A bioactive oligopeptide and its extraction method and application

Abstract

The present invention provides a cyclic oligopeptide. The oligopeptide has significant inhibitory effects on lymphoma, leukemia, breast cancer, lung cancer, liver cancer, or myeloma. It also exhibits some inhibitory effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella Paratyphi B. Furthermore, it has a certain uric acid-lowering effect. Therefore, this compound is of great significance for the preparation of antitumor drugs, antibacterial drugs, and uric acid-lowering drugs.

Description

Technical Field

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

[0002] Xuefeng Cordyceps is the dried complex of the fruiting bodies and larval bodies of the fungus Ophiocordyceps xuefengensis (T.C. Wen, R.C. Zhu, J.C. Kang, & K.D. Hyde), a member of the Nematode family. It parasitizes the larvae of insects belonging to the family Bat Moth and is derived from the fungus. Fungal taxonomy and molecular biology studies have shown that Xuefeng Cordyceps is the closest relative of Cordyceps sinensis to the same genus. It nourishes the lungs and kidneys, relieves coughs and reduces phlegm. It is primarily used to treat kidney deficiency, spermatorrhea, impotence, low back pain, and chronic coughs and asthma. It is used in folk medicine to enhance immunity, strengthen the body, and prevent and treat cancer, with remarkable efficacy.

[0003] Modern pharmacological studies have demonstrated that Cordyceps sinensis possesses antitumor and antioxidant activities. Its ethyl acetate fraction exhibits IC50 inhibition against NB4 and U937 cells of 3.76 and 1.91 μg / mL, respectively, demonstrating superior in vitro antitumor activity compared to the positive control, cisplatin. Animal studies have also demonstrated that the ethyl acetate fraction possesses potent in vivo antitumor activity. Studies have shown that the ethyl acetate fraction contains a large number of oligopeptides, which have become a hot topic in the development of antitumor and antibacterial drugs, playing an essential role in the discovery of new drug leads.

[0004] Bioactive oligopeptides are a class of short peptides composed of a small number of amino acids. They have attracted considerable attention due to their unique bioactivity and broad application prospects. Due to their significant anti-tumor, antibacterial, anti-inflammatory, and immunomodulatory bioactivities, as well as their simple structure, high bioavailability, and good safety, bioactive oligopeptides are considered an important source for the development of next-generation drugs. They are also being used to develop foods and nutritional supplements with antioxidant, anti-fatigue, and immune-enhancing properties. Furthermore, oligopeptides with antimicrobial activity can be used to develop antimicrobial coatings and medical devices, reducing the risk of infection and improving medical safety.

[0005] Therefore, carrying out targeted separation, purification and structural identification of oligopeptide compounds in Cordyceps sinensis, clarifying their anti-tumor, antibacterial and other biological activities and action characteristics, 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 with 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 with biological activity.

[0009] An oligopeptide having the following structure:

[0010]

[0011] An oligopeptide preparation comprising the oligopeptide according to 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 drug 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 cancer includes 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 downregulating 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 can be used to prepare drugs for inhibiting xanthine oxidase.

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

[0024] S1: crushing the Xuefeng Cordyceps herb, adding 95% ethanol, thoroughly mixing, and then reflux extraction, cooling, filtering, obtaining a filtrate and a filter residue, and retaining the filtrate;

[0025] S2: adding the filter residue to 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 freeze-drying it in vacuum 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: concentrating the ethyl acetate extract from step S4 under reduced pressure, and freezing it in a vacuum to obtain an ethyl acetate extract;

[0029] S6: dissolving the ethyl acetate extract of 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 reduced pressure 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. It also has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella Paratyphi B. It also has a certain uric acid-lowering effect. Therefore, this 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 compounds provided by the present invention;

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

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

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

[0041] Figure 5The HMBC nuclear magnetic resonance spectrum of the bioactive 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 The immunoblot band diagram of the bioactive 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 NorA molecule.

[0045] Figure 9 Provided is a two-dimensional diagram of the docking of the biologically active oligopeptide compound and NorA molecule;

[0046] Figure 10 A three-dimensional diagram of the docking of the bioactive oligopeptide compound provided by the present invention and the xanthine oxidase molecule;

[0047] Figure 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 discovered that Cordyceps sinensis is rich in oligopeptides consisting of 6-15 amino acids, and cell fishing technology has confirmed that these compounds have strong anti-tumor activity. Therefore, isolating, purifying, and identifying oligopeptides from Cordyceps sinensis, and studying their anticancer, antibacterial, and uric acid-lowering mechanisms, will facilitate the discovery of new compounds with these properties and their preparation into corresponding formulations.

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

[0050] 1) Grind the herbal medicine of Xuefeng Cordyceps into a fine powder. Weigh 150 g of the powder and place it in a round-bottom flask. Add 1200 ml of 95% ethanol, mix thoroughly, and reflux for 2 hours. Cool to room temperature, filter, and obtain a filtrate and a residue. The filtrate should be retained.

[0051] 2) The filter residue from step 1) was again added to 900 ml of 95% ethanol, refluxed for 2 hours, cooled to room temperature, and filtered to obtain 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 filtrate volume is approximately 100 ml, and then freezing the filtrate in a -50° C. vacuum freeze dryer to obtain 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 from step 4) at 60° C. under reduced pressure (vacuum degree ≤ 0.095 MPa), and freezing in a vacuum freeze dryer at -50° C. to obtain an ethyl acetate extract;

[0055] 6) Dissolve the ethyl acetate extract from step 5) in methanol. Pour the sample into an ODS chromatography column and elute with 80% methanol at a flow rate of 1 mL / min. Twenty fractions were collected and analyzed using HPLC-Q-TOF-MS, and the fractions were combined. The HPLC-Q-TOF-MS parameters were as follows:

[0056] Chromatographic conditions: Inertsil ODS-2 column (4.6×250 mm, 5 μm); flow rate 0.5 mL / min; column temperature 30°C; injection volume 20 μL; detection wavelength 230 nm, full wavelength scan range 190 nm to 400 nm; mobile phase methanol (B)-formic acid water (0.1%) (A); gradient elution: 0–30 min, 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 mode: positive and negative ion full scan mode; scanning range: m / z 100 to 1000 Da.

[0058] After performing HPLC-Q-TOF-MS analysis on the 20 fractions separated in 6), the inventors found that fractions 10-14 all contained a compound with the same retention time and mass-to-charge ratio. Therefore, the inventors combined the separated fractions 10-14 and concentrated them under reduced pressure at 60° C. (vacuum degree ≤ 0.095 MPa) to a solution with a volume of 1 ml. The concentrated solution was further separated and purified 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 (9.4 mm × 250 mm, 5 μm); mobile phase: methanol (A)-water (B); mobile phase filtered through a 0.22 μm microporous membrane and ultrasonically degassed for 15 minutes before use; elution conditions: isocratic elution, mobile phase ratio: A:B = 75:25; detection wavelength: 230 nm; column temperature: 30°C; mobile phase flow rate: 2.5 mL / min; injection volume: 50 μL. The isolated compound I had a purity of 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), suggesting that the molecular formula of the compound is C 48 H 84 N4O 12 .

[0062] The 1H NMR spectrum (600 MHz, CDCl3-d) of compound I showed two high-field d peaks at δH 0.67 (3H, d) and δH 0.87 (3H, d). Two-dimensional spectra indicated that the two methyl groups were attached to carbon atoms, suggesting that they correspond to leucine. The δH 0.55-1.05 ppm region integrated approximately 16 methyl groups, with a total of 48 protons, suggesting eight dimethyl residues. The δH 4.43-5.65 ppm region, defined as the α-H region, integrated eight protons, suggesting eight α-H residues. Four major s peaks were observed in the δH 2.85-3.25 ppm region, typical of methyl groups from methoxy or methylamine groups. Based on the residue probability, four residues were inferred to be aminomethyl substituted. No signals were observed in the δH 6.5-8.0 ppm region, confirming the absence of aromatic rings, active hydrogen (-NH / OH), and COOH residues.

[0063] The 13C spectrum (600MHz, 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 quaternary carbon signals of the carbonyl 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 distributed in a narrow range, suggesting that the possible group environments are similar; DEPT-90 spectrum showed that the compound had 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], which are very close in position and partially overlap, among which δC31.75, 31.00, 30.66, 29.74 are nitrogen methyl groups (N-CH3). Based on 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 combined with the hydrogen spectrum, it is confirmed that there are many terminal methyl and methylene groups connected to the alkyl group. 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 (δH 4.43, 1H, dd) in fragment 1 (N-Me-Leu-1) was the first α-H, HSQC correlated with C-2 (δC 57.63, CH), H-2 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) in the HMBC spectrum, and C-2 correlated with H-7 (δH 2.89, 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 δH 2.89ppm; in the 1H-1H COSY spectrum, H-2 and H-3 (δH The HSQC site was near δC38.08 (C-3), and the -CH2-methylene structure was seen in combination with DEPT. The alkyl carbons related to H-2 (δH 4.43, 1H, dd) in HMBC were δC 38.08 (C-3), δC 25.04 (C-4), and δC 20.75 (C-6). DEPT90 confirmed that δC 25.04 was a tertiary methyl structure -CH-, and δC 20.75 was a methyl group. Therefore, the fragment was analyzed to be an N-Me-Leu residue. Structural elucidation 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 downfield, 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 fragment is characterized by a δC 14.87 residue, C-12 (δC 18.87), H-11 (δH 0.55, 3H, d) correlated with C9 and C-10, and H-12 correlated with C-10 and C-11, confirming that the fragment is a S-α-HOIva residue. The 1H-1H COSY spectrum shows that H-9 is connected to H-10, and H-10 is connected to H-11, further confirming 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). Meanwhile, 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, indicating that this fragment is another N-Me-Leu residue. The 1H-1H COSY spectrum showed that 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 elucidation 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 shift to the downfield, 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 The fragment is related to another S-α-HOIva residue (δ 1.02, 3H, s, H-24; δ C 18.33, CH3, C-24). The 1H-1H COSY spectrum shows that H-21 is connected to H-22, and H-22 is connected to H-23, further confirming the structure of this fragment.

[0071] Structural elucidation 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 group (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). In the HMBC spectrum, H-27 is associated with one CH (δH 1.61, 1H, m, H-28; δC 24.88, CH, C-28). H-27 is also associated with two CH3 residues (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, this fragment is 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 associated with C-32 (δC 170.63, C), C-32 is associated with a CH (δH 5.51, 1H, d, H-33; δC 74.37, CH, C-33), and the δH and δC of the CH are shifted to the downfield, confirming the existence of ester -O-α-CH-; H-33 is associated with 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 correlates with C-36, and H-36 correlates with C-34, confirming that this fragment is the third S-α-HOIva residue. The 1H-1H COSY spectrum shows that H-33 is connected to H-34, and H-34 is connected to H-35, further confirming the structure of this fragment.

[0073] Structural elucidation 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, in addition to being correlated with C-43, is also correlated with a CH2 (δH 1.76, 2H, m, H-39; δC 53.42, CH, C-39). H-39 is also correlated with another CH (δH 1.42, 1H, s, H-40; δC The fragment is analyzed to be the fourth N-Me-Leu residue. The H-1H COSY spectrum shows that H-38 is connected to H-39, H39 to H-40, and H-40 to H-42, further confirming the structure of this fragment.

[0074] Structural elucidation of fragment 8 (S-α-HOIva-4). In the HMBC spectrum, H-38 is associated with C-44 (δC 171.14, C), C-44 is associated with 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 downfield, confirming the presence of ester -O-α-CH-; H-45 is associated with C-43, 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 associated with C-43; H-48 (δH 0.93, 3H, s) correlated with C-45, confirming that the fragment is the fourth S-α-HOIva residue. The 1H-1H COSY spectrum showed that H-45 is connected to H-46, further confirming the structure of this fragment.

[0075] Based on the above inferences, the compound is deduced to be a cyclic octapeptide structure, formed by the alternating condensation of four leucines and four oxo-substituted valines / α-hydroxyisovaleric acid. Due to the spatial configuration, the NMR spectrum exhibits distinct peaks, but the overall peaks are similar. The planar structure of the compound is shown below.

[0076]

[0077] A SciFinder search revealed that the compound was a new compound not reported in the literature and was named Xuefenglastatin A. The present invention used the following methods 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 a rate of 1.25×10 5 The cells were seeded at a density of 100 μL / mL into a 96-well culture plate, with 90 μL added to each well. Normal saline was used as the negative control group, and cisplatin was used as the positive control group. The amount of each sample added was 10 μL per well.

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

[0083] The formula for calculating the inhibition rate is: inhibition rate (%) = (OD value of control group - OD value of test group) / OD value of control group. Samples with an inhibition rate greater than 50% were rescreened and IC was calculated. 50 The results are shown in Table 2.

[0084] Table 2

[0085]

[0086] Furthermore, since most cancers are caused by blocked apoptosis, inducing apoptosis in cancer cells is helpful for cancer treatment. Therefore, the present invention compared the effects of Compound I on apoptosis in lung cancer cells.

[0087] Specifically, to further explore the effect of Compound I on lung cancer cell apoptosis and to reveal the potential mechanism of its anti-tumor effect, the present invention first observed the effect of Compound I on cell morphology using phase contrast microscopy and evaluated its effect on cell nuclei using Hoechst 33258 staining to verify its ability to induce lung cancer cell apoptosis. 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 Well-growing cells (A549 cells) were seeded at a density of 1:1 to ensure uniform cell distribution. Subsequently, the six-well plate was placed in an environment of 37°C, 5% CO2 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 cultured for 24 hours.

[0092] (3) Cell morphology observation

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

[0094] The results showed that under high concentration treatment, the cell morphology treated with compound I became rounded and gradually showed signs of detachment 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, and the cells were washed three times with pre-cooled PBS. 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, and the cells were washed three times with PBS. The results were 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. The staining results became brighter at high concentrations, indicating that the cell nuclei shrank, which is a typical nuclear change characteristic during cell apoptosis, indicating that compound I can effectively induce apoptosis in 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] Cells treated in the same manner were collected and prepared into a single-cell suspension of 1 × 10^6 cells / mL. A negative control and a blank control were set up. The cells were washed with PBS, the supernatant discarded, and 100 μL of PBS was retained to disperse the cells. The cells were then fixed overnight in pre-chilled 75% ethanol while vortexing. The fixed cells were centrifuged, washed once with PBS, centrifuged again, and the supernatant discarded. The cells were then stained with the prepared IP staining solution for 10 minutes. Finally, the cells were analyzed by flow cytometry.

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

[0102] (2) Apoptosis protein detection

[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 buffer was added to lyse the cells. 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 the proteins in the gel were then 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 the 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 the secondary antibody for 1 hour. After the incubation was completed, it was washed again with TBST three times for 5 minutes each time. Finally, it was processed with an enhanced chemiluminescence detection kit and the target protein blot was acquired 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), apoptosis-related protein 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. Aberrant expression of Nrf2, Bcl-2, and Caspase-3 is correlated with lung cancer progression and malignant biological behavior. Combined with previously observed cell morphology and nuclear changes, these results confirm that Compound I can induce apoptosis in lung cancer cells in a dose-dependent manner.

[0105] Monomer antibacterial activity detection:

[0106] The MIC values of the corresponding strains were detected by the test tube dilution method, 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 filled with 2.0 mL of liquid culture medium. After adding 5 mg / mL of the test drug to the first tube and mixing thoroughly with the liquid culture medium, 2 mL was added to the second tube. Then, 2 mL was taken from the second tube and added to the third tube, and so on. The final concentrations of the test drug in each tube were 0.25, 0.125, 0.0625, 0.0313, 0.0157, 0.0078, 0.0039, and 0.0020 mg / mL, respectively (the total volume of each tube was 2 mL). Each test tube was diluted with 0.1 mL of a logarithmically growing culture of the respective strain at a concentration of 1 × 10^7 CFU / mL. A control was also established with an equal volume of culture medium containing no drug or bacteria, as well as a blank control containing bacteria but no drug. Bacteria were incubated at 37°C for 24 hours, and the growth of the different strains was observed. The highest drug dilution that did not show bacterial growth was designated as the minimum inhibitory concentration (MIC). 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 MIC determination results of compound I

[0110]

[0111] Note: "+" indicates turbidity and bacterial growth within the test tube as observed by naked eye; "-" indicates clearness and no bacterial growth within the test tube as observed by naked eye. Similarly, the inventors further employed molecular docking to explore the antibacterial mechanism and active groups of Compound I. NorA was selected as the target protein. The receptor protein was retrieved from the PDB database (http: / / www.rcsb.org / pdb). The receptor protein was subjected to operations such as dehydration and ligand removal using PyMOL 2.3.4 software. Modifications such as hydrogenation and charge balancing were performed using AutoDock Tools software. The receptor protein was processed using the Grid Option tool, enabled by the Grid Box command within the Grid program. A docking pocket was generated based on the target protein's own ligand, and the receptor protein was converted to pdbqt format. Molecular docking was performed using AutoDock Vina 1.1.2, using Compound I as the ligand and NorA as the receptor. The affinity of the receptor-ligand complex was evaluated by calculating a comprehensive score based on steric effects, repulsion, hydrogen bonding, hydrophobic interactions, and molecular flexibility, ultimately resulting in an affinity score. 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 shows that compound I can prevent bacteria from transporting it from inside the bacteria to outside the bacteria, thereby prolonging its antibacterial time.

[0112] To evaluate the advantages of Compound I in its antibacterial effect, the inventors combined its binding results with NorA to further evaluate its antibacterial duration. The specific operating method is the same as the above antibacterial test, that is, the revived and activated test strain is inoculated into the corresponding culture medium, cultured with shaking at 37°C until the logarithmic growth phase, and then diluted with sterile saline to a certain concentration so that the bacterial solution concentration reaches 1×10^6 CFU / mL. Different experimental groups and control groups are set up in 96-well plates. The experimental groups are added with different concentrations of Compound I and bacterial solution, and the control groups are added with equal amounts of sterile water and bacterial solution. Three replicates are set up for each concentration. The 96-well plate is placed in a constant temperature incubator and incubated at 37°C. From 0h to 48h, the 96-well plate is removed every 1 hour, and the bacterial solution in each well is serially diluted using the viable bacteria counting method and spread onto the plate culture medium. After incubation at 37°C for 24h, the number of colonies on the plate is counted. The duration for which Compound I maintains the minimum inhibitory concentration of the drug is evaluated. The results are shown in Table 4.

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

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

[0115] Monomer uric acid-lowering activity assay:

[0116] Cancer patients may experience hyperuricemia, primarily due to the massive proliferation of tumor cells, which leads to increased production of metabolites and, consequently, elevated uric acid levels. Furthermore, cancer patients may also receive treatments such as chemotherapy and radiotherapy, which can lead to massive tumor cell destruction or kidney damage, further increasing uric acid production and decreasing excretion, thus causing elevated 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 and incubated at 25°C in the dark for 30 minutes. 0.6 mL of xanthine solution was then added to initiate the reaction, and the reaction was continued at 25°C for 30 minutes. 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 of the reaction system with sample added; A 对照 : Absorbance value of the reaction system with sample added but no xanthine solution added; A 空白 Absorbance of the reaction system without sample addition. Repeat the experiment three times. Use Origin Pro 9.1 to plot a logarithmic curve, with the logarithmic value of sample concentration as the horizontal axis and the inhibition rate as the vertical axis. Set Y = 50 in the statistics page to obtain the sample concentration that results in a 50% inhibition rate.

[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 the target protein. The specific operation was the same as that of anti-tumor molecular docking. The results showed that Compound I had a strong binding interaction with xanthine oxidase through hydrogen bonding and hydrophobic interactions, with an affinity of -8.5 kcal / mol. Figure 10-11 shown.

[0123] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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. A method for extracting oligopeptides, characterized in that: The steps include: S1: crushing the Xuefeng Cordyceps herb, adding 95% ethanol, thoroughly mixing, and then reflux extraction, cooling, filtering, obtaining a filtrate and a filter residue, and retaining the filtrate; S2: adding the filter residue to 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 freeze-drying it in vacuum 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: concentrating the ethyl acetate extract from step S4 under reduced pressure, and freezing it in a vacuum to obtain an ethyl acetate extract; S6: dissolving the ethyl acetate extract of 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; The structure of the oligopeptide is as follows: 。 2. The method for extracting oligopeptides according to claim 1, wherein: The reflux extraction time is 2 hours.

3. The method for extracting oligopeptides according to claim 1, wherein: The temperature of the condensation and reduced pressure concentration is 60°C; The vacuum degree of the condensation and reduced pressure concentration is ≤0.095MPa; The vacuum freeze-drying temperature is -50°C.

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