High-purity zedoaria lectin, preparation method and application

Abstract

The present invention provides a preparation method and application of a highly pure product of Curcuma longifolia lectin, which is characterized by comprising the following steps: 1. sample processing: peeling and crushing the fresh rhizome of Curcuma longifolia, and extracting at low temperature with Tris-HCl buffer; 2. crude product preparation: solid ammonium sulfate precipitation, dialysis; 3. semi-finished product preparation: the crude product is separated and purified using a cellulose anion exchange chromatography DEAE-52 column; 4. pure product preparation: the semi-finished product is separated and purified using a dextran gel chromatography Sephadex G-75 column to obtain a highly pure Curcuma longifolia lectin WLT. The pure Curcuma longifolia lectin prepared by the present invention has a molecular weight of approximately 28 KDa, is a mannose-binding natural protein, and has broad-spectrum anti-tumor activity. Therefore, the lectin product prepared by the present invention can be used as a new drug source for the research and development of anti-tumor drugs, which is conducive to accelerating the development of novel natural protein drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a high-purity zedoaria lectin, a preparation method and an application thereof. Background Art

[0002] Wen Yujin Curcuma wenyujin YHChen & C.Ling is a plant of the genus Curcuma in the Zingiberaceae family, a local medicinal material in Zhejiang Province. Its underground rhizomes and tubers can be processed in various ways to produce three traditional Chinese medicines: Curcuma zedoaria, Curcuma longa, and Curcuma aromatica. Modern research indicates that the volatile oil (curcuma oil) extracted from the rhizomes of Curcuma zedoaria contains terpenes such as curdiene, curcumol, turmericdiol, curcumene, δ-elemene, and β-elemene, demonstrating antiviral, anti-inflammatory, analgesic, and anti-tumor properties. However, the effective terpenoid content in Curcuma zedoaria is low, and exploring new medicinal sources for Curcuma zedoaria is a future development trend.

[0003] Plant lectins are a class of non-immunogenic proteins with numerous sugar-specific binding sites on their surface. Recent studies have shown that plant lectins can specifically bind to sugar molecules on the surface of cancer cell membranes, thereby inducing apoptosis or inhibiting tumor growth. This represents a new area of ​​drug development and medical applications. Our previous high-throughput genomic sequencing revealed that the rhizomes of Curcuma wenyujin contain numerous genes encoding lectin proteins at high expression levels, suggesting that these genes are rich in plant-derived lectin proteins. Therefore, the extraction and preparation of high-purity Curcuma wenyujin lectin products holds great promise for future applications.

[0004] The Chinese patent document "A Method for Preparing a Mixture of Crude and Pure Wenyujin Lectin and PGRP Strain, and Wenyujin Fertilizer" (Patent No.: CN 109553672A) discloses a method for extracting Wenyujin lectin, including pulping, enzymatic hydrolysis, ammonium sulfate precipitation, and dialysis. The resulting product is a crude lectin. This technology product is primarily used in conjunction with a plant rhizome growth-promoting bacterial strain (PGRP) to produce a biofertilizer. However, the patent utilizes organic solvents such as acetone and petroleum ether, and the technology product cannot be directly used as a source of new biopharmaceuticals. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a high-purity zedoaria lectin, a preparation method and an application thereof.

[0006] The technical solution adopted by the present invention is as follows: a method for preparing a high-purity product of Curcuma agglutinin comprises the following steps:

[0007] S1: Sample processing: Wash, peel, and crush fresh rhizomes of Curcuma aromatica. Add Tris-HCl buffer and extract for 12-16 h. Centrifuge and collect the supernatant.

[0008] S2: Crude product preparation: solid ammonium sulfate was added to the supernatant obtained in step S1 and stirred thoroughly until the saturation reached 60-70%. The mixture was allowed to stand for 10-12 hours, centrifuged, and the precipitate was dissolved in Tirs-HCl buffer, dialyzed, and freeze-dried to obtain the crude product of Curcuma agglutinin;

[0009] S3: Preparation of semi-finished product: The crude product of Curcuma australis lectin obtained in step S2 was dissolved in Tris-HCl buffer, separated and purified using a DEAE-52 cellulose anion exchange column, and gradient eluted using Tris-HCl buffer containing 0.2-1.0 mol / l NaCl. The protein peaks with coagulant activity were combined and collected, and freeze-dried to obtain the Curcuma australis lectin semi-finished product;

[0010] S4: Preparation of high-purity product: The semi-finished product of Curcuma australis lectin obtained in step S3 was dissolved in Tris-HCl buffer, separated and purified using a Sephadex G-75 column, and gradient elution using Tris-HCl buffer containing 0.2-1.0 mol / l NaCl was performed to collect the test tube liquid with the coagulation activity protein peak, combine the liquid, and freeze-dry to obtain a high-purity product of Curcuma australis lectin.

[0011] The leaching in step S1 and the standing in step S2 are both performed at 4-6°C.

[0012] The material-liquid ratio of the mass of the pulverized material to the volume of the buffer solution in step S1 is 1:4-6.

[0013] The crude lectin in step S3 and the semi-finished product in step S4 are both dissolved in Tris-HCl buffer with a weight / volume ratio of 1:8 to 15.

[0014] The gradient elution process in steps S3 and S4 includes a step of using an ultraviolet spectrophotometer to monitor the absorbance of the effluent at 280 nm in real time to determine the protein peak of the eluate until the absorbance of the effluent approaches a normal value.

[0015] The specific parameters of the centrifugation in steps S1 and S2 are as follows: temperature 4-6°C, rotation speed 5000-8000 rpm, and centrifugation time 10-15 min.

[0016] The Tris-HCl buffer has a concentration of 10-12 mM and a pH of 7-9.

[0017] The high-purity product of Curcuma australis lectin prepared by the preparation method described above is obtained.

[0018] The high-purity product of Curcuma australis lectin as described above is used for preparing anti-tumor drugs.

[0019] An anti-tumor drug comprising the above-mentioned high-purity Wenyujin lectin.

[0020] The beneficial effects of the present invention are as follows: the Wenyujin lectin prepared by the present invention has high purity and low impurity protein, which can ensure the medicinal value of the product. Furthermore, the high-purity Wenyujin lectin provided by the present invention has broad-spectrum anti-tumor activity, with an inhibitory concentration (IC) of 50% against lung cancer H460, lung cancer A549, breast cancer BT-549, liver cancer SMMC-772, colon cancer HCT-116 and other cells. 50 ) were 146.60 ug / ml, 113.38 ug / ml, 141.96 ug / ml, 138.10 ug / ml, and 98.46 ug / ml, respectively. The pure lectin obtained in the present invention can be used as a new source for the development of anti-tumor drugs, which is conducive to accelerating the development of novel natural protein drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying any creative work, other drawings obtained based on these drawings still fall within the scope of the present invention.

[0022] Figure 1 This is the elution curve of the zedoaria agglutinin on a DEAE-52 column using anion exchange chromatography in Example 1.

[0023] Figure 2 This is the elution curve of the semi-finished product of Curcuma agglutinin in Example 1 after chromatography on a Sephadex G-75 column.

[0024] Figure 3 This is an SDS-PAGE image of the pure Wenyujin lectin WLT prepared in Example 1.

[0025] Figure 4 This is a graph showing the coagulation activity of the pure WLT Wenyujin lectin prepared in Example 1-3.

[0026] Figure 5 This is a graph showing the anti-lung cancer A549 cell activity of the pure WLT Wenyujin lectin prepared in Example 1-3.

[0027] Figure 6 This is a graph showing the activity of the pure WLT Wenyujin lectin prepared in Example 1-3 against lung cancer H460 cells.

[0028] Figure 7This is a graph showing the activity of the pure WLT Wenyujin lectin prepared in Example 1-3 against breast cancer BT-549 cells.

[0029] Figure 8 This is a graph showing the activity of the pure WLT Wenyujin lectin prepared in Example 1-3 against liver cancer SMMC-772 cells.

[0030] Figure 9 This is a graph showing the activity of the pure WLT Wenyujin lectin prepared in Example 1-3 against liver cancer and colon cancer HCT-116 cells. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0032] Example 1

[0033] A high-purity product of Curcuma longa lectin is prepared by the following preparation process:

[0034] S1: Sample processing: Wash and peel 100 g of fresh Curcuma aromatica rhizomes, prepare a homogenate using a grinder, add 500 ml of Tris-HCl buffer (pH = 7.5, concentration of 10 mM), place in a 4 °C refrigerator for 12 h, centrifuge at 7000 rpm at 4 °C for 15 min, and collect the supernatant.

[0035] S2: Preparation of crude product: The above supernatant was placed on a magnetic stirrer and solid ammonium sulfate was added and stirred thoroughly until the saturation reached 65%. The supernatant was placed in a 4°C refrigerator for 10 h. The supernatant was centrifuged at 6000 rpm at 4°C for 10 min. The precipitate was dissolved in Tirs-HCl buffer (pH = 7.5, concentration: 10 mM), dialyzed for desalination, and freeze-dried after 24 h to obtain the crude Curcuma agglutinin.

[0036] S3: Preparation of semi-finished product: 0.5 g of the above crude lectin was dissolved in 6 ml of Tris-HCl buffer (pH = 7.5, concentration of 10 mM). The dissolved solution was separated and purified on a DEAE-52 cellulose anion exchange column. Gradient elution was performed using Tris-HCl buffer (pH = 7.5, 10 mM) containing 0.3 mol / l NaCl. The peristaltic pump controlled the flow rate at 0.5 ml / min and collected 5 ml in each tube. Furthermore, the absorbance of the effluent at 280 nm was monitored by an ultraviolet spectrophotometer. The elution was performed until the absorbance of the effluent approached normal values. In this example, four protein peaks (such as Figure 1 ), the eluate was further evaluated for activity using 2% rabbit red blood cells to determine the protein peak with coagulation activity (such as Figure 1In this example, peak 2 was obtained, and the test tube liquids with the coagulation activity protein peak were combined and freeze-dried to obtain the semi-finished product of Curcuma agglutinin.

[0037] S4: Preparation of high-purity products: 200 mg of the above-mentioned lectin semi-finished product was dissolved in 3 ml of Tris-HCl buffer (pH = 7.5, concentration of 10 mM), and the dissolved solution was separated and purified on a Sephadex G-75 column. Gradient elution was performed using a Tris-HCl buffer (pH = 7.5, 10 mM) containing 0.3 mol / l NaCl. The peristaltic pump controlled the flow rate to 0.5 ml / min, and 5 ml was collected in each tube. Furthermore, the absorbance of the effluent at 280 nm was monitored by an ultraviolet spectrophotometer. The elution was performed until the absorbance of the effluent approached normal values, and the activity of the effluent was evaluated using 2% rabbit red blood cells to determine the protein peak with coagulation activity. The protein peak with coagulation activity (such as Figure 2 ) in the test tube, combined and freeze-dried to obtain the high-purity WLT of Curcuma agglutinin.

[0038] SDS-PAGE electrophoresis of WLT revealed that the protein showed only a single band (e.g. Figure 3 ), indicating that WLT is a pure lectin substance with a molecular weight of approximately 28 KDa.

[0039] Example 2:

[0040] A high-purity product of Curcuma longa lectin, the preparation process of which is as follows:

[0041] S1: Sample processing: Wash and peel 60 g of fresh Curcuma aromatica rhizomes, crush them in a homogenizer, add 250 ml of Tris-HCl buffer (pH = 9, concentration of 12 mM), place in a 6 °C refrigerator for 16 h, centrifuge at 5 °C, 8000 rpm for 10 min, and collect the supernatant.

[0042] S2: Preparation of crude product: The supernatant was placed on a magnetic stirrer and solid ammonium sulfate was added and stirred thoroughly until the saturation reached 70%. The supernatant was placed in a 6°C refrigerator for 10 h. The supernatant was centrifuged at 7000 rpm at 5°C for 10 min. The precipitate was dissolved in Tirs-HCl buffer (pH = 9, concentration: 12 mM), dialyzed for desalination, and freeze-dried after 24 h to obtain the crude Curcuma agglutinin.

[0043] S3: Preparation of semi-finished product: Dissolve 0.2 g of the crude lectin in 2 ml of Tris-HCl buffer (pH = 9, concentration 12 mM). Apply the dissolved solution to a DEAE-52 cellulose anion exchange column for separation and purification. Use Tris-HCl buffer (pH = 9, 12 mM) containing 0.5 mol / l NaCl for gradient elution. Use a peristaltic pump to control the flow rate at 0.5 ml / min. Collect 5 ml in each tube. Combine the test tubes with the coagulation activity protein peak and freeze-dry to obtain the lectin semi-finished product.

[0044] S4: Preparation of High-Purity Product: 100 mg of the semi-finished lectin was dissolved in 1 ml of Tris-HCl buffer (pH = 9, 12 mM). The solution was separated and purified on a Sephadex G-75 column using a gradient elution method using Tris-HCl buffer (pH = 9, 12 mM) containing 0.5 mol / l NaCl. The flow rate was controlled by a peristaltic pump at 0.5 ml / min. 5 ml of the solution was collected from each tube. The test tubes containing the coagulation activity protein peak were collected, combined, and freeze-dried to obtain the highly pure lectin WLT. The results of SDS-PAGE electrophoresis of the WLT protein were the same as those in Example 1.

[0045] Example 3:

[0046] A high-purity product of Curcuma longa lectin, the preparation process of which is as follows:

[0047] S1: Sample processing: Wash and peel 300 g of fresh Curcuma aromatica rhizomes, crush them in a homogenizer, add 1800 ml of Tris-HCl buffer (pH = 8, concentration of 10 mM), place in a 5°C refrigerator for 14 h, centrifuge at 6°C, 6000 rpm for 12 min, and collect the supernatant.

[0048] S2: Preparation of crude product: The supernatant was placed on a magnetic stirrer and solid ammonium sulfate was added and stirred thoroughly until the saturation reached 60%. The supernatant was placed in a 5°C refrigerator for 12 h. The supernatant was centrifuged at 6000 rpm for 12 min at 6°C. The precipitate was dissolved in Tirs-HCl buffer (pH = 8, concentration: 10 mM), dialyzed for desalination, and freeze-dried after 24 h to obtain the crude Curcuma agglutinin.

[0049] S3: Preparation of semi-finished product: Dissolve 1 g of the crude lectin in 8 ml of Tris-HCl buffer (pH = 8, concentration 10 mM). Apply the dissolved solution to a DEAE-52 cellulose anion exchange column for separation and purification. Use Tris-HCl buffer (pH = 8, 10 mM) containing 1.0 mol / l NaCl for gradient elution. Use a peristaltic pump to control the flow rate at 0.5 ml / min. Collect 5 ml in each tube. Combine the test tube liquids with the coagulation activity protein peak and freeze-dry to obtain the lectin semi-finished product.

[0050] S4: Preparation of a Highly Pure Product: 500 mg of the semi-finished lectin was dissolved in 7.5 ml of Tris-HCl buffer (pH 8, 10 mM). The solution was then purified on a Sephadex G-75 column using a gradient elution method using Tris-HCl buffer (pH 8, 10 mM) containing 1.0 mol / l NaCl. The flow rate was controlled by a peristaltic pump at 0.5 ml / min. 5 ml of the solution was collected from each tube. The tubes containing the coagulation-active protein peak were collected, combined, and freeze-dried to obtain a highly pure lectin WLT. The WLT protein was analyzed by SDS-PAGE electrophoresis, and the results were the same as in Example 1.

[0051] The high-purity Wenyujin lectin WLT prepared by the present invention is a mannose-binding lectin, which has obvious inhibitory effects on human liver cancer, lung cancer, breast cancer and colon cancer cells, and can be used as a new source of anticancer drugs.

[0052] In order to further understand the characteristics of Wenyujin lectin WLT, its sugar inhibition and anti-tumor activity are described in detail below.

[0053] Example 4: Wenyujin lectin WLT coagulation activity detection and sugar inhibition experiment

[0054] Prepare a 0.5 mg / ml solution of the WLT lectin mentioned above. Add 50 μl of physiological saline (containing 10 mM Ca) to a 96-well plate. 2+ ), add 50 μl of lectin solution to the first well of each row, and then add 50 μl of 2% rabbit red blood cell suspension to each well, mix well, and observe the results after standing at room temperature for 2 hours. Figure 4 , indicating that WLT has strong coagulant activity.

[0055] A 50 mM sugar solution of D-glucose, D-fructose, D-mannose, D-galactose, D-arabinose, and L-rhamnose was prepared. 30 μL of each sugar solution and 30 μL of a 2% rabbit red blood cell suspension were added to a 96-well plate and mixed thoroughly. Then, 40 μL of the lectin solution was added to the first well of each row. The plate was incubated at room temperature for 2 hours and the coagulation effect was observed. The results of the sugar inhibition test are shown in Table 1. D-mannose showed the most significant inhibition on WLT lectin activity, while the other sugar solutions had a minor inhibitory effect. This indicates that D-mannose can compete with red blood cells for WLT binding, and therefore WLT is a mannose-binding lectin.

[0056]

[0057] Example 5: Activity detection of WLT anti-tumor cell line

[0058] The anti-tumor activity of WLT was detected by CCK-8 assay. The tumor cell lines used were lung cancer A549, lung cancer H460, breast cancer BT-549, colon cancer HCT-116, and liver cancer SMMC-772. Cells in the logarithmic growth phase were obtained and the culture medium of each cell line was prepared to a concentration of 2×10 6 Cells were plated at 400 μg / ml, 200 μl was taken from each well and added to a 96-well plate for culture until adhered. A 10 mg / ml stock solution of Wenyujin lectin (WLT) was prepared and added to a 96-well plate. The solution was diluted to 0, 20, 50, 100, and 200 μg / mL of lectin in the drug-containing medium and cultured for 48 h. Subsequently, 10 μl of CCK-8 solution was added to each well and the cells were incubated in an incubator for 30 min. The absorbance was read at 450 nm on a microplate reader. The experiment was repeated three times and the cell inhibition rate was calculated. The blank control was the medium containing CCK-8 solution; the control group was the medium containing cells and CCK-8 solution (without drug addition). The tumor cell growth inhibition rate was calculated according to the following formula: Tumor cell growth inhibition rate (%) = , IC 50 is the drug concentration at which the cell growth inhibition rate is 50%.

[0059] The results are as follows Figures 5 to 9 As shown in the results, WLT has a significant inhibitory effect on tumor cell lines. With the increase of WLT dosage, the cell growth inhibition rate increases, indicating that the inhibition rate is in a dose-effect relationship. The 50% inhibitory concentration (IC) of WLT on lung cancer H460 cells, lung cancer A549 cells, breast cancer BT-549 cells, liver cancer SMMC-772 cells, and colon cancer HCT-116 cells is 50) were 146.60 ug / ml, 113.38 ug / ml, 141.96 ug / ml, 138.10 ug / ml, and 98.46 ug / ml, respectively. In particular, WLT had the most significant inhibitory effect on colon cancer HCT-116 cells.

[0060] Unless otherwise specified, the materials and reagents used in the above examples can be purchased from commercial channels.

[0061] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing a high-purity product of Curcuma agglutinin, characterized in that: The following steps are involved: S1: Sample processing: Wash, peel, and crush fresh rhizomes of Curcuma aromatica. Add Tris-HCl buffer and extract for 12-16 h. Centrifuge and collect the supernatant. S2: Crude product preparation: solid ammonium sulfate was added to the supernatant obtained in step S1 and stirred thoroughly until the saturation reached 60-70%. The mixture was allowed to stand for 10-12 hours, centrifuged, and the precipitate was dissolved in Tirs-HCl buffer, dialyzed, and freeze-dried to obtain the crude product of Curcuma agglutinin; S3: Preparation of semi-finished product: The crude product of Curcuma australis lectin obtained in step S2 was dissolved in Tris-HCl buffer, separated and purified using a DEAE-52 cellulose anion exchange column, and gradient eluted using Tris-HCl buffer containing 0.2-1.0 mol / l NaCl. The protein peaks with coagulant activity were combined and collected, and freeze-dried to obtain the Curcuma australis lectin semi-finished product; S4: Preparation of a high-purity product: The semi-finished product of Curcuma australis lectin obtained in step S3 was dissolved in Tris-HCl buffer, separated and purified using a Sephadex G-75 column, and gradient eluted using Tris-HCl buffer containing 0.2-1.0 mol / l NaCl. The test tubes containing the protein peak with coagulation activity were collected, combined, and freeze-dried to obtain a high-purity product of Curcuma australis lectin; The crude lectin in step S3 and the semi-finished product in step S4 are both dissolved in Tris-HCl buffer with a weight / volume ratio of 1:8 to 15; The gradient elution process in steps S3 and S4 includes a step of monitoring the absorbance of the effluent at 280 nm in real time using an ultraviolet spectrophotometer to determine the protein peak of the eluate until the absorbance of the effluent approaches a normal value; During the gradient elution in steps S3 and S4, the peristaltic pump controlled the flow rate to be 0.5 ml / min.

2. The method for preparing a high-purity product of Curcuma agglutinin according to claim 1, characterized in that: The leaching in step S1 and the standing in step S2 are both performed at 4-6°C.

3. The method for preparing a high-purity product of Curcuma agglutinin according to claim 1, characterized in that: The material-liquid ratio of the mass of the pulverized material to the volume of the buffer solution in step S1 is 1:4-6.

4. The method for preparing a high-purity product of Curcuma agglutinin according to claim 1, characterized in that: The specific parameters of the centrifugation in steps S1 and S2 are as follows: temperature 4-6°C, rotation speed 5000-8000 rpm, and centrifugation time 10-15 min.

5. The method for preparing a high-purity product of Curcuma agglutinin according to claim 1, characterized in that: The Tris-HCl buffer has a concentration of 10-12 mM and a pH of 7-9.

6. Use of the high-purity Wenyujin lectin prepared by the method for preparing the high-purity Wenyujin lectin according to any one of claims 1 to 5 for preparing an anti-tumor drug, wherein the tumor is one of lung cancer, breast cancer, liver cancer, and colon cancer.

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