CN105456298A - Anti-senile dementia activity of bryostatins and application thereof - Google Patents

Abstract

The invention relates to the technical field of medicine, in particular to the anti-senile dementia activity of total lactones of Bryophora and its application. The total lactones of the bryozoan (BRYS) of the present invention can significantly shorten the swimming time through the mouse water maze test, and have obvious improvement effects on cholinesterase (AChE) and acetylcholine Ach in the mouse brain. The content of acetylcholinetransferase (ChAT) and malondialdehyde (MDA) was significantly increased, the effect was significant, and the difference was statistically significant. It can significantly increase the activity of SOD in serum (P<0.01), and increase the levels of serum glutathione (GSH) and serum glutathione peroxidase (GSH-PX). In addition, the total lactones of Bryonysus had no significant effect on the body weight and organ index of mice, indicating that the toxicity was low. The experimental results show that the total lactones of Bryonymus have significant anti-senile dementia activity and low toxicity, and can be applied to the development of anti-senile dementia drugs.

Description

Anti-senile dementia activity and application of total lactones of bryozoa

technical field

The invention relates to the technical field of medicine, in particular to the anti-senile dementia activity of total lactones of bryozoa and its application in the field of medicine.

Background technique

Marine bryozoans, commonly known as bryozoans, sea mats, false corals and bryozoans, belong to true coelomates and are one of the important components of marine benthic animals. There are many types, about 4,000 species, the most common of which are Bryoplasma totalis. Bugulaneritina Linnaeus is widely distributed in the waters of the world, and it is found in the saline waters along the coast of my country from the Bohai Sea to the Xisha Islands in the South China Sea. The group is reddish-brown or purple-brown, in the shape of tufts, 40-150mm high, and each branch has two rows of worms, arranged alternately. Its groups often attach to kelp, agarose, shellfish, and underwater facilities such as ship bottoms, docks, and navigation marks, affecting marine aquaculture, national defense, and marine traffic, and is one of the main marine fouling organisms. Until 1968, in the extensive research of marine invertebrates and vertebrates by the Pettit research group of Arizona State University, the anti-cancer activity of total synapses was discovered for the first time. After more than ten years of hard work, in 1982, the Pettit research group successfully isolated the first monomer bryostatin1 with anti-cancer activity from the total bryostatin collected in the California sea area, and determined its structure by X-ray diffraction. It is a macrolide compound. Since then, the group has been working on the research of macrolide components with anti-cancer activity in the total B. So far, 20 active monomers have been obtained from it, namely bryostatin 1-20 (LinHW, YiYH, LiWL, YaoXS, WuHM. Chinese J. Marine Drugs 1998, 65, 1; PettitGR. Fortschritteder Chemie Organischer Naturstoffe, 1991, 57, 153). Bryostatin1 and bryostatin4 have been bioidentified by the National Cancer Institute (NCI) and have been put into clinical trials. These compounds have the characteristics of low toxicity and strong activity. Its novel mechanism of action on protein kinase C (PKC) has inspired researchers to conduct extensive and in-depth studies on its mechanism of action. In view of the limited natural resources of bryozoan and the tiny content of bryolides, the natural resources of bryozoans have been extensively investigated.

Alzheimer's disease (mainly Alzheimer's disease) is a progressive and fatal neurodegenerative disease. Symptoms and behavioral disturbances. The disease can appear at any age, but usually occurs between the ages of 60-70. When diagnosed with the disease, about 3.3 years left to live. Prevalence research shows that the prevalence rate of 60-year-old population is 1%, and the prevalence rate increases by 2 times for every 5 years of age increase. At present, my country is accelerating into an aging society. According to the prediction of relevant experts, by 2020, the population over the age of 60 in my country will reach 400 million, and there will be more than 10 million people with Alzheimer's disease. According to the latest report, there are already 120,000 Alzheimer's patients in Shanghai.

Alzheimer's disease is a group of primary degenerative brain degenerative diseases with unknown etiology. It usually occurs in old age, with latent onset, slow and irreversible course of disease, and mainly intellectual impairment in clinical practice. The main pathological changes are diffuse atrophy of the cortex, widening of the sulci, enlarged ventricles, a large number of neurons, senile plaques (SP), neurofibrillary knots (NFT) and other lesions, and the content of choline acetylase and acetylcholine is significantly reduced. The etiology of Alzheimer's disease is not very clear at present, and there are many subtypes, and the organs that undergo pathological changes may have other organs besides the brain. There are two main pathogenesis recognized at present: 1. Due to the abnormality of amyloid precursor protein, the protein components leak out of the cell membrane, resulting in neurofibrillary tangles and cell death. The gene is located on chromosome 21. In AD patients, acetylcholine is obviously lacking in the brain, and the activity of acetylcholinesterase and choline-acyltransferase is reduced, especially in the hippocampus and temporal lobe cortex. 2. It is related to the gene of apolipoprotein E (APO-E4).

Since the exact etiology of Alzheimer's disease has not yet been fully elucidated, the treatment method mainly uses drugs to act on different neurotransmitter systems to enhance the high-level activity of the central nervous system, relieve various symptoms during the disease process, and delay the further development of dementia. . The commonly used clinical treatment mainly increases the content of acetylcholine in the patient's body by inhibiting acetylcholinesterase, and improves the clinical symptoms of Alzheimer's disease. At present, most of the therapeutic drugs in clinical use are acetylcholinesterase inhibitors (such as tacrine, donepezil, rivastigmine, galantamine, huperzine A), NMDA receptor antagonists (memantine) and antioxidants (dimension). Lygiline, vitamin E, melatonin, ginkgo extract) and other drugs for cognitive dysfunction are often used in the treatment of cognitive dysfunction, but the clinical curative effect is not accurate enough, oral absorption is poor, it is difficult to pass through the blood-brain barrier, and the specificity is not strong. Or toxic and side effects are big, especially shortcoming such as treating the symptoms but not the disease. Research and development of novel and highly effective anti-senile dementia drugs is an urgent problem to be solved in current medicine and health care. But there is still a big gap between this research field and similar foreign researches. Finding anti-senile dementia chemical components from natural products is a new hotspot and development direction in natural medicine research. Especially marine natural products, due to their special chemical structure and physiological activity, the discovery of anti-senile dementia chemical components will have a broader prospect.

There is no report on the anti-senile dementia activity of total lactones of bryozoan at home and abroad.

Contents of the invention

The object of the present invention is to provide the total lactone of bryozoan and its preparation method; another object of the present invention is to provide a new medical application of the total lactone of bryozoan.

The first aspect of the present invention provides a total bryozoan lactone. The total bryozoan lactone is extracted and purified by conventional methods to obtain the total bryozoan total lactone from the total bryozoan.

The total lactones of bryozoans according to the present invention, through high performance liquid chromatography analysis, mainly contain more than 10 kinds of lactone components such as bryozolides 5, 10, 18 and 4, wherein bryozolides 4 highest content. Bryostatin4 accounts for more than 60% (mass percentage) of the total lactones of Bryostatin.

Preferably, the bryozoa are collected from the southern sea area of my country.

The second aspect of the present invention provides a method for preparing total lactones of bryozoan.

A kind of preparation method of total lactone of bryozoan, is characterized in that, the preparation method of described total lactone of bryozoan is as follows:

The freshly collected Burgelaneritina Linnaeus was extracted with 95% ethanol at room temperature, and the ethanol extract was recovered under reduced pressure to obtain the extract;

Suspend and disperse the extract with 90% methanol, extract with n-hexane, discard the n-hexane extract; add water to the aqueous methanol layer to form an 80% methanol aqueous solution, extract with _CCl4 , and obtain the _CCl4 extract;

The CCl ₄ extract was subjected to 200-300 mesh silica gel column chromatography to obtain the active components; and then SephadexLH-20 gel column chromatography, eluting with CH ₂ Cl ₂ :MeOH (1:1), with a flow rate of 60mL/h, to obtain active ingredient A and active ingredient B;

Active component A and active component B were subjected to gel column chromatography twice, the first time was eluted with Hexane:CH ₂ Cl ₂ :MeOH (4:5:1), and the second time was eluted with Hexane:CH ₂ Cl ₂ : MeOH (10:10:1) was eluted to obtain active component C and active component D respectively;

The active component C and the active component D were subjected to ODS flash column chromatography, and the gradient elution of 50% and 80% MeOH was used to obtain the total lactones of Bryocarpus.

In a preferred embodiment of the present invention, the preparation method of described bryozoan total lactones is as follows:

Freshly collected Bugulaneritina Linnaeus raw materials were leached with 95% ethanol at room temperature for one week, extracted 4 times in total, combined ethanol extracts, and recovered ethanol under reduced pressure to obtain extract. The extract was suspended and dispersed with 90% methanol, extracted 5 times with n-hexane to obtain the n-hexane extract, which was screened for in vitro anticancer activity and discarded. Add water to the aqueous methanol layer to make 80% methanol aqueous solution, and extract 5 times with CCl ₄ to obtain CCl ₄ extract, which is the active site. The active part is subjected to fast silica gel column chromatography (200-300 mesh) to obtain the active component. After SephadexLH-20 gel column chromatography, eluting with CH ₂ Cl ₂ :MeOH (1:1) at a flow rate of 60mL/h, two active components A and B were obtained. The two active components were subjected to gel column chromatography twice, followed by Hexane-CH ₂ Cl ₂ -MeOH (4:5:1) and Hexane:CH ₂ Cl ₂ :MeOH (10:10:1) After elution, two components C and D with higher purity and stronger activity were obtained respectively. The two components C and D still contain more green pigment, so the two are combined, and ODS flash column chromatography is carried out, and the gradient elution of 50% and 80% MeOH is used to obtain light yellow total bryozolide (BRYS) .

The amount of said 95% ethanol is 20-30 times (weight ratio) of the raw material.

The n-hexane extraction is performed 5 times, and the dosage is 4-5L each time.

The CCl ₄ is extracted 5 times, and the dosage is 1-2L each time.

The third aspect of the present invention provides a new medical application of the above-mentioned total lactones of bryozoan.

The invention provides the application of total lactones of bryozoan in the preparation of medicines or health care products for preventing or treating senile dementia.

In the application of the total bryozolides of the present invention in the preparation of medicines or health care products for preventing or treating senile dementia, in the medicines or health care products: the total lactones of bryozoans are used as the only active ingredient, or contain grass Composition of total lactones in bryozoa.

In the medicine or health care product, the content of total lactones of the bryozoan is 0.1-99wt%, preferably 0.5-90wt%.

Among the medicines, the total bryozolides and the pharmaceutical composition containing the total bryozolides can be prepared into pharmaceutical preparations with conventional pharmaceutical excipients in pharmacy.

The pharmaceutical preparations can be tablets, granules, dispersible tablets, capsules, soft capsules, dropping pills, injections, powder injections, or aerosols and the like.

The research of the present invention found that the total lactones of bryozoa have anti-senile dementia biological activity:

Male ICR mice were randomly divided into 9 groups with 6 mice in each group. A control group is set up, and BRYS drugs are set at: 0.0025mg/kg, 0.005mg/kg, 0.01mg/kg groups, anti-aging positive drug (vitamin E). BRYS and vitamin E were given by injection for a total of 60 days. One hour after the last administration, all the mice were subjected to the water maze test for one week, and the time for the mice to find the platform was recorded. In the mouse water maze experiment, compared with the model group, the medium concentration group and high concentration group can significantly shorten the swimming time. One week later, the samples were processed according to the kit instructions, and the brain homogenate acetylcholinesterase activity, acetylcholine transferase content and serum SOD activity, malondialdehyde content, glutathione peroxidase activity, acetylcholine content, glutathione content were detected. content. The experimental data were processed by SPSS13.0 software, and the data were t-tested. The experimental results showed that the total lactones of Bryonymus had obvious anti-senile dementia activity. In addition, the total lactones of Bryophora had no significant effect on body mass and visceral index, indicating low toxicity.

The above experimental results show that the total lactones of Bryonymus have significant anti-senile dementia activity and low toxicity, and can be used to develop anti-senile dementia medicines.

Because, among the total lactones of the bryostatin of the present invention, the content of bryostatin 4 is the highest, and Bryostatin 4 accounts for more than 60% of the total lactones of the bryostatin. Therefore, the present invention also provides the application of Bryostatin4 in the preparation of medicaments or health care products for preventing or treating senile dementia.

The present invention finds safe and effective anti-senile dementia medicine.

detailed description

The present invention will be further explained below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1. Extraction and purification of total lactones of Bryophora

The freshly collected Burgelaneritina Linnaeus raw material (10kg, dry weight) was leached with 95% ethanol at room temperature for one week, and extracted 4 times in total (300L/time, 4 times). The ethanol extract was combined, and the ethanol was recovered under reduced pressure to obtain Extract 320g. The extract was suspended and dispersed with 90% methanol, extracted 5 times with 5 L each time with n-hexane to obtain 56 g of n-hexane extract, which was inactive through in vitro anticancer activity screening. Add water to the aqueous methanol layer to make 80% methanol aqueous solution, extract with CCl ₄ (1 L/time, 5 times), and obtain 9 g of CCl ₄ extract (IC50=7 μg/mL, P388) as the active site. The active fraction was subjected to flash silica gel column chromatography (200-300 mesh) to obtain the active component 2BH-10 (0.41g, IC50=2.4g/mL, P388). 2BH-10 was subjected to SephadexLH-20 gel column chromatography, eluted with CH ₂ Cl ₂ :MeOH (1:1) at a flow rate of 60mL/h to obtain two active components A (0.17g, IC50=0.3μg/mL , P388) and B (0.11 g, IC50 = 0.8 g/mL, P388). The two active components were subjected to gel column chromatography twice, followed by Hexane-CH ₂ Cl ₂ -MeOH (4:5:1) and Hexane:CH ₂ Cl ₂ :MeOH (10:10:1) After elution, two components C (0.07g, IC50=0.1g/ml, P388) and D (0.05g, IC50=810-2g/mL, P388) with higher purity and stronger activity were obtained respectively. The two components C and D still contain more green pigment, so the two are combined, and ODS flash column chromatography is carried out, and the gradient elution of 50% and 80% MeOH is used to obtain light yellow total bryozolide (BRYS) 0.01 g.

Example 2. Anti-senile dementia biological activity experiment of bryozolide

1 Experimental materials

1.1 Experimental animals: ICR mice, male, 6-8 weeks old, 18-22g, Dalian Medical University, 1.2 Reagents and drugs: acetylcholinesterase (AChE) kit (Nanjing Jiancheng Bioengineering Institute), acetylcholine transferase ( ChAT) kit (Nanjing Jiancheng Bioengineering Research Institute), glutathione peroxidase test kit (Nanjing Jiancheng Bioengineering Research Institute), trace reduced glutathione kit (Nanjing Jiancheng Bioengineering Research Institute), Acetylcholine kit (Nanjing Jiancheng Bioengineering Research Institute), SOD kit (Nanjing Jiancheng Bioengineering Research Institute), malondialdehyde test kit (Nanjing Jiancheng Bioengineering Research Institute), vitamin E (Dalian Tianyu Aosen Pharmaceutical Co., Ltd.), Tested drug: total lactones (BRYS) obtained in Example 1.

1.3 Instruments: microplate reader, centrifuge.

2 Experimental methods

2.1 Experimental scheme: Mice were randomly divided into 9 groups, with 6 mice in each group. A control group was set up, and the total lactones of Bryocarpus set: 0.0025mg/kg, 0.005mg/kg, 0.01mg/kg groups, anti-aging test positive drug group (vitamin E, VE). BRYS and vitamin E were given by injection for a total of 60 days. One hour after the last administration, all the mice were subjected to the water maze test for one week, and the time for the mice to find the platform was recorded. For the water maze test method, please refer to the literature: Wang Weigang et al., Application of Morris water maze test in mouse phenotype analysis, Chinese Journal of Cell Biology, 2011, (1): 8-14.

One week later, the samples were processed according to the instructions of each kit, and various indicators were tested:

Acetylcholinesterase (AChE) kit to detect the activity of acetylcholinesterase in brain homogenate;

Acetylcholine transferase (ChAT) kit, detect the content of acetylcholine transferase;

Glutathione peroxidase test kit to detect glutathione peroxidase activity;

Trace reduced glutathione kit to detect glutathione content;

Acetylcholine kit, detect the content of acetylcholine;

SOD kit, detect serum SOD activity;

Malondialdehyde test kit, detect the content of malondialdehyde.

2.2 Experimental data inspection: SPSS13.0 software was used to process the data, and the data were t-tested.

2.3 Experimental results: see Table 1-4.

Table 1 Mouse water maze X±SD

Compared with the model group *P<0.05,**P<0.01

Table 2 Activity of AChE in 10% mouse brain homogenate, content of ChAT, content of MDA X±SD

Compared with the model group *P<0.05, **P<0.01

Table 3 SOD activity in mouse serum, MDA, Ach, GSH content, GSH-PX activity X±SD

Table 4 Mouse body weight, brain, liver, spleen, thymus, testis index (X±SD)

2.4 Conclusion

In the mouse water maze experiment, compared with the model group, the middle concentration group and the high concentration group could significantly shorten the swimming time; cholinergic neurons play an important role in the process of learning and memory, and acetylcholine ( Ach) is an important central neurotransmitter released from the end of cholinergic neurons. Cholinesterase (AChE) and acetylcholine transferase (ChAT) jointly maintain the stability of Ach level. (AChE) is a hydrolase of acetylcholine. The level of AChE can indirectly reflect the change of Ach. Compared with the model group, the BRYS high concentration group had a significant improvement on AChE and Ach (P<0.05). Brain tissue acetylcholine transferase (ChAT), BRYS compared with the model group, the high concentration group has a better improvement (P<0.05). The content of malondialdehyde (MDA) in the brain tissue of each BRYS group was significantly improved, and the high concentration group had a significant effect, and the difference was statistically significant (P<0.05). Serum superoxide dismutase (SOD) plays a key role in regulating the balance of oxidation and anti-oxidation in the body. It can scavenge superoxide anion free radicals, reduce lipid peroxidation, maintain the structure and function of biofilm, and protect cells. BRYS can significantly increase the activity of SOD in serum (P<0.01), increase the levels of serum glutathione (GSH) and serum glutathione peroxidase (GSH-PX). In addition, the total lactones of Bryophora had no significant effect on body mass and visceral index, indicating low toxicity.

The above experimental results show that the total lactones of Bryonymus have significant anti-senile dementia activity and low toxicity, and can be used to develop anti-senile dementia medicines.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. Modifications or replacements, these equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (10)

1. The application of total lactones of bryozoan in the preparation of drugs or health care products for preventing or treating senile dementia. 2. the application of the total lactones of bryozoans according to claim 1 in the preparation of medicines or health products for preventing or treating senile dementia, characterized in that, the total lactones of bryozoans is based on total bryozoans Raw materials are extracted and purified by conventional methods to obtain total lactones of Bryocarpus. 3. The application of the total lactones of bryostatin according to claim 1 in the preparation of medicines or health care products for preventing or treating senile dementia, characterized in that, Bryostatin4 accounts for more than 60% of the total lactones of bryostatin. 4. the application of total lactones of bryozoan according to claim 1 in the preparation of prevention or treatment of senile dementia medicine or health products, is characterized in that, the preparation method of total lactones of described bryozoan is as follows: The freshly collected Burgelaneritina Linnaeus was extracted with 95% ethanol at room temperature, and the ethanol extract was recovered under reduced pressure to obtain the extract; Suspend and disperse the extract with 90% methanol, extract with n-hexane, discard the n-hexane extract; add water to the aqueous methanol layer to form an 80% methanol aqueous solution, extract with _CCl4 , and obtain the _CCl4 extract; The CCl ₄ extract was subjected to 200-300 mesh silica gel column chromatography to obtain the active component; and then SephadexLH-20 gel column chromatography, eluted with CH ₂ Cl ₂ : MeOH at a ratio of 1:1, and the flow rate was 60mL/h to obtain the active component. component A and active component B; Active component A and active component B were subjected to gel column chromatography twice, the first time was eluted with Hexane:CH ₂ Cl ₂ :MeOH at a ratio of 4:5:1, and the second time was eluted with Hexane:CH ₂ Cl ₂ : MeOH is eluted at 10:10:1 to obtain active component C and active component D respectively; The active component C and the active component D were subjected to ODS flash column chromatography, and the gradient elution of 50% and 80% MeOH was used to obtain the total lactones of Bryocarpus. 5. The application of the total lactones of bryozoan according to any one of claims 1 to 4 in the preparation of medicines or health products for preventing or treating senile dementia, characterized in that, in the medicine or health products, the bryozoan Total lactones as the sole active ingredient, or compositions containing total lactones of B. 6. The application of total lactones of bryozoans according to claim 5 in the preparation of medicines or health products for preventing or treating senile dementia, characterized in that, in the medicine, total lactones of bryozoans, including bryozoans The pharmaceutical composition of pyretalactone can be prepared into pharmaceutical preparations with conventional pharmaceutical auxiliary materials in pharmacy. 7. the application of total lactones of bryozolin according to claim 6 in the preparation of prevention or treatment of senile dementia medicine or health products, is characterized in that, described pharmaceutical preparation is tablet, granule, dispersible tablet, capsule formulations, soft capsules, dripping pills, injections, powder injections, or aerosols. 8. Application of Bryostatin4 in the preparation of drugs or health products for preventing or treating senile dementia. 9. a preparation method of total lactones of bryozoan, is characterized in that, the preparation method of described total lactones of bryozoan is as follows: The freshly collected Burgelaneritina Linnaeus was extracted with 95% ethanol at room temperature, and the ethanol extract was recovered under reduced pressure to obtain the extract; Suspend and disperse the extract with 90% methanol, extract with n-hexane, discard the n-hexane extract; add water to the aqueous methanol layer to form an 80% methanol aqueous solution, extract with _CCl4 , and obtain the _CCl4 extract; The CCl ₄ extract was subjected to 200-300 mesh silica gel column chromatography to obtain the active component; and then SephadexLH-20 gel column chromatography, eluted with CH ₂ Cl ₂ : MeOH at a ratio of 1:1, and the flow rate was 60mL/h to obtain the active component. component A and active component B; Active component A and active component B were subjected to gel column chromatography twice, the first time was eluted with Hexane:CH ₂ Cl ₂ :MeOH at a ratio of 4:5:1, and the second time was eluted with Hexane:CH ₂ Cl ₂ : MeOH is eluted at 10:10:1 to obtain active component C and active component D respectively; The active component C and the active component D were subjected to ODS flash column chromatography, and the gradient elution of 50% and 80% MeOH was used to obtain the total lactones of Bryocarpus. 10. the preparation method of a kind of bryozoan total lactone according to claim 9 is characterized in that, with 95% ethanol room temperature leaching, the consumption of 95% ethanol is 20% of the total bryozoan raw material of fresh collection. -30 times; the n-hexane extraction is 5 times in total, and the amount of n-hexane is 4-5L each time; the CCl ₄ extraction is 5 times, and the amount of CCl ₄ is 1-2L each time.