Ganoderma lucidum oligopeptide with anti-tumor effect and preparation method thereof

Abstract

The invention provides lucid ganoderma oligopeptide with an anti-tumor effect and a preparation method thereof. The three oligopeptides are separated and purified from the ganoderma lucidum for the first time, and the oligopeptides have broad-spectrum anti-tumor activity, can effectively inhibit tumor cell proliferation, have good biological compatibility and have great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment techniques, and more specifically, to a Ganoderma lucidum oligopeptide with anti-tumor efficacy and a preparation method thereof. Background Art

[0002] Ganoderma lucidum is a fungus of the Ganodermataceae family and the Ganoderma genus. Most Ganoderma lucidum fruiting bodies are annual, and a few are perennial, with a stalk and a lateral small stalk. Ganoderma lucidum is distributed on all continents of the world, and most of it grows in tropical, subtropical and temperate regions. China is a place where Ganoderma lucidum fungal resources are widely distributed.

[0003] Ganoderma lucidum is one of the traditional precious Chinese medicinal materials and can be used as both medicine and food. Ganoderma lucidum has various functions such as antioxidant, immune regulation, anti-tumor, blood pressure lowering, blood lipid lowering, and antiviral, and can be used for the prevention and treatment of various diseases. The active ingredients of Ganoderma lucidum mainly concentrate on polysaccharides, nucleosides, furans, sterols, alkaloids, triterpenoids, oils, various amino acids and proteins, enzymes, organic germanium and various trace elements, etc.

[0004] Among them, Ganoderma lucidum polypeptide is another substance that can repair damaged cranial nerves following nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), and glial cell line-derived neurotrophic factor (GDNF). In the prior art, the separation, purification and identification of the active oligopeptide components in Ganoderma lucidum polypeptide have always been the technical hotspots in this industry. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art and to effectively explore the potential of Ganoderma lucidum active oligopeptides, the present invention provides a Ganoderma lucidum oligopeptide with anti-tumor efficacy and a preparation method thereof. The present invention first isolates and purifies three oligopeptides from Ganoderma lucidum. The oligopeptides have broad-spectrum anti-tumor activity, can effectively inhibit the proliferation of tumor cells, and have great application prospects.

[0006] Specifically, the present invention first provides a preparation method of Ganoderma lucidum oligopeptide, including the following steps:

[0007] 1) Preparation of materials: After cleaning Ganoderma lucidum, cut it into pieces and dry it in the air at room temperature for later use;

[0008] 2) Physical pulverization: Put the Ganoderma lucidum pieces dried in the air at room temperature in step 1) into a wall breaker and pulverize them into fine particles;

[0009] 3) Complex enzymatic hydrolysis: The fine Ganoderma lucidum particles from step 2) are added to 5 times the volume of ultrapure water for dissolution, and compound protease is added at 20% (v / v). After continuous stirring and enzymatic hydrolysis, the supernatant is obtained by centrifugation to obtain the Ganoderma lucidum oligopeptide enzymatic hydrolysate;

[0010] 4) Filtration and concentration: The Ganoderma lucidum oligopeptide enzymatic hydrolysate from step 3) is filtered using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1000 - 3500 Da; The filtered Ganoderma lucidum oligopeptide is concentrated under vacuum to obtain the Ganoderma lucidum oligopeptide concentrate;

[0011] 5) Identification of Ganoderma lucidum oligopeptide: Take the Ganoderma lucidum oligopeptide concentrate from step 4) for mass spectrometry sequencing, and the amino acid sequences of the Ganoderma lucidum oligopeptides obtained through identification are shown as SEQ ID NO.1 - 3 in sequence;

[0012] 6) Purity identification of Ganoderma lucidum oligopeptide: The Ganoderma lucidum oligopeptide in step 5) is analyzed by high performance liquid chromatography for its purity;

[0013] 7) Transfer the Ganoderma lucidum oligopeptide concentrate with qualified purity to a spray dryer, with an inlet air temperature of 80 - 100 °C and an outlet air temperature of 60 - 80 °C for concentration until the water content of the Ganoderma lucidum oligopeptide dry powder is 5 - 9%, and collect the dry powder to obtain the Ganoderma lucidum oligopeptide.

[0014] Preferably, in step 1), the size of the fragments is fragments with a length, width, and height of 0.5 - 2.0 cm.

[0015] Preferably, the wall-breaking condition in step 2) is wall-breaking at 50000 - 100000 r / min for 5 - 30 min.

[0016] Preferably, the compound protease in step 3) is a compound of neutral protease and papain, and the dosage ratio of the two is 2 - 5:1.

[0017] Preferably, the enzymatic hydrolysis condition in step 3) is continuous stirring and enzymatic hydrolysis for 5 - 12 h at a temperature of 25 - 37 °C.

[0018] Preferably, step 3) further includes heating the enzymatic hydrolysate to 90 - 100 °C and keeping it at a constant temperature for 10 - 30 min to inactivate the enzyme.

[0019] Preferably, the centrifugation condition in step 3) is centrifugation at 3000 - 5000 r / min for 30 - 60 min.

[0020] Preferably, the vacuum concentration in step 4) is concentrated to a concentration of 10 - 18 °Bé.

[0021] Preferably, the amino acid sequences of the Ganoderma lucidum oligopeptides obtained through identification in step 5) are shown as SEQ ID NO.1 - 3 in sequence.

[0022] Another object of the present invention is to provide an anti-tumor drug, which takes Ganoderma lucidum oligopeptide as the main active ingredient, and the amino acid sequence of the Ganoderma lucidum oligopeptide is any one or a combination of SEQ ID NO.1-3.

[0023] Preferably, the Ganoderma lucidum oligopeptide is a Ganoderma lucidum oligopeptide obtained by combining three different sequences of SEQ ID NO.1-3 in the amino acid sequence.

[0024] Furthermore, the present invention also provides the use of the Ganoderma lucidum oligopeptide in the preparation of an anti-tumor drug, and the amino acid sequence of the Ganoderma lucidum oligopeptide is any one or a combination of SEQ ID NO.1-3.

[0025] Preferably, the tumors include but are not limited to breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, gastric cancer, esophageal cancer, colon cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer, uterine cancer, etc.

[0026] The advantages of the present invention are as follows: The present invention first isolates and purifies three kinds of oligopeptides from Ganoderma lucidum. The oligopeptides have broad-spectrum anti-tumor activity, can effectively inhibit the proliferation of tumor cells, have good biocompatibility, and have great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph showing the analysis results of the proliferation activity of tumor cells by the Ganoderma lucidum oligopeptide of the present invention;

[0028] Figure 2 It is a graph showing the analysis results of the tumor killing effect of the Ganoderma lucidum oligopeptide of the present invention on model mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The following will further elaborate on the present invention in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0030] The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0031] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are all conventional means well-known to those skilled in the art.

[0032] Example 1 Preparation, Isolation and Identification of Ganoderma lucidum Oligopeptide

[0033] The preparation method of Ganoderma lucidum oligopeptide includes the following steps:

[0034] 1) Preparation of materials: After cleaning Ganoderma lucidum, cut it into small pieces and air-dry at room temperature for later use;

[0035] 2) Physical pulverization: Put the Ganoderma lucidum pieces air-dried at room temperature in step 1) into a wall breaker and pulverize them into fine particles;

[0036] 3) Composite enzymatic hydrolysis: Add 10 mg of the Ganoderma lucidum fine particles obtained in step 2) to 50 mL of ultrapure water for dissolution, and add 20% (v / v) composite protease. Continuously stir and hydrolyze for 5 h, then heat up to 90 °C and keep it at a constant temperature for 15 min to inactivate the enzyme. Subsequently, centrifuge to separate the supernatant to obtain the Ganoderma lucidum oligopeptide enzymatic hydrolysate; among them, the composite protease is a compound of neutral protease and papain, and the mass ratio of the two is 2:1; the enzymatic hydrolysis condition is continuous stirring and enzymatic hydrolysis at 37 °C for 10 h.

[0037] 4) Filtration and concentration: Filter the Ganoderma lucidum oligopeptide enzymatic hydrolysate in step 3) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1000 - 3500 Da; Vacuum concentrate the filtered Ganoderma lucidum oligopeptide to a concentration of 14 °Bé to obtain the Ganoderma lucidum oligopeptide concentrated solution;

[0038] 5) Identification of Ganoderma lucidum oligopeptide: Take the Ganoderma lucidum oligopeptide concentrated solution in step 4) for sequencing with an Obitrap Fusion Lumos mass spectrometer;

[0039] 6) Purity identification of Ganoderma lucidum oligopeptide: Transfer the Ganoderma lucidum oligopeptide concentrated solution to a spray dryer, with an inlet air temperature of 100 °C and an outlet air temperature of 80 °C for concentration until the water content of the Ganoderma lucidum oligopeptide dry powder is 6%. Collect the dry powder to obtain Ganoderma lucidum oligopeptide.

[0040] The results show that: A total of 3 active peptides were obtained through sequencing and identification analysis, and the sequences are shown as SEQ ID NO.1 - 3 in sequence. After purity determination, the purity of the obtained Ganoderma lucidum polypeptide is 95.6%.

[0041] Example 2 Analysis of the anti-tumor effect of Ganoderma lucidum oligopeptide

[0042] 2.1 Analysis of the proliferation activity of Ganoderma lucidum oligopeptide on tumor cells

[0043] Culture breast cancer cells MCF7, pancreatic cancer cells PANC-1, lung cancer cells A549 and hepatocellular carcinoma cells HEPG2 respectively, at 1×10 4Cells were inoculated into a 96-well cell culture plate at a density of [quantity] cells per well, with 100 μL per well. The plate was placed in an incubator and incubated for 6 h for cell attachment. Then, Ganoderma lucidum oligopeptide solutions with a final concentration of 10 μg / mL, as shown in SEQ ID NO.1-3, were prepared using cell culture medium and added to the 96-well plate one by one, 100 μL per well, and co-incubated for 12 h. Among them, treatment groups 1-3 were Ganoderma lucidum oligopeptides shown in SEQ ID NO.1-3 in sequence, treatment group 4 was a mixture of Ganoderma lucidum oligopeptides shown in SEQ ID NO.1-3 (a mixture of three Ganoderma lucidum oligopeptides with different sequences in a mass ratio of 1:1:1), the control group was not treated with Ganoderma lucidum oligopeptide, and the blank group was pure culture medium. An MTT cell proliferation and cytotoxicity detection kit (purchased from Shangbao Biotechnology, product number 20311) was used to analyze the proliferation activity of tumor cells.

[0044] The results are as Figure 1 shown. The three Ganoderma lucidum oligopeptides prepared by the present invention all have good inhibitory activity against tumor cell proliferation. Especially when the three Ganoderma lucidum oligopeptides are used in combination, their inhibitory effect on tumor cell proliferation is the most obvious.

[0045] 2.2 In vivo anti-tumor activity of Ganoderma lucidum oligopeptide

[0046] Taking lung cancer cell A549 as an example, 1×10 6 A549 cells were injected into the abdomen of immunodeficient NSG mice to establish a mouse lung cancer tumor model. When the tumor volume reached 80 mm 3 , 100 μg / kg of the mixed Ganoderma lucidum oligopeptide (treatment group 4) was administered via the tail vein. The abdominal tumor volume was monitored weekly, and a tumor growth curve was made based on the tumor volume. As Figure 2 can be seen, compared with the blank group, the mixed Ganoderma lucidum oligopeptide (a mixture of three Ganoderma lucidum oligopeptides with different sequences in SEQ ID NO.1-3 in a mass ratio of 1:1:1) can significantly inhibit tumor growth and reduce the tumor volume. This shows that the Ganoderma lucidum oligopeptide isolated by the present invention has good anti-tumor activity in vivo. The in vivo anti-tumor activity results for other tumor model mice also similarly show that Ganoderma lucidum oligopeptide has a general killing activity against solid tumors and can effectively inhibit tumor growth.

[0047] It is necessary to point out here that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not further limitations on the technical solution of the present invention. The method of the present invention is only a preferred implementation, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Ganoderma lucidum oligopeptide, characterized in that: The following steps are involved: 1) Preparation: Wash the Ganoderma lucidum, cut it into small pieces, and dry it with air at room temperature for later use; 2) Physical crushing: Put the Ganoderma lucidum pieces dried by air at room temperature in step 1) into a wall-breaking machine and crush them into fine particles; 3) Composite enzymatic hydrolysis: add 5 times the volume of ultrapure water to dissolve the Ganoderma lucidum fine particles obtained in step 2), and add 20% (v / v) composite protease, continue stirring for enzymatic hydrolysis, and centrifuge to separate the supernatant to obtain Ganoderma lucidum oligopeptide enzymatic hydrolyzate; 4) Filtration and concentration: the enzymatic hydrolyzate of the Ganoderma lucidum oligopeptide in step 3) is filtered by an ultrafiltration membrane, with a molecular weight cutoff of 1000-3500Da polypeptide molecules; the filtered Ganoderma lucidum oligopeptide is vacuum concentrated to obtain a Ganoderma lucidum oligopeptide concentrate; 5) Identification of Ganoderma lucidum oligopeptides: The Ganoderma lucidum oligopeptide concentrate obtained in step 4) was subjected to mass spectrometry sequencing, and the amino acid sequences of the Ganoderma lucidum oligopeptides obtained by identification were shown in SEQ ID NO. 1-3 in sequence; 6) Purity identification of the Ganoderma lucidum oligopeptide: The Ganoderma lucidum oligopeptide in step 5) is subjected to high performance liquid chromatography to analyze its purity; 7) The Ganoderma lucidum oligopeptide concentrate with qualified purity is transferred to a spray dryer, and the air inlet temperature is 80-100° C. and the air outlet temperature is 60-80° C. to concentrate the Ganoderma lucidum oligopeptide dry powder to a moisture content of 5-9%, and the dry powder is collected to obtain Ganoderma lucidum oligopeptide.

2. The method according to claim 1, characterized in that In step 1), the size of the fragments is 0.5 to 2.0 cm in length, width and height.

3. The method according to claim 1, characterized in that Step 2) The wall breaking condition is 50000-100000 r / min for 5-30 min.

4. The method according to claim 1, characterized in that Step 3) the composite protease is a combination of neutral protease and papain, with the mass ratio of the two being 2-5:

1.

5. The method according to claim 1, characterized in that The enzymatic hydrolysis conditions in step 3) are continuous stirring at a temperature of 25-37° C. for 5-12 hours; and the centrifugal conditions are centrifugation at 3000-5000 r / min for 30-60 minutes.

6. The method according to claim 1, step 3) further comprises heating the enzymatic hydrolysate to 90-100°C and maintaining the temperature for 10-30 minutes to inactivate the enzyme.

7. The method according to claim 1, characterized in that The vacuum concentration in step 4) is carried out to a concentration of 10-18° Bé.

8. An anti-tumor drug, characterized in that: The drug uses Ganoderma lucidum oligopeptide as a main active ingredient, and the amino acid sequence of the Ganoderma lucidum oligopeptide is any one or a combination of SEQ ID NO.1-3.

9. Use of Ganoderma lucidum oligopeptide in the preparation of anti-tumor drugs, characterized in that: The amino acid sequence of the Ganoderma lucidum oligopeptide is any one or a combination of SEQ ID NO. 1-3.

10. The medicine according to claim 8 or the use according to claim 9, characterized in that: The tumor includes, but is not limited to, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, gastric cancer, esophageal cancer, colon cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer or uterine cancer.

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