Application of mulberry leaf extract in preparation of products for treating diabetic complications
Patent Information
- Application Number
- CN202510587967.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-06-24
Smart Images
Figure CN120189455A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to health products, and in particular to the use of mulberry leaf extract in the preparation of products for treating diabetic complications. Background Art
[0002] Diabetes not only leads to metabolic disorders, but is also associated with a variety of serious complications. Cerebrovascular lesions and abnormal cerebral blood perfusion in diabetic patients are important components of diabetic complications. The cerebrovascular lesions and abnormal cerebral blood perfusion in diabetic patients have multiple effects on diabetic patients, involving multiple aspects such as physiology, psychology, and quality of life. These lesions and abnormalities are mainly caused by vascular endothelial dysfunction, vascular sclerosis, and microcirculation disorders due to long-term hyperglycemic state, resulting in changes in cerebral blood flow and damage to brain tissue.
[0003] First, cerebrovascular lesions in diabetic patients significantly increase the risk of stroke. The hyperglycemic state caused by diabetes leads to the accelerated development of atherosclerosis, affecting the main blood supply arteries of the brain, such as the carotid artery and the vertebrobasilar artery. The stenosis and sclerosis of these arteries increase the incidence of ischemic stroke. In addition, diabetes is also associated with an increased risk of hemorrhagic stroke because hyperglycemia weakens the integrity of the blood vessel wall and increases the likelihood of blood vessel rupture.
[0004] Second, chronic hyperglycemic state can lead to microvascular lesions, affecting the microcirculation of the brain. This kind of microvascular lesion can lead to white matter lesions of the brain and decline in cognitive function. Research shows that diabetic patients are more likely to have cognitive impairment or even dementia. This decline in cognitive function is related to abnormal cerebral blood perfusion because some regions of the brain may be damaged due to insufficient blood supply, especially the regions responsible for memory and executive function.
[0005] Vascular endothelial cells play a key role in maintaining vascular health and function, and the disorder of their proliferation and function is one of the core mechanisms of diabetic vascular complications. The hyperglycemia and insulin resistance caused by diabetes lead to vascular endothelial cell dysfunction, which in turn affects angiogenesis and repair ability. In addition, the chronic low-grade inflammation and oxidative stress caused by diabetes further damage vascular function, resulting in reduced cerebral blood flow and affecting cognitive and emotional functions.
[0006] In summary, there is an urgent need for a product for preventing or treating microvascular lesions and abnormal cerebral blood perfusion caused by diabetes. Summary of the Invention
[0007] Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to provide the use of mulberry leaf extract in the preparation of products for treating diabetic complications. Another purpose of the present invention is to provide a preparation method of the mulberry leaf extract.
[0008] One of the objectives of the present invention is achieved through the following technical solutions:
[0009] Use of mulberry leaf extract in the preparation of a product for treating diabetic complications, wherein the diabetic complication is cerebrovascular disease or abnormal cerebral blood perfusion; the product includes a food or a pharmaceutical composition.
[0010] Specifically, the mulberry leaf extract is used to prepare a food or a pharmaceutical composition for promoting the proliferation of vascular endothelial cells.
[0011] Specifically, the mulberry leaf extract is used to prepare a food or a pharmaceutical composition for promoting angiogenesis, cell migration or metabolic reaction efficiency in a hyperglycemic and hyperlipidemic environment.
[0012] Specifically, the mulberry leaf extract is used to prepare a food or a pharmaceutical composition for promoting cerebral microvascular angiogenesis or increasing cerebral blood perfusion volume.
[0013] Specifically, the mulberry leaf extract is used to prepare a food or a pharmaceutical composition for reducing the anxiety level or promoting cognitive function.
[0014] Specifically, the mulberry leaf extract is used to prepare a food or a pharmaceutical composition for preventing or treating diabetic encephalopathy, diabetic cerebrovascular disease, diabetes-related vascular dementia, diabetic cognitive dysfunction, diabetic anxiety, diabetic depression, diabetes-related mood disorder, diabetes-related memory loss, diabetes-related chronic cerebral ischemia, diabetes-related cerebral microvascular disease or post-diabetic stroke encephalopathy.
[0015] Another objective of the present invention is achieved through the following technical solutions:
[0016] A preparation method of mulberry leaf extract, comprising the following steps:
[0017] A. A step of extracting adult mulberry leaves or adult mulberry leaf powder with water, adding 10 - 20 times the weight of water based on the weight of the raw material, extracting at 60 - 100 °C for 1 - 3 times, combining the extraction liquids and passing through a sieve to obtain a filtrate;
[0018] B. A step of passing the filtrate through a cation resin chromatography column at a flow rate of 0.5 - 2 resin volumes / h, removing impurities with deionized water or 0.05 - 0.5 mol / L ammonia water, and using 0.5 - 2 mol / L ammonia water as the eluent at a flow rate of 1.5 column volumes / h;
[0019] C. A step of collecting the eluent, concentrating, spray drying, and pulverizing through an 80 - 100 mesh sieve.
[0020] Specifically, the adult mulberry leaf powder is obtained by pulverizing adult mulberry leaves and passing through a 20 - 40 mesh sieve.
[0021] Specifically, the combined extraction liquid is passed through a 200 mesh sieve.
[0022] Specifically, the cation exchange resin chromatography column is a 732 cation exchange resin chromatography column.
[0023] The beneficial effects of the present invention are as follows:
[0024] The present invention demonstrates that the mulberry leaf extract has a promoting effect on cerebral microvascular angiogenesis and cerebral blood perfusion in diabetic mice, the mulberry leaf extract promotes the proliferation of vascular endothelial cells, the activating effect of the mulberry leaf extract on angiogenesis, cell migration and glycolipid metabolism pathways in the diabetic model, and the enhancing effect of the mulberry leaf extract on the migration ability of vascular endothelial cells and mitochondrial membrane potential; that is, the mulberry leaf extract can prevent and treat microvascular lesions caused by diabetes and improve related diseases caused by abnormal cerebral blood perfusion;
[0025] And it is found that the mulberry leaf extract has the effect of improving the anxiety-like behavior and cognitive function of diabetic mice. Description of the Drawings
[0026] Figure 1 It is a diagram showing the promotion of the proliferation of vascular endothelial cells by the mulberry leaf extract.
[0027] Figure 2 It is a diagram showing the activating effect of the mulberry leaf extract on angiogenesis, cell migration and glycolipid metabolism pathways in the diabetic model.
[0028] Figure 3 It is a diagram showing the enhancing effect of the mulberry leaf extract on the migration ability of vascular endothelial cells and mitochondrial membrane potential.
[0029] Figure 4 It is a diagram showing the promoting effect of the mulberry leaf extract on cerebral microvascular angiogenesis and cerebral blood perfusion in diabetic mice.
[0030] Figure 5 It is a diagram showing the improvement of the anxiety-like behavior and cognitive function of diabetic mice by the mulberry leaf extract. Detailed Embodiments
[0031] The following is a further description in combination with the detailed embodiments:
[0032] Example 1
[0033] The adult mulberry leaves are crushed and screened through a 40-mesh sieve, then mixed with water at a material-liquid ratio of 1:12, and water-bathed at 65°C for 30 min. The crude extraction filtrate is kept warm at 60°C for standby. The crude extraction filter residue is added to boiling water of equal weight, refluxed and extracted at 95°C for 2 h, and then screened through a 200-mesh sieve to combine the filtrates. The filtrate is passed through a chromatography column filled with 732 cationic resin at a speed of 1 resin volume / h, deionized water of 3 resin volumes is used to remove impurities at a speed of 1 resin volume / h, and the active components are eluted with 1 mol / L ammonia water at a speed of 1.5 column volumes / h, and the eluate is collected. The eluate is concentrated and spray-dried, and the finished product is crushed and screened through an 80-mesh sieve.
[0034] Example 2
[0035] The adult mulberry leaves are crushed and screened through a 20-mesh sieve, added with water at 100°C which is 10 times the weight of the raw materials, and refluxed and extracted twice, 1 h each time, and then screened through a 200-mesh sieve to combine the filtrates. The filtrate is passed through a chromatography column filled with 732 cationic resin at a speed of 0.5 resin volume / h, 0.5 mol / L ammonia water of 2 resin volumes is used to remove impurities, and the active components are eluted with 2 mol / L ammonia water at a speed of 1.5 column volumes / h, and the eluate is collected. The eluate is concentrated and spray-dried, and the finished product is crushed and screened through a 100-mesh sieve.
[0036] Example 3
[0037] Water which is 20 times the weight of the raw materials is added to the adult mulberry leaves and kept warm at 60°C for extraction three times, 1 h each time, and then screened through a 200-mesh sieve to combine the filtrates. The filtrate is passed through a chromatography column filled with 732 cationic resin at a speed of 2 resin volumes / h, 0.05 mol / L ammonia water of 2 resin volumes is used to remove impurities, and the active components are eluted with 0.5 mol / L ammonia water at a speed of 1.5 column volumes / h, and the eluate is collected. The eluate is concentrated and spray-dried, and the finished product is crushed and screened through an 80-mesh sieve.
[0038] After inspection, the content of 1-deoxynojirimycin in the mulberry leaf extracts obtained in Examples 1-3 is 5 wt%, and the mulberry leaf extracts obtained in Examples 1-3 are all commercial Mulberry leaf extract.
[0039] Verification experiment
[0040] I. Experiment on promoting the proliferation of vascular endothelial cells by mulberry leaf extract
[0041] The human umbilical vein endothelial cell line EA.hy926 was used as an experimental model to evaluate the effects of the mulberry leaf extract prepared in Example 1 on cell proliferation and toxicity at different concentrations. The experimental design included concentration gradients of the mulberry leaf extract at 0, 0.0005, 0.002, 0.005, 0.05, 0.2, 0.4, 0.8, 2, and 4 mg / ml. Cell proliferation and toxicity were detected using a CCK8 kit after 24 hours and 48 hours, respectively. The results showed that among all the tested concentrations, the mulberry leaf extract at 0.05 mg / ml and 0.2 mg / ml significantly promoted the proliferation of EA.hy926 cells, and no cytotoxic effect was observed at any concentration( Figure 1 a,b). This finding suggests that the mulberry leaf extract of the present invention can support the healthy proliferation of vascular endothelial cells at appropriate concentrations.
[0042] II. Experiment on the activation effect of mulberry leaf extract on angiogenesis, cell migration, and glycolipid metabolism pathways in a diabetes model
[0043] By simulating a diabetic environment with high glucose and high fat, the effects of the mulberry leaf extract prepared in Example 1 on the functions of vascular endothelial cells were evaluated. The experiment was divided into three groups: (1) negative control group (Control); (2) palmitic acid group: a group treated with 300 μM sodium palmitate to simulate a diabetes model; (3) mulberry leaf & palmitic acid group: a group treated with 0.05 mg / ml mulberry leaf extract (the mulberry leaf extract prepared in Example 1) while being treated with sodium palmitate to evaluate the intervention effect of the mulberry leaf extract on diabetes.
[0044] Through transcriptomic sequencing analysis, it was found that the three groups of samples were significantly separated in the principal component analysis (PCA), indicating that different treatment conditions had a significant impact on cell gene expression. In particular, the samples in the mulberry leaf & palmitic acid group were closer to the negative control group in the PCA, suggesting that the mulberry leaf extract could partially restore the gene expression pattern of cells in the diabetes model( Figure 2 a). Further GO signaling pathway enrichment analysis showed that compared with the palmitic acid group, the pathways related to vascular endothelial cell proliferation and angiogenesis (such as blood vessel endothelial cell proliferation involved in sprouting angiogenesis) were significantly enriched in the mulberry leaf & palmitic acid group. In addition, the pathways related to glycolipid metabolism (such as glycolipid biosynthetic process and fatty acid oxidation) were also significantly enriched( Figure 2 b).
[0045] The results of Gene Set Enrichment Analysis (GSEA) further supported this finding. Compared with the negative control group, the palmitic acid group showed significant downregulation of the positive regulation of angiogenesis, cellular response to glucose stimulus, cell migration, and wnt signaling pathway ( Figure 2 c-j). This indicates that the high-glucose and high-fat environment has a negative impact on angiogenesis, cell migration, and metabolic responses. However, in the mulberry leaf & palmitic acid group, these pathways were significantly upregulated compared with the palmitic acid group, demonstrating the potential of the mulberry leaf extract of the present invention in reversing the inhibitory effect of the diabetic environment on vascular endothelial cells ( Figure 2 c-j).
[0046] III. Experiment on the enhancing effect of mulberry leaf extract on the migration ability of vascular endothelial cells and mitochondrial membrane potential
[0047] EA.hy926 cells were treated with 300 μM sodium palmitate for 48 hours to simulate the high-glucose and high-fat diabetic environment, and 0.05 mg / ml of mulberry leaf extract (the mulberry leaf extract prepared in Example 1) was added for intervention. Subsequently, a scratch test was performed, and the cell migration at 0 hour and 24 hours after scratching was recorded by microscopic photography. The results showed that the migration rate of cells in the sodium palmitate treatment group (Palmitic acid) was approximately 40%, while the migration rate of cells in the mulberry leaf extract intervention group (Mulberry leaf & Palmitic acid) was significantly increased to 80% ( Figure 3 a, b). This result indicates that the mulberry leaf extract of the present invention can significantly promote the migration ability of vascular endothelial cells, probably by enhancing the motility and repair ability of cells to promote angiogenesis. In addition, mitochondrial membrane potential is an important indicator of cell metabolic activity and functional state. The mitochondrial membrane potential was detected using JC-1 fluorescent dye, and the results showed that compared with the mitochondrial membrane potential of cells in the sodium palmitate treatment group, the mitochondrial membrane potential of the mulberry leaf extract intervention group was significantly enhanced, with an approximately 4-fold increase ( Figure 3 c, d). This finding indicates that the mulberry leaf extract of the present invention can support cell proliferation and migration activities by enhancing mitochondrial function and increasing the cell energy metabolism level.
[0048] IV. Experiment on the promoting effect of mulberry leaf extract on cerebral microvascular angiogenesis and cerebral blood perfusion in diabetic mice
[0049] Male C57BL / 6J mice at 4 months of age were fed a high-fat diet for 3 months to induce obesity and insulin resistance. Subsequently, the mice were fasted and intraperitoneally injected with 60 mg / kg streptozotocin (STZ) for 5 consecutive days to induce a type 2 diabetes model. Five days later, the establishment of the diabetes model was confirmed by detecting the fasting blood glucose level of the mice. Mice with a fasting blood glucose level exceeding 11.1 mmol / L were considered to have successfully established the diabetes model.
[0050] After successful modeling, the mice were randomly divided into two groups: (1) the control group (Control), given normal drinking water; (2) the mulberry leaf extract group (Mulberry leaf), and each mouse was given 100 mg / kg / day of the mulberry leaf extract (the mulberry leaf extract prepared in Example 1) through drinking water. The experiment lasted for one month to evaluate the long-term effect of the mulberry leaf extract. One month later, the brain regions of the mice were imaged by a laser speckle contrast imaging system (LSCI). LSCI is a non-invasive imaging technique that can measure blood flow dynamics in real time. The pseudo-color images and dynamic curves of blood flow in the brain regions of the mice were collected to visually display the formation of brain microvessels and calculate the cerebral blood perfusion volume.
[0051] The results showed that the formation of brain microvessels in the mulberry leaf extract group of mice was significantly increased, and the cerebral blood perfusion volume was significantly improved. In addition, the cerebral blood perfusion volume of the control group mice was relatively low, while that of the mulberry leaf extract group mice was significantly increased, manifested as higher blood flow velocity and a more extensive microvascular network. Further quantitative analysis showed that the cerebral blood perfusion volume of the mulberry leaf extract group mice was significantly higher than that of the control group, indicating that the mulberry leaf extract of the present invention has a significant effect on improving the cerebral blood supply in diabetic mice ( Figure 4 a,b). This result indicates that the mulberry leaf extract of the present invention can effectively promote the generation and expansion of brain microvessels in diabetic mice, thereby improving the cerebral blood supply. The increased cerebral blood perfusion volume may protect against cerebrovascular complications caused by diabetes by enhancing the supply of oxygen and nutrients to the brain and improving the functional state of nerve cells.
[0052] V. Experiment on the improvement of anxiety-like behavior and cognitive function in diabetic mice by mulberry leaf extract
[0053] Male C57BL / 6J mice at 4 months of age were fed a high-fat diet for 3 months to induce obesity and insulin resistance. Subsequently, the mice were fasted and intraperitoneally injected with 60 mg / kg streptozotocin (STZ) for 5 consecutive days to induce a type 2 diabetes model. Five days later, the establishment of the diabetes model was confirmed by detecting the fasting blood glucose level of the mice. Mice with a fasting blood glucose level exceeding 11.1 mmol / L were considered to have successfully established the diabetes model.
[0054] After successful modeling, the mice were randomly divided into two groups: (1) the control group (Control), given normal drinking water; (2) the mulberry leaf extract group (Mulberry leaf), and each mouse was given 100 mg / kg / day of mulberry leaf extract (the mulberry leaf extract prepared in Example 1) through drinking water. The experiment lasted for one month to evaluate the long-term effect of the mulberry leaf extract. After one month, the open field test and the novel object recognition test were used to systematically evaluate the improvement effect of the mulberry leaf extract on the anxiety-like behavior and cognitive function of diabetic model mice.
[0055] The open field test is a classic behavioral test used to evaluate the anxiety level and spontaneous activity ability of experimental animals. In this experiment, after one month of intervention with the mulberry leaf extract, diabetic mice were placed in the open field for testing. The results showed that the moving distance of the mice in the mulberry leaf extract group exceeded 30 meters within 2 minutes, while that of the control group mice was only 20 meters ( Figure 5 a, b). This indicates that the mulberry leaf extract of the present invention can significantly improve the activity level of mice. In addition, the residence time of the mice in the mulberry leaf extract group in the central area of the open field was significantly prolonged, exceeding 15 seconds, while that of the control group mice was only 7 seconds ( Figure 5 c). An increase in the residence time of mice in the central area is usually interpreted as a decrease in the anxiety level, because mice tend to stay in the edge area of the field when they feel uneasy. Thus, it can be seen that the mulberry leaf extract of the present invention can effectively relieve the anxiety symptoms of diabetic mice.
[0056] The novel object recognition test is used to evaluate the cognitive function of mice, especially its ability to remember and explore new things. The experiment is divided into two stages: on the first day, two identical objects were placed for the mice to adapt, and on the second day, one of the objects was replaced with a new object, and the exploration time of the mice for the new object was detected. The experimental results showed that the exploration time of the control group mice for the new object was less than 10% of the total time, indicating that their cognitive function and exploration desire were relatively low. While the exploration time of the mice in the mulberry leaf extract group for the new object increased significantly to 50% ( Figure 5d, e), indicating that the mulberry leaf extract of the present invention can significantly enhance the cognitive function of diabetic mice. The improved exploratory behavior and cognitive ability may be related to the improvement of cerebral blood perfusion and neuroprotective effects of the mulberry leaf extract.
[0057] VI. Experimental Summary
[0058] The verification experiment systematically revealed the mechanism of action of the mulberry leaf extract in improving diabetes-related cognitive impairment and anxiety symptoms by integrating in vitro cell experiments and diabetic animal models, and regulating vascular function and metabolic homeostasis through multiple dimensions.
[0059] Experiment 1 confirmed that the mulberry leaf extract obtained in the present invention can promote the healthy proliferation of vascular endothelial cells under certain concentration conditions. Its mechanism involves the coordinated regulation of angiogenesis and metabolic pathways. Experiment 2 confirmed through transcriptomic sequencing analysis that the mulberry leaf extract obtained in the present invention can up-regulate the expression of the diabetic model cells in the pathways related to vascular endothelial cell proliferation and angiogenesis, as well as the pathways related to glycolipid metabolism; it also confirmed through gene set enrichment analysis that the mulberry leaf extract obtained in the present invention can up-regulate the expression of the positive regulatory pathway, the pathway of cell response to glucose stimulation, the cell migration pathway, and the wnt signaling pathway. Experiment 3 confirmed through scratch experiments that the mulberry leaf extract of the present invention can significantly promote the migration ability of vascular endothelial cells, and may promote angiogenesis by enhancing cell motility and repair ability; it also confirmed through the detection of mitochondrial membrane potential that the mulberry leaf extract of the present invention can support cell proliferation and migration activities by enhancing mitochondrial function and improving the energy metabolism level of cells.
[0060] In summary, it can be inferred that the mulberry leaf extract obtained in the present invention can treat or relieve cerebrovascular lesions caused by diabetes through the proliferation of vascular endothelial cells and the migration of vascular endothelial cells.
[0061] Experiment 4 confirmed through non-invasive imaging techniques that the mulberry leaf extract of the present invention can effectively promote the generation and expansion of brain microvessels in diabetic mice, thereby improving cerebral blood supply. The increased cerebral blood perfusion volume may improve the functional state of nerve cells by enhancing the supply of oxygen and nutrients to the brain. Experiment 5 confirmed through open field experiments that the mulberry leaf extract of the present invention can effectively relieve the anxiety symptoms of diabetic mice and can also significantly enhance the cognitive function of diabetic mice.
[0062] In summary, it can be inferred that the mulberry leaf extract obtained in the present invention can treat or relieve abnormal cerebral blood perfusion, anxiety, and cognitive impairment caused by diabetes through the generation and expansion of brain microvessels.
[0063] In addition, the validation experiment for the first time constructed a theoretical framework for the "vascular-metabolic-neural" cascade regulation, and clarified the multi-target action mode of mulberry leaf extract to achieve neuroprotective effects by activating the angiogenesis ability of endothelial cells, restoring mitochondrial energy metabolism, and promoting the reconstruction of cerebral microcirculation. It is worth noting that the extract did not show cytotoxicity in the concentration range of 0.05 - 4 mg / ml, and showed bioavailability equivalent to the dose of human dietary supplements in animal experiments, providing a basis for its clinical translation in terms of safety and feasibility.
[0064] The above embodiments and descriptions in the specification only illustrate the principles and the best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.
Claims
1. Use of mulberry leaf extract in preparing a product for treating diabetic complications, characterized in that: The diabetic complications are cerebrovascular disease or abnormal cerebral blood perfusion; the products include food or pharmaceutical compositions.
2. The use according to claim 1, characterized in that: The mulberry leaf extract is used for preparing a food or a pharmaceutical composition for promoting the proliferation of vascular endothelial cells.
3. The use according to claim 1, characterized in that: The mulberry leaf extract is used for preparing a food or pharmaceutical composition for promoting angiogenesis, cell migration or metabolic reaction efficiency in a high-sugar and high-fat environment.
4. The use according to claim 1, characterized in that: The mulberry leaf extract is used for preparing a food or medicine composition for promoting cerebral microangiogenesis or increasing cerebral blood perfusion.
5. The use according to claim 1, characterized in that: The mulberry leaf extract is used for preparing a food or pharmaceutical composition that reduces anxiety levels or promotes cognitive functions.
6. The use according to claim 1, characterized in that: The mulberry leaf extract is used to prepare a food or pharmaceutical composition for preventing or treating diabetic encephalopathy, diabetic cerebrovascular disease, diabetic-related vascular dementia, diabetic cognitive dysfunction, diabetic anxiety, diabetic depression, diabetic-related mood disorders, diabetic-related memory loss, diabetic-related chronic cerebral ischemia, diabetic-related cerebral microangiopathy or diabetic post-stroke encephalopathy.
7. The method for preparing the mulberry leaf extract for use according to any one of claims 1 to 6, characterized in that: The following steps are involved: A. the step of adding water to the adult mulberry leaves or adult mulberry leaf powder for extraction, adding water 10-20 times the weight of the raw material and extracting at 60-100° C. for 1-3 times, combining the extracts and sieving to obtain a filtrate; B. passing the filtrate through a cationic resin chromatography column at a flow rate of 0.5-2 times the resin volume / h, removing impurities with deionized water or 0.05-0.5 mol / L ammonia water, and using 0.5-2 mol / L ammonia water as the eluent at a flow rate of 1.5 times the column volume / h; C. The eluate is collected, concentrated, spray-dried, and crushed to pass through a 80-100 mesh sieve.
8. The preparation method according to claim 7, characterized in that: The adult mulberry leaf powder is obtained by crushing adult mulberry leaves and then passing them through a 20-40 mesh sieve.
9. The preparation method according to claim 7, characterized in that: The combined extract was sieved through a 200-mesh sieve.
10. The preparation method according to claim 7, characterized in that: The cationic resin chromatography column is a 732 cationic resin chromatography column.