levels.

[0117] Xanthine oxidase inhibitory activity was used to determine the uric acid-lowering activity of compound I. 1.8 mL of phosphate buffer or sample solution and 0.6 mL of xanthine oxidase mixture were placed in a brown centrifuge tube in sequence and incubated at 25 °C in the dark for 30 min. 0.6 mL of xanthine solution was added to start the reaction, and then the reaction was continued at 25 °C for 30 min. Finally, 0.3 mL of hydrochloric acid solution was added to terminate the reaction, and the absorbance (A) was measured at 295 nm. The inhibition rate was calculated according to the following formula.

[0118] Inhibition rate (%) = [1-(A 样品 -A 对照 ) / A 空白 ]×100%

[0119] Where: A 样品 : absorbance value of the reaction system with added sample; A 对照 : Absorbance value of the reaction system with sample added but no xanthine solution added; A 空白 : Absorbance value of the reaction system without adding sample. The operation was repeated three times. The logarithmic curve was drawn using Origin Pro 9.1 with the logarithmic value of sample concentration as the horizontal axis and the inhibition rate as the vertical axis. In the statistics page, Y=50 was set to obtain the sample concentration when the inhibition rate was 50%.

[0120] Table 5 IC of compound I against xanthine oxidase inhibitory activity 50 Value (mg / mL)

[0121]

[0122] Similarly, the inventors further used molecular docking technology to explore the uric acid-lowering mechanism and active groups of compound I. Xanthine oxidase was selected as its target protein. The specific operation was the same as anti-tumor molecular docking. The results showed that compound I had a strong binding effect with xanthine oxidase through hydrogen bonding and hydrophobic interaction, with an affinity of -8.5 kcal / mol. Figure 10-11 shown.

[0123] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. An oligopeptide, characterized in that: It has the following structure:

2. An oligopeptide preparation, characterized in that: Comprising the oligopeptide as claimed in claim 1.

3. The oligopeptide preparation according to claim 2, characterized in that: The oligopeptide preparation includes a lyophilized preparation of the oligopeptide.

4. The use of the oligopeptide according to claim 1, characterized in that: for use in the preparation of drugs for treating cancer; or For use in the preparation of a medicament for treating bacterial infection; or For use in the preparation of drugs for treating cancer and bacterial infections; or Used in the preparation of uric acid-lowering drugs.

5. The use of the oligopeptide according to claim 4, characterized in that: The cancer includes lymphoma, leukemia, breast cancer, lung cancer and / or liver cancer or myeloma; The bacteria include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and / or Salmonella paratyphi B.

6. The use of the oligopeptide according to claim 1, characterized in that: Used in the preparation of drugs for down-regulating the expression of Nrf2 in tumor cells; or Used in the preparation of drugs for down-regulating the expression of Bcl-2 in tumor cells; or Used in the preparation of drugs for activating the expression of Caspase 3 in tumor cells; or Used in the preparation of drugs for inhibiting the production of NorA protein; or It is used to prepare drugs for inhibiting xanthine oxidase.

7. The method for extracting oligopeptides according to claim 1, wherein: The steps include: S1. Crush the Xuefeng Cordyceps herb and add 95% ethanol, mix thoroughly, reflux and extract, cool, filter, obtain a filtrate and a filter residue, and retain the filtrate; S2: adding the filter residue into 95% ethanol again, extracting under reflux, cooling, filtering, and obtaining a filtrate; S3: combining the filtrates obtained in step S1 and S2, condensing and concentrating under reduced pressure, and then freezing the filtrate and performing vacuum freeze drying to obtain an extract; S4: dissolving the extract described in step S3 in water and extracting with ethyl acetate to obtain an ethyl acetate extract; S5: concentrate the ethyl acetate extract of step S4 under reduced pressure, and freeze it in vacuum to obtain ethyl acetate extract; S6: dissolving the ethyl acetate extract in step S5 in methanol, pouring the sample into an ODS chromatography column, eluting with methanol, collecting 20 fractions, and separating the fractions to obtain the oligopeptide.

8. The method for extracting oligopeptides according to claim 7, characterized in that: The ratio of the Xuefeng Cordyceps to the 95% ethanol is 150:1200 ml.

9. The method for extracting oligopeptides according to claim 7, wherein: The reflux extraction time is 2 hours.

10. The method for extracting oligopeptides according to claim 7, characterized in that: The temperature of the condensation and reduced pressure concentration is 60°C; The vacuum degree of the condensation and decompression concentration is ≤0.095MPa; The freeze-drying temperature is -50°C.

Images