CN112472725A

CN112472725A - Brown algae extract and its application

Info

Publication number: CN112472725A
Application number: CN201910861869.8A
Authority: CN
Inventors: 袁宁生
Original assignee: Ningbo Huxin Drug Technology Co ltd
Current assignee: Ningbo Huxin Drug Technology Co ltd
Priority date: 2019-09-11
Filing date: 2019-09-11
Publication date: 2021-03-12

Abstract

A brown algae extract is prepared by soaking brown algae in water, cleaning, drying, adding acetone solution, heating under reflux to obtain extractive solution, recovering solvent under reduced pressure, concentrating, and drying the concentrated solution. Animal experiments prove that the brown algae extract has the functions of improving the nervous system, such as: but not limited to, the prevention and treatment of Alzheimer disease, Parkinson disease and primary lateral sclerosis, and the improvement of memory (especially spatial memory), and has a prospect of being developed as health-care products and medicaments.

Description

Brown algae extract and its application

Technical Field

The invention relates to an extract from plants, in particular to an extract from algae and application thereof in preparing a composition for preventing and treating neurodegenerative diseases.

Background

Alzheimer's Disease (AD), commonly known as Alzheimer's disease or senile dementia, is a neurodegenerative disease whose symptoms are mainly manifested by a variable loss of memory and cognitive abilities of the patient and a dysfunction of the behavioral activities. The pathogenesis of AD is not well understood and may be related to various factors such as cellular aging, genetic damage, neurotransmitter damage, and inflammation. At present, the anti-AD drugs applied clinically or in clinical research are mainly used for improving the Ach level in cranial nerves, recovering the Ach nerve conduction, improving the memory, cognition and behavior ability of patients and delaying the development of diseases. One class of drugs is the cholinergic drugs, and the other class of drugs is the drugs that promote the release of Ach in the cranial nerves.

The commonly used preventive and therapeutic drugs comprise central stimulants, cholinergic-improving substances, cerebral blood circulation improving agents and the like, but the drugs have the defects of uncertain curative effect, weak specificity or large toxic and side effects, poor oral absorption, temporary solution and permanent solution, and the like, so the application of the drugs is limited.

Chinese patent ZL200710099008.8 discloses an fucoidan polysaccharide sulfate with the function of preventing and treating Parkinson's disease. Pulverizing herba Zosterae Marinae, extracting with distilled water, filtering the extractive solution with diatomite, dialyzing the filtrate with tap water for one day, dialyzing with distilled water for one day, concentrating the dialysate, adding ethanol to obtain 75% precipitate, and drying the precipitate to obtain crude fucoidan sulfate. The polysaccharide sulfate mainly comprises fucose and galactose, wherein the fucose content is 31%, the sulfate group content is 32%, and the average molecular weight is 180 KD. The fucoidan polysaccharide sulfate has certain neuroprotective effect on DA-nervoussoirs of PD cell models and animal models.

Intensive research around brown algae, especially in the prevention and treatment of neurodegenerative diseases, is being attempted to find molecules or compositions with exact chemical structures.

Disclosure of Invention

The invention aims to provide a brown algae extract which has pharmacological action of preventing and treating neurodegenerative diseases.

Another object of the present invention is to provide a brown algae extract having pharmacological effects of improving memory (including spatial memory).

It is still another object of the present invention to provide a brown algae extract having pharmacological effects of improving alzheimer's disease, parkinson's disease and primary lateral sclerosis.

The invention also aims to provide an application of the brown algae extract as an active ingredient in preparing a medicament for preventing and treating neurodegenerative diseases.

The invention also aims to provide an application of the brown algae extract as an active ingredient in preparing foods or health-care products for preventing neurodegenerative diseases.

The "brown algae" referred to herein are multicellular plants, and typical species thereof include plants of the Sargassaceae family of the phylum Phaeophyta. Sargassum fusiforme (Harv.) Setchel belongs to Sargassaceae of Phaeophyta, is a representative brown algae, also called sea barley, antler tip and seaweed, and is used as a medicine by using whole dried algae, and has bitter, salty and cold properties without toxicity, and has the main effects of softening and resolving hard mass, eliminating phlegm, cooling and detoxifying, removing blood stasis, inducing diuresis and relieving edema, and can be used for treating goiter, testicular swelling and pain, and phlegm-fluid edema. It mainly grows in the warm water areas in the west of the north pacific, and is widely distributed in China, and is distributed in the shallow sea areas of the Shandong and Zhejiang, Nandinjian and Guangdong, in the peninsula of Liaodong. Others are as follows: but are not limited to, sargassum pallidum, kelp, carrageen, kelp, wakame seaweed, and the like. In the present invention, it refers to one of sargassum pallidum, kelp, carrageen, kelp, wakame and hizikia fusiforme or one of sargassum pallidum, kelp, carrageen, kelp, wakame and hizikia fusiforme.

The composition of the invention also comprises various pharmaceutical excipients which are suitable for the contained compound or the composition and are prepared into dosage forms which are beneficial to drug delivery (such as: but not limited to aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder spray, sustained release agent, controlled release agent, etc. These pharmaceutical excipients may be those conventionally used in various formulations, such as: but are not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrating agents, lubricants, and the like; it may also be selected for use in accordance with the substance, such as: the auxiliary materials can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate, absorption rate and the like of the compounds, thereby improving the metabolism of various compounds in organisms and further enhancing the administration effect of the composition. In addition, specific administration purposes or modes may be achieved, such as: sustained release administration, controlled release administration, pulse administration, and the like, and used auxiliary materials such as: but are not limited to gelatin, albumin, chitosan, polyether and polyester-based polymer materials, such as: but are not limited to, polyethylene glycol, polyurethane, polycarbonate, copolymers thereof, and the like. The main indications of so-called "facilitated administration" are: but not only improving the treatment effect, improving the bioavailability, reducing the toxic and side effects, improving the compliance of patients and the like.

In aqueous injection, the adjuvant generally comprises isotonic agent, buffer, necessary emulsifier (such as Tween-80, Pluronic and Poloxamer), solubilizer, bacteriostatic agent, etc. In addition, the pharmaceutical composition also comprises other pharmaceutically acceptable pharmaceutical excipients, such as: antioxidants, pH modifiers, analgesics, and the like.

The adjuvants used for preparing oral liquid preparation generally include solvent, and necessary correctant, bacteriostat, emulsifier and colorant, etc.

The excipients used for the preparation of tablets generally include fillers (e.g., starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, mannitol, etc.), binders (e.g., ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, gelatin solution, sucrose solution, and an aqueous or alcoholic solution of polyvinylpyrrolidone, etc.), disintegrants (e.g., dry starch, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose, crosslinked polyvinylpyrrolidone, and crosslinked sodium carboxymethylcellulose), and lubricants (e.g., magnesium stearate, colloidal silica, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, magnesium lauryl sulfate, etc.), and the like.

The adjuvants used for preparing emulsion are generally water, oil (such as fatty acid), emulsifier, and necessary antiseptic and correctant.

The excipients used to make granules are similar to tablets, but the granulation process is different. Mixing the obtained granule with glidant, and encapsulating to obtain capsule.

The term "organism", "animal" or "patient" as used herein refers to humans, wild animals and Livestock (Livestock). The wild animal is an animal which is not artificially domesticated in a natural state. Livestock are animals that are artificially raised to provide a food source, such as: but are not limited to, dogs, cats, mice, rats, hamsters, pigs, rabbits, cows, buffalos, bulls, sheep, goats, geese, chickens, and the like. The "patient" or "organism" to which treatment is administered is preferably a mammal, particularly a human.

As used herein, "prevention" refers to any means or measure, including medical, physical or chemical means, for preventing the onset or progression of a disease, before the disease is identified by clinical criteria, to prevent or reduce the onset or progression of the various symptoms of the disease.

As used herein, "treating" or "treatment" refers to inhibiting, suppressing, reducing, ameliorating, slowing, stopping, delaying or reversing the progression or exacerbation of a disease or condition in order to arrest or reduce the occurrence or progression of the disease, and the various indications of the disease, disorder or pathological condition described as maintaining and/or administering include alleviating or reducing symptoms or complications, or curing or eliminating the disease, disorder or condition.

The term "food" as used herein is intended to encompass any individual compound or composition made edible by the various compounds, compositions or extracts provided herein. The production and manufacture of such single compounds or compositions should meet relevant food safety standards, which, however, are not limiting to the present invention.

The term "health product" as used herein refers to a composition or composition prepared from various compounds, compositions or extracts provided by the present invention and administered to a patient for the purpose of preventing and treating diseases. Which belongs to the food product of the present invention, but which should also be manufactured, manufactured and sold in conformity with various relevant requirements, standards and specifications.

The term "drug" as used herein refers to a single compound, a composition comprising a plurality of compounds, a Chinese medicinal material or an extract thereof, or a composition or preparation (formulation) comprising a single compound as a main active ingredient, or a composition or preparation comprising a plurality of compounds as active ingredients, which can be used for preventing or treating a disease. "medicament" is understood to mean not only the product approved and approved for production by the regulatory agency established in accordance with the state of law, but also the forms of the various substances formed in order to obtain the approved and approved production, which contain the single compound as active ingredient. "Forming" is understood to mean obtaining it by chemical synthesis, biotransformation or purchase.

The invention provides a brown algae extract which is prepared by the following method:

soaking brown algae in water, cleaning and drying;

then adding acetone solution (such as but not limited to 20 v/v% -95 v/v%, 20 v/v% -70 v/v%, 50 v/v% -90 v/v% or 30 v/v% -65 v/v%, etc.), heating and refluxing (such as but not limited to 0.5-6 hours, 1-4 hours, 0.5-3 hours or 2-5.5 hours, etc.) for extraction, and filtering to obtain filtrate;

then, concentrating the obtained filtrate to a concentrated solution with the relative density of 1.10-1.50;

and finally, drying the concentrated solution to obtain the brown algae extract.

The other brown algae extract provided by the invention is prepared by the following method:

soaking brown algae in water, cleaning, and drying (such as air drying or sun drying or oven drying);

then, adding 20 v/v% -95 v/v% acetone solution with volume being 5-40 times of the weight of the brown algae, heating, refluxing and extracting for 0.5-6 hours, and filtering to obtain filtrate;

then, concentrating (e.g., reducing pressure) the obtained filtrate to a concentrated solution with no acetone smell and a relative density of 1.10-1.50;

finally, drying the concentrated solution (such as vacuum drying under reduced pressure or spray drying) to obtain brown algae extract.

The acetone solution of the present invention is understood to be a solution with acetone as solute, such as: but not limited to, acetone aqueous solution, and solutions of acetone dissolved in ethanol, methanol, ethyl acetate, chloroform, organic acids, inorganic acids, organic bases, inorganic bases, etc. of various concentrations, and acetone solution obtained by mixing acetone as a solute with organic or inorganic solvents in proportion is used as an extraction solvent for preparing the brown algae extract.

In the preparation of the brown algae extract of the present invention, the time for soaking brown algae in water is 0.5 to 5 hours.

In the preparation of the brown algae extract, the residual material obtained after the separation from the filtrate is extracted by acetone solution, and the extraction is repeated for a plurality of times, and the filtrates are combined and concentrated to the liquid with the relative density of 1.10-1.50.

In addition to direct extraction or extraction, the extract or extracts may be further refined and purified by various purification methods, such as: but are not limited to, normal and reverse phase chromatography techniques, ultrafiltration techniques, membrane techniques, and the like, using various chromatographic packing materials or media, such as: but are not limited to, macroporous resins, gels, silica gels, small pore resins, anion exchange resins, cation exchange resins, molecular sieves, cellulose, activated carbon, alumina, hollow fibers, dialysis bags and dialysis membranes of various cut-off specifications, and the like.

The invention provides a pharmaceutical composition, which takes the brown algae extract provided by the invention as an active ingredient.

The invention also provides another food, which takes the brown algae extract provided by the invention as an active ingredient.

The invention provides another health product, which takes the brown algae extract provided by the invention as an active ingredient.

The technical scheme of the invention has the following beneficial effects:

the various extracts provided by the invention have the function of improving the nervous system, such as: but are not limited to, alzheimer's disease, parkinson's disease and primary lateral sclerosis, and memory (especially spatial memory).

Detailed Description

The technical solution of the present invention is described in detail below. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Reagents used in the present invention were purchased from Sigma-Aldrich (Sigma-Aldrich) unless otherwise specified.

EXAMPLE 1 extraction of Brown algae

Soaking brown algae in water for 0.5-5 hr, washing and drying; then, adding 20 v/v% -95 v/v% acetone solution with volume being 5-40 times of the weight of the brown algae, heating, refluxing and extracting for 0.5-6 hours, and filtering to obtain filtrate; then, concentrating the obtained filtrate under reduced pressure to a concentrated solution which has no acetone smell and has a relative density of 1.10-1.50; and finally, drying the concentrated solution under reduced pressure and vacuum to obtain the brown algae extract.

Example 2

Step one, soaking a sargassum fusiforme medicinal material (1000g) in 20 times of water, stirring and cleaning, changing water for three times, filtering, drying in the sun, adding 10L of 20% acetone solution, heating, refluxing and extracting for 2 hours, filtering, adding 8L of 20% acetone solution into filter residue, heating, refluxing and extracting for 2 hours, filtering, and combining filtrates.

And step two, taking the filtrate, recovering acetone under reduced pressure until no acetone smell exists, obtaining a concentrated solution, wherein the relative density is 1.10, drying by distillation in a water bath at 95 ℃, and drying in an oven at 70 ℃ overnight to obtain the test sample 1.

Example 3

Step one, soaking a sargassum fusiforme medicinal material (1000g) in 20 times of water, stirring and cleaning, changing water for three times, filtering, drying in the sun, adding 10L of 30% acetone solution, heating and refluxing for 2 hours, filtering, adding 8L of 30% acetone solution into filter residue, heating and refluxing for 2 hours, filtering, and combining filtrates.

And step two, taking the filtrate, recovering acetone under reduced pressure until no acetone smell exists, obtaining a concentrated solution, wherein the relative density is 1.15, drying by distillation in a water bath at 95 ℃, and drying in an oven at 70 ℃ overnight to obtain the test sample 2.

Example 4

Step one, soaking a sargassum fusiforme medicinal material (1000g) in 20 times of water, stirring and cleaning, changing water for three times, filtering, drying in the sun, adding 10L of 50% acetone solution, heating, refluxing and extracting for 2 hours, filtering, adding 8L of 50% acetone solution into filter residue, heating, refluxing and extracting for 2 hours, filtering, and combining filtrates.

And step two, taking the filtrate, recovering acetone under reduced pressure until no acetone smell exists, obtaining a concentrated solution, wherein the relative density is 1.2, drying by distillation in a water bath at 95 ℃, and drying in an oven at 70 ℃ overnight to obtain the test sample 3.

Example 5

Step one, soaking a sargassum fusiforme medicinal material (1000g) in 20 times of water, stirring and cleaning, changing water for three times, filtering, drying in the sun, adding 10L of 70% acetone solution, heating, refluxing and extracting for 2 hours, filtering, adding 8L of 70% acetone solution into filter residues, heating, refluxing and extracting for 2 hours, filtering, and combining filtrates.

And step two, taking the filtrate, recovering acetone under reduced pressure until no acetone smell exists, obtaining a concentrated solution, wherein the relative density is 1.30, drying by distillation in a water bath at 95 ℃, and drying in an oven at 70 ℃ overnight to obtain the test sample 4.

Example 6

Step one, soaking a sargassum fusiforme medicinal material (1000g) in 20 times of water, stirring and cleaning, changing water for three times, filtering, drying in the sun, adding 10L of 90% acetone solution, heating and refluxing for extraction for 2 hours, filtering, adding 8L of 90% acetone solution into filter residues, heating and refluxing for extraction for 2 hours, filtering, and combining filtrates.

And step two, taking the filtrate, recovering acetone under reduced pressure until no acetone smell exists, obtaining a concentrated solution, wherein the relative density is 1.45, drying by distillation in a water bath at 95 ℃, and drying in an oven at 70 ℃ overnight to obtain the test sample 5.

Example 7 Effect of test 2 on sodium nitrite-induced memory reproduction disorder in mice

In this example, the acetone extract of brown algae was used as sample 2.

Sodium nitrite is a cerebral anoxia agent, and it is believed that dysmnesia caused by sodium nitrite is caused by hypoxia of various tissues of the whole body due to oxidation of hemoglobin in vivo into methemoglobin after the sodium nitrite enters the body, so that the hemoglobin loses the functions of oxygen carrying and releasing, thereby causing cerebral anoxia dysmnesia. The medicine for improving the learning memory can reduce the number of animals which are shocked after the mice with poor memory consolidation caused by sodium nitrite are 24 hours, reduce the error frequency within 5 minutes and shorten the incubation period of the first time when the mice jump down the platform.

Male ICR mice (Shanghai Seaprol-Bikai laboratory animals Co., Ltd., animal certification number: SCXK 2008. 0016) were administered by oral gavage (i.g.), wherein the blank control group and the model group were each administered with an equal volume of 0.5% sodium carboxymethylcellulose (CMC-Na), the administration group was administered with 120mg/kg of test article 2, 10 mice per group, 1 time/day, 10ml/kg of body weight, and they were administered continuously for 3 weeks. 1 hour after the last dose, the mice were placed in a diving platform apparatus for acclimation for 5 minutes. Then electrifying, wherein the normal reaction of the animals after being shocked is to jump back to the platform to avoid the injurious stimulation, most animals can jump to the copper grid again or for many times, and quickly jump back to the platform after being shocked, training is carried out for 5 minutes, and the shocking times of each mouse are recorded. Immediately after training, 120mg/kg (0.1mL/10g) of sodium nitrite was injected subcutaneously, and the blank control group was given an equal volume of physiological saline. After 24 hours, the test is repeated, the screen analysis system photographs and automatically records all relevant data of the animals, and the experimental results are shown in the table 1.

TABLE 1 Effect of test article 2 on sodium nitrite-induced memory consolidation disorder in mice after 3 weeks of administration

P < 0.01, P < 0.05 compared to model group.

Test article 2 had no significant effect on mouse body weight three weeks after administration. In the model test of the mouse memory consolidation disorder caused by sodium nitrite, the difference of the latency period of the test article 2 compared with the model group has statistical significance.

Example 8 Effect of test article 3 on ethanol-induced mouse memory reproduction disorder

In this example, the acetone extract of brown algae was used as sample 3.

Ethanol is used as central depressant, and can inhibit nerve function activity of cerebral cortex, inhibit conditioned reflex process of animals, and change systems such as protein and RNA synthesis in brain, cholinergic system and dopamine system. Given prior to retesting, memory reproduction can be significantly disturbed. The ethanol is cheap and easily available, and under the appropriate concentration, the result is stable and easy to repeat, and has no obvious influence on the center and general motor functions. The medicine for improving learning and memory can reduce the latency and the error frequency from the light room to the dark room of a mouse with memory reproduction disorder caused by ethanol after 24 hours. Male ICR mice (Shanghai Seaprol-Bikai laboratory animals Co., Ltd., animal certification number: SCXK 2008. 0016) were administered by oral gavage (i.g.), wherein the blank control group and the model group were each administered with an equal volume of 0.5% sodium carboxymethylcellulose (CMC-Na), the administration group was administered with 3 samples at a dose of 120mg/kg, and each group was administered with 10 mice at a dose of 1 time/day at a dose of 10ml/kg for 3 weeks. The last time of administration is 1 hour, training once by dark method. The mouse is placed in a dark avoiding box, the mouse is placed in a bright room with the back facing to the opening, the visual screen analysis device is started at the same time, the animal passes through the opening and enters the dark room to be subjected to electric shock, and the timing is automatically stopped. The mice were removed and screen analysis recorded the time required for each mouse to encounter an electric shock from placement in the bright room to entry in the dark room, which is the latency period. The test was performed 24 hours later, and 30 minutes prior to the test, 45% ethanol solution (0.1ml/10g) was administered by oral gavage, and the blank control group was administered with an equal volume of physiological saline. The video analysis system will photograph and automatically record all relevant data of the animals, and the experimental results are shown in table 2.

TABLE 2 Effect of test substance 3 on ethanol-induced memory reproduction disorder in mice

P < 0.01, P < 0.05 compared to model group.

There was no significant effect on mouse body weight 3 weeks after administration. In the sample test, the difference of the latency of the test article 3 is statistically significant compared with the model group, and the difference of the error times of the test article 3 is significantly statistically significant compared with the model group.

Example 9 Effect of test article 5 on ethanol-induced mouse memory reproduction disorder

In this example, the acetone extract of brown algae was used as sample 5.

Ethanol is used as central depressant, and can inhibit nerve function activity of cerebral cortex, inhibit conditioned reflex process of animals, and change systems such as protein and RNA synthesis in brain, cholinergic system and dopamine system. Given prior to retesting, memory reproduction can be significantly disturbed. The ethanol is cheap and easily available, and under the appropriate concentration, the result is stable and easy to repeat, and has no obvious influence on the center and general motor functions. The medicine for improving learning and memory can reduce the latency and the error frequency from the light room to the dark room of a mouse with memory reproduction disorder caused by ethanol after 24 hours. Male ICR mice (Shanghai Seaprol-Bikai laboratory animals Co., Ltd., animal certification number: SCXK 2008. 0016) were administered by oral gavage (i.g.), wherein the blank control group and the model group were each administered with an equal volume of 0.5% sodium carboxymethylcellulose (CMC-Na), the administration group was administered with 120mg/kg of test article 5, 10 mice per group, 1 time/day, 10ml/kg of body weight, and the administration was continued for 3 weeks. The last time of administration is 1 hour, training once by dark method. The mouse is placed in a dark avoiding box, the mouse is placed in a bright room with the back facing to the opening, the visual screen analysis device is started at the same time, the animal passes through the opening and enters the dark room to be subjected to electric shock, and the timing is automatically stopped. The mice were removed and screen analysis recorded the time required for each mouse to encounter an electric shock from placement in the bright room to entry in the dark room, which is the latency period. The test was performed 24 hours later, and 30 minutes prior to the test, 45% ethanol solution (0.1ml/10g) was administered by oral gavage, and the blank control group was administered with an equal volume of physiological saline. The video analysis system will photograph and automatically record all relevant data of the animals, and the experimental results are shown in table 3.

TABLE 3 Effect of test article 5 on ethanol-induced memory reproduction disorder in mice

P < 0.01, P < 0.05 compared to model group.

There was no significant effect on mouse body weight 3 weeks after administration. In the sample test, the difference between the latency of the test article 5 and the model group is statistically significant, and the difference between the error frequency and the number of the shocked animals of the test article 5 and the model group is statistically significant.

Claims

1. A brown algae extract is characterized by being prepared by the following method:

soaking brown algae in water, cleaning and drying;

then, adding an acetone solution, and heating, refluxing and extracting to obtain a filtrate;

2. The brown algae extract of claim 1, wherein the concentration of the acetone solution is 20 v/v% to 95 v/v%.

3. The brown algae extract according to claim 1, wherein the acetone is added in a volume 5 to 40 times the weight of the brown algae.

4. The brown algae extract according to claim 1, wherein the drying is vacuum drying under reduced pressure or spray drying.

5. The brown algae extract of claim 1, wherein the filtrate is concentrated to no acetone odor.

6. The brown algae extract according to claim 1, wherein the time for soaking the brown algae in water is 0.5 to 5 hours.

7. The brown algae extract of claim 1, wherein the remaining material obtained after separation from the filtrate is extracted with acetone solution and repeatedly extracted in this way.

8. Use of the brown algae extract according to any one of claims 1 to 7 in the preparation of a medicament, food or health product for improving nervous system function.

9. Use of the brown algae extract according to any one of claims 1 to 7 for the preparation of a medicament, food or health product for preventing and treating alzheimer's disease, parkinson's disease, primary lateral sclerosis, and memory.

10. A pharmaceutical composition comprising the brown algae extract according to any one of claims 1 to 7 as an active ingredient.