CN109589400B - Composition with neuroprotective effect - Google Patents
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- CN109589400B CN109589400B CN201811648298.1A CN201811648298A CN109589400B CN 109589400 B CN109589400 B CN 109589400B CN 201811648298 A CN201811648298 A CN 201811648298A CN 109589400 B CN109589400 B CN 109589400B
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- 230000002936 tranquilizing effect Effects 0.000 description 1
- 229940036248 turpentine Drugs 0.000 description 1
- 125000005314 unsaturated fatty acid group Chemical group 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 231100000889 vertigo Toxicity 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 0.000 description 1
Images
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8988—Gastrodia
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
Abstract
The invention relates to a composition with neuroprotective effect. The composition essentially comprises: rhizoma Gastrodiae extract and semen Juglandis peptide. The preparation method comprises the steps of extracting, filtering, concentrating and drying. The composition has good free radical scavenging activity, can enhance memory, promote nervous system recovery and protect nervous tissue, and can be developed into food, health food or medicine for brain health and preventing, improving or treating related neurological diseases such as Alzheimer's disease and Parkinson's disease.
Description
Technical Field
The invention belongs to the field of health products, and relates to a preparation method for processing a composition by taking natural plants as raw materials, in particular to a composition with neuroprotective effect. The composition of the present invention can be used as a food, a health food, or a medicine.
Background
With the aging population, environmental pollution, stress and other problems, the incidence of brain diseases such as stroke, alzheimer Disease (AD), parkinson Disease (PD), depression and schizophrenia is increasing, which is a major problem seriously harming human health and is highly regarded. These brain diseases are common pathological processes in which nerve cell damage exists. For nervous system diseases, TCM has always paid attention to and paid attention to. The effective components in the traditional Chinese medicine play a key role, are the core of the efficacy of the traditional Chinese medicine, and play a role in preventing or delaying the apoptosis of nerve cells, the accumulation of Nitric Oxide (NO), inflammatory injury, oxidative stress injury, the overload of calcium ions in cells, autophagy and the like, thereby blocking the continuous injury of the nerve cells and achieving the effect of treating nervous system diseases. Therefore, the traditional Chinese medicine composition with the neuroprotective effect is developed to achieve the related application of preventing, improving or treating the brain diseases.
Gastrodia elata Bl, a dried tuber of Gastrodia elata Bl of Orchidaceae, also known as rhizoma Gastrodiae, ganoderma lucidum, dioscorea opposita, plectranthus unicolor, lysimachia christinae Hance, etc., is a perennial parasitic plant, whose host is Armillariella mellea, and Armillariella mellea hypha and secretion are used as nutrition sources for growth and development, and are mainly distributed in Yunobu plateau, sichuan, tibet, etc. It is sweet in flavor, mild in nature, entering liver meridian, and has the functions of extinguishing wind, relieving spasm, suppressing liver yang, dispelling wind and dredging collaterals. It is used clinically for infantile convulsion, epilepsy, convulsion, headache, vertigo, numbness of limbs, and arthralgia due to wind-dampness. Gastrodia elata is listed as the superior product in Shen nong Ben Cao Jing, and the book Ben Cao gang mu records that Gastrodia elata can cause various kinds of arthralgia due to wind-cold, can tonify qi after being taken for a long time, is light and long-lived, is a traditional famous and precious traditional Chinese medicinal material, and has been recorded and recorded in the Chinese pharmacopoeia (part of 2005 edition). Modern medicine shows that the gastrodia elata has pharmacological activities of calming, sleeping, resisting convulsion, eliminating free radicals, regulating immunity of an organism, improving memory and the like. It is reported that gastrodia elata contains various active ingredients such as saccharides, glycosides, gastrodin, sitosterol and the like. The gastrodin and total glycosides of rhizoma Gastrodiae have effects of tranquilizing, improving sleep, relieving pain, resisting inflammation, improving phagocytic ability of macrophage in vivo, and removing free radicals in vivo, delaying cell aging, lowering blood pressure, and inhibiting bacteria.
Walnut (Juglans regia L.) is also called walnut, belongs to one of four nuts, and has high nutritional and medicinal values. In ancient medical books of China, there are clear records, li Shizhen (compendium of materia Medica) describing "tonifying qi and nourishing blood, moistening dryness and resolving phlegm, benefiting life gate, benefiting triple energizer, warming lung and moistening intestines. It is indicated for lung and intestine moistening. For deficiency cold cough and asthma, severe pain of waist and feet, hernia pain in heart and abdomen, bloody dysentery with intestinal wind, and swelling and toxin, the walnut (walnut) carried by Songhua (Kaibao materia Medica) has sweet, mild and nontoxic taste. Eating it can nourish the body, moisten the skin and blacken the hair, take out the flesh and burn to make it black, without cutting off the smoke, and grind on the turpentine to remove scrofula. In Tang Hua Meng (Chinese materia medica), walnut kernel can dredge meridians and blacken beard and hair, and is often used to make bone and meat fine and smooth. From Shi Jing (classic of food) of Yu Tong, it says "Ding Shi Li and Wu Zhi. The evaluation of the book of Yi Lin Du Yao (treatise on medical forest) is that it can tonify kidney, moisten Mingmen, secure essence, moisten large intestine, clear heat, and stop cold diarrhea and deficient diarrhea. "and the like. The walnut contains rich nutritional ingredients such as protein and fat, has relatively balanced content, belongs to relatively ideal high-protein and high-fat food, and reportedly contains 52-70% of grease in walnut kernel, wherein most of the grease is unsaturated fatty acid, and also contains about 24% of protein, 12-16% of carbohydrate, 1.5-2% of cellulose and 1.7-2% of mineral substances. The walnut is rich in amino acids necessary for human bodies, the proportion of the amino acids is reasonable, the contents of glutamic acid, aspartic acid and arginine which have important functions on the physiological action of the human bodies are higher, and the glutamic acid is also an important functional substance which influences the intelligence and memory development of the human bodies, particularly adolescents.
The invention aims to research and develop a composition which takes natural plants as sources and has better neuroprotective effect, the gastrodia elata extract and the walnut peptide are compounded according to the optimal proportion to obtain the composition with the neuroprotective effect, and the composition is confirmed by biological activity experiments.
Disclosure of Invention
The invention aims to solve the problems that: the invention also aims to provide a preparation method of the composition with the neuroprotective effect, which is simple, environment-friendly and suitable for large-scale production.
The invention is realized by the following technical scheme:
the invention provides a composition with neuroprotective effect, which is characterized in that: the composition consists of gastrodia elata extract and walnut peptide.
Preferably, 40-95 wt% of gastrodia elata extract and 5-60 wt% of walnut peptide.
Preferably, 50-90 wt% of gastrodia elata extract and 10-50 wt% of walnut peptide.
More preferably, 65-85 wt% of gastrodia elata extract and 15-35 wt% of walnut peptide; or 75-85 wt% of gastrodia elata extract and 15-25 wt% of walnut peptide; or 80-85 wt% of gastrodia elata extract and 15-20 wt% of walnut peptide.
Most preferably, 65wt% of gastrodia elata extract and 35wt% of walnut peptide; 85wt% of gastrodia elata extract and 15wt% of walnut peptide.
The invention also provides a preparation method of the composition with the neuroprotection, which is characterized by comprising the following steps: firstly, preparing the gastrodia tuber extract and the walnut peptide respectively, and then mixing the gastrodia tuber extract and the walnut peptide according to the optimal percentage.
The gastrodia elata extract is characterized by being prepared by the following method: pulverizing rhizoma Gastrodiae, sequentially extracting, filtering, concentrating, and drying to obtain rhizoma Gastrodiae extract.
The walnut peptide is characterized in that the peptide is prepared by the following method: after the walnut is shelled and squeezed, the walnut peptide is obtained after protein extraction, biological enzyme enzymolysis, filtration, concentration and drying in sequence.
The preparation method of the gastrodia elata extract is characterized by comprising the following steps: the extraction can be water extraction, organic solvent extraction, water extraction followed by organic solvent extraction, mixed solution extraction of organic solvent and water, supercritical carbon dioxide extraction, or biological enzyme extraction, preferably biological enzyme extraction.
The preparation method of the walnut peptide is characterized by comprising the following steps: the protein extraction is an alkali extraction and acid precipitation method.
The preparation method of the composition is characterized by comprising the following steps: the biological enzyme can be pectinase (the enzyme activity is more than or equal to 1 ten thousand u/g), glycanase (the enzyme activity is more than or equal to 1000 u/g), cellulase (the enzyme activity is more than or equal to 1 ten thousand u/g), neutral protease (the enzyme activity is more than or equal to 30 ten thousand u/g), papain (the enzyme activity is more than or equal to 40 ten thousand u/g), bromelain (the enzyme activity is more than or equal to 30 ten thousand u/g), alkaline protease (the enzyme activity is more than or equal to 20 ten thousand u/g), acid protease, pepsin (the enzyme activity is more than or equal to 50 ten thousand u/g), pancreatin (the enzyme activity is more than or equal to 3000 u/g), ficin (alpha-amylase)
The enzyme activity is more than or equal to 5 ten thousand u/g), fructus momordicae protease (the enzyme activity is more than or equal to 5 ten thousand u/g) or a mixture thereof, and preferably food grade neutral protease is used.
The preparation method of the walnut peptide is characterized by comprising the following steps: the filtration can be membrane filtration, centrifugal filtration, screen filtration, plate-and-frame filtration, and preferably membrane filtration is used.
The preparation method of the composition is characterized by comprising the following steps: the concentration method can be vacuum concentration, atmospheric concentration, membrane concentration, and preferably vacuum concentration.
The preparation method of the composition is characterized by comprising the following steps: the drying method may be vacuum drying, heat drying, air drying, freeze drying and spray drying, and preferably spray drying is used.
The preparation method of the gastrodia elata extract is characterized by comprising the following steps of: taking a certain amount of gastrodia elata medicinal material, crushing, mixing with water according to a weight ratio of 1-1.
The preparation method of the walnut peptide is characterized by comprising the following steps:
pretreatment of walnut meal: removing shells of walnuts, squeezing and degreasing to obtain walnut meal;
protein extraction: mixing a certain amount of degreased walnut meal with water according to a weight ratio of 1-1;
enzymolysis: heating the walnut protein liquid to 40-55 ℃, adjusting the pH value to be neutral, adding biological enzyme accounting for 0.5-2% of the weight of the walnut meal, stirring for enzymolysis for 3-6 h, boiling for inactivation for 30min, centrifuging, and obtaining supernatant fluid which is protein enzymolysis liquid.
And (3) filtering: filtering the protein enzymolysis liquid by using a microfiltration membrane with the aperture of 0.1-0.5 mu m, treating the permeate by using a 2000-20000 Dalton ultrafiltration membrane, concentrating the permeate at 50-80 ℃ until the relative density is 1.0-1.5 g/mL, drying to obtain walnut peptide powder, wherein the content is over 80wt% according to the determination method of the peptide content in GB/T22492-2008 appendix B and GB/T22729-2008, and the molecular weight of more than 95% of the walnut peptide is less than 1500Dalton according to the high-efficiency gel filtration chromatography (GPC) described in GB/T22492-2008 appendix A and GB/T22729-2008 appendix A.
The invention also provides a composition, which contains the composition and edible or medicinal auxiliary materials; preferably, the composition is tablets, dispersible tablets, capsules, soft capsules, microcapsules, granules, injections, powder injections, freeze-dried powder injections, micro-pills, dropping pills, syrups, powders, extracts, soft extracts, oral liquid preparations and other formulations acceptable in the pharmaceutical or food fields.
The composition containing the above extract composition can be used for preparing food, health food, or medicine for preventing, improving or treating symptoms caused by excessive free radicals; used for preparing food, health food or medicine for preventing, improving or treating hypomnesis; for preparing a food, a health food, or a pharmaceutical product for preventing, improving, or treating Parkinson's disease and Alzheimer's disease; for preparing a food, a health food, or a pharmaceutical product for preventing, improving, or treating Parkinson's disease and Alzheimer's disease; can be used for preparing food, health food or medicine for preventing, improving or treating nerve related diseases.
It will be understood by those skilled in the art that when the amount of the gastrodia elata extract or the walnut peptide is the corresponding preferred amount of the component, the amount of the other component in the composition, i.e., the cistanche deserticola extract or the walnut peptide, can be calculated according to the sum of the amounts of the two in the composition being equal to 100 wt%.
In addition, as can be understood by those skilled in the art, if the composition only contains the gastrodia elata extract or the walnut peptide, the composition still has a certain neuroprotective effect as proved by a zebra fish neuroprotective efficacy experiment.
Drawings
FIG. 1: evaluation of the Central neuroprotective Effect of TM, HT, Z-1 and Z-2 combinations on Zebra Fish
Examples
The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that the simple modification of the preparation method of the present invention based on the concept of the present invention falls within the scope of the present invention claimed. All the raw materials and solvents used in the examples are commercially available products unless otherwise specified.
Preparation example 1:
(1) Taking 100kg of gastrodia elata medicinal material, crushing, adding 2000L of water, heating to 45 ℃, adjusting the pH value to 7-8, adding 100g of food-grade neutral protease with the enzyme activity of 30 ten thousand u/g, extracting for 2 hours, heating to more than 85 ℃, keeping for 2min, carrying out centrifugal filtration, and carrying out spray drying to obtain 55.4 kg of gastrodia elata extract (with the batch number of TM) with the sugar content of 86.2%.
(2) Crushing 100kg of squeezed and degreased walnut meal, adding 1000L of water, adjusting the pH to 9-10, extracting at room temperature for 2 hours, extracting for 2 times, combining the two extracting solutions, adjusting the pH to 5, standing for 6 hours, discarding the supernatant, finally adding water with the volume ratio of 1. Heating the walnut protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 1kg of neutral protease (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 6h, boiling for inactivation for 30min, centrifuging, and obtaining the supernatant fluid as protein enzymolysis liquid. Filtering the protein enzymolysis solution with a microfiltration membrane with the aperture of 0.1 mu m, treating the permeate with a 5000Dalton ultrafiltration membrane, concentrating at 65 ℃ to the relative density of 1.2g/mL, and spray drying to obtain the walnut peptide (batch number is HT) with the yield of 22.6wt%. The content is 81.8wt% according to the determination method of the peptide content described in GB/T22492-2008 appendix B and GB/T22729-2008, and the molecular weight of 96.9% of the walnut peptide is less than 1500Dalton according to the high performance gel filtration chromatography (GPC) method described in GB/T22492-2008 appendix A and GB/T22729-2008 appendix A.
And (3) uniformly mixing 13kg of gastrodia elata extract and 7kg of walnut peptide to obtain a composition (with the batch number of Z-1), wherein the mass percentages of the extracts are as follows: 65wt% of gastrodia elata extract and 35wt% of walnut peptide.
Preparation example 2:
(1) Taking 100kg of gastrodia elata medicinal material, crushing, adding 2000L of water, heating to 45 ℃, adjusting the pH value to 7-8, adding 100g of food-grade neutral protease with the enzyme activity of 30 wumu/g, extracting for 2 hours, heating to more than 85 ℃, keeping for 2min, carrying out centrifugal filtration, and carrying out spray drying to obtain 52.6 kg of gastrodia elata extract, wherein the measured sugar content is 87.4wt%.
(2) Crushing 100kg of squeezed and degreased walnut meal, adding 1000L of water, adjusting the pH value to 9-10, extracting for 2 times at room temperature, combining the two extracting solutions, adjusting the pH value to 5, standing for 6 hours, discarding the supernatant, finally adding water with the volume ratio of 1. Heating the walnut protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 1kg of neutral protease (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 6h, boiling for inactivation for 30min, centrifuging, and obtaining the supernatant fluid as protein enzymolysis liquid. Filtering the protein enzymolysis solution with a microfiltration membrane with the aperture of 0.1 mu m, treating the permeate with a 20000Dalton ultrafiltration membrane, concentrating at 55 ℃ to the relative density of 1.1g/mL, and spray drying to obtain the walnut peptide with the yield of 22.1wt%. The content is 80.4wt% according to the determination method of the peptide content described in GB/T22492-2008 appendix B and GB/T22729-2008, and the molecular weight of 97.7% walnut peptide is less than 1500Dalton according to the high performance gel filtration chromatography (GPC) described in GB/T22492-2008 appendix A and GB/T22729-2008 appendix A.
And (3) uniformly mixing 17kg of gastrodia elata extract and 3kg of walnut peptide to obtain a composition (batch number is Z-2), wherein the mass percentages of the extracts are as follows: 85wt% of gastrodia elata extract and 15wt% of walnut peptide.
Biological activity example 1:
preparation of PBS buffer: weighing 8g of sodium chloride, 0.2g of potassium chloride, 0.24 g of potassium dihydrogen phosphate and 3.62g of disodium hydrogen phosphate dodecahydrate, putting the materials into a 1000mL beaker, adding 800mL of distilled water, stirring to dissolve the distilled water, adjusting the pH value to 7.4 by using hydrochloric acid or sodium hydroxide, transferring the solution into a 1000mL volumetric flask, adding distilled water to dilute the solution to a scale, and shaking uniformly for later use.
ABTS + Preparation of a storage solution: ABTS is weighed to precision + Placing the mixture into a 20mL brown volumetric flask, adding 15mL distilled water, carrying out ultrasonic treatment for 5min, fixing the volume to the scale with the distilled water, and shaking up. Accurately weighing about 76mg of potassium persulfate, placing the potassium persulfate in a 2mL brown volumetric flask, adding 1mL of distilled water, performing ultrasonic dissolution, fixing the volume to the scale with the distilled water, and shaking up. Add 352. Mu.L of potassium persulfate solution to the ABTS solution with precision, shake up, and stand overnight.
ABTS + Preparation of working solution: precisely aspirate 1mL of the storage solution, add about 65mL of PBS buffer, and shake well.
Preparing a test solution: and (3) precisely weighing a proper amount of the composition, placing the composition into a 20mL brown volumetric flask, adding 15mL PBS buffer solution, carrying out ultrasonic treatment for 5min, fixing the volume to a scale with the PBS buffer solution, and shaking up to obtain the composition.
The method comprises the following operation steps: accurately sucking 0.5mL of test solution and 5mL of ABTS working solution, and uniformly mixing; accurately sucking 0.5mL of test solution and 5mL of PBS buffer solution, and uniformly mixing; accurately pipette 5mL of ABTS working solution and 0.5mL of PBS buffer, mix well, immediately measure absorbance at 734 nm, and calculate the radical clearance according to the following formula:
IR％＝[1-(Ai-Aj)/A0]*100％；
wherein Ai represents the absorbance of the solution after the solution to be tested and the ABTS are mixed;
aj represents the absorbance of the solution after the solution to be detected and the solvent are mixed;
a0 represents the absorbance of the solution after mixing ABTS and the solvent.
The compositions (Z-1 and Z-2) of preparation examples 1 and 2 were formulated at a concentration of 500. Mu.g/mL, with vitamin C as a positive control (at a concentration of 100. Mu.g/mL), and the results are shown in Table 1:
TABLE 1 antioxidant Activity of the compositions
As can be seen from Table 1, the gastrodia and walnut peptide composition prepared by the method has a good effect of eliminating ABTS free radicals.
Biological activity example 2:
experimental animals: wild type AB strain zebrafish, in a natural mated mating breeding mode. The age is 1 day after fertilization, and 30 animals in each experimental group are used for evaluating the central nervous protection effect of the composition on the zebra fish.
Sample preparation: the composition is prepared into 20mg/mL mother solution by using fish culture water, and is diluted as required and prepared as required.
1. Determination of Maximum Tolerated Concentration (MTC) of Zebra fish
Wild type AB strain zebra fish 1 day (1 dpf) after fertilization are randomly selected to be placed in a six-hole plate, 30 zebra fish are processed in each hole (experimental group), and the zebra fish central nerve injury is induced by mycophenolate mofetil. TM, HT, Z-1 and Z-2 sample dilutions were dissolved in water to give different concentrations, and a normal control group (zebrafish treated with water for fish farming) and a model control group were set to have a volume of 3mL per well. After the sample and mycophenolate mofetil are treated together for 24 hours, the phenotype and death condition of the zebra fish are observed and recorded, and the MTC value of the sample to the zebra fish is respectively determined.
TABLE 1 "concentration-lethality" results after TM, HT, Z-1 and Z-2 treatment
From the experimental results of table 1, the following conclusions can be drawn:
(1) After the TM and the mycophenolate mofetil are treated together for 24 hours, the TM does not generate any obvious toxic or side effect on the zebra fish and does not cause death of the zebra fish at the concentration of 125-500 mu g/mL, so that the TM is determined to be 500 mu g/mL on the MTC of the zebra fish.
(2) After the HT and the mycophenolate mofetil are jointly processed for 24 hours, the HT does not generate any obvious toxic or side effect on the zebra fish and does not cause the death of the zebra fish at the concentration of 125-2000 mug/mL; HT was therefore determined to be 2000. Mu.g/mL for zebrafish MTC.
(3) After the Z-1, the Z-2 and the mycophenolate mofetil are jointly treated for 24 hours, the Z-1 and the Z-2 do not generate any obvious toxic or side effect on the zebra fish and do not cause the death of the zebra fish when the concentration is 125-1000 mu g/mL; therefore, Z-1 and Z-2 were determined to be 1000. Mu.g/mL for Zebra fish MTC.
From the above results, it can be seen that the MTC value of HT on zebra fish can reach at least 2000 mug/mL, which indicates that the safe dosage of HT is quite high, while the MTC value of TM on zebra fish does not exceed 500 mug/mL at most, but the MTC reaches 1000 mug/mL after being compounded with HT according to a certain proportion.
2. Evaluation of protective action on central nerve of zebra fish
Randomly selecting wild AB strain zebra fish 1 day after fertilization into a six-hole plate, treating 30 zebra fish in each hole, and inducing a zebra fish central nerve damage model by mycophenolate mofetil. TM, HT, Z-1 and Z-2 sample diluents with different concentrations are respectively dissolved in water, the concentration of a positive control drug Glutathione (GSH) is 154 mu g/mL, and a normal control group (zebra fish treated by water for fish culture) and a model control group are simultaneously arranged, and the volume of each hole is 3mL. Respectively treating the test sample and mycophenolate mofetil for 24 hours, dyeing with acridine orange, randomly selecting 10 zebra fish from each experimental group after dyeing, photographing under a fluorescence microscope, storing pictures, carrying out image analysis by NIS-Elements D3.10 advanced image processing software, calculating the fluorescence intensity (S) of apoptotic cells of central nerves (brain and spinal cord) of the zebra fish, and evaluating the protective effect of the test sample on the central nerves of the zebra fish according to the statistical analysis result of the fluorescence intensity of the apoptotic cells. Statistical treatment results are expressed as mean ± SE, and the central nervous system protective effect is calculated by the formula: central nervous protective effect (%) = (S) Model control group -S Test article group )× 100％/(S Model control group -S Normal control group ). Statistical analysis by analysis of variance and Dunnett's T-test, p<0.05 indicated significant differences. The measurement results are shown in Table 2.
TABLE 2 evaluation results of central neuroprotective effects of TM, HT, Z-1 and Z-2 on zebrafish (n = 10)
Note: compared with the model control group, ** p<0.01， *** p<0.001
from the experimental results of table 2, the following conclusions can be drawn:
(1) Comparing the fluorescence intensity of the central nerve apoptotic cells of the zebra fish in the model control group with that of the normal control group, wherein p is less than 0.001, which shows that the model is successfully established; the protective effect of the positive control drug GSH 154 mug/mL group on the central nerve of the zebra fish is 93%, which shows that the GSH has obvious protective effect on the central nerve of the zebra fish.
(2) The central nervous protection effects of TM on zebra fish are respectively 29%, 64% and 74% at the concentrations of 56, 167 and 500 mug/mL, which shows that TM has obvious protection effect on the central nervous of zebra fish at the concentration in the experiment, and the TM is increased along with the increase of the concentration along with the dependence of the concentration.
(3) The protective effect of HT on the central nerve of the zebra fish is 42%, 28% and-18% respectively at the concentrations of 222, 667 and 2000 mug/mL, which shows that HT has obvious protective effect on the central nerve of the zebra fish at medium and low concentration, and also presents concentration dependence, and decreases with the increase of the concentration.
(4) When the concentration of the composition Z-1 is 111, 333 and 1000 mug/mL, the central nerve protection effect on the zebra fish is respectively 26%, 30% and 47%, which indicates that the composition Z-1 has obvious protection effect on the central nerve of the zebra fish under the experimental concentration; the central nerve protection effect of the composition Z-2 on the zebra fish is 10 percent, 46 percent and 82 percent respectively at the concentration of 333 and 1000 mu g/mL, which indicates that the composition Z-2 has obvious protection effect on the central nerve of the zebra fish at the concentration of 333 and 1000 mu g/mL. Both compositions exhibited concentration dependence with increasing activity as concentration increased.
The conclusion shows that the safe dosage of the gastrodia elata extract is 500 mug/mL, and the safe dosage reaches 1000 mug/mL after the gastrodia elata extract is compounded with walnut peptide. Meanwhile, after compounding, the neuroprotective effect is improved from the highest activity of 74 percent of the single gastrodia elata extract to 82 percent of the compound, the phenomenon that the activity of the single walnut peptide is reduced along with the increase of the concentration is not shown, and the activity is enhanced along with the increase of the concentration. Therefore, the compound product has higher extraction safety dosage than the original independent gastrodia elata and wider action concentration than the original independent walnut peptide on the premise of ensuring the enhancement of the protection effect.
Claims (3)
1. A composition having neuroprotective effects characterized by: the composition consists of a gastrodia elata extract and walnut peptides, wherein the gastrodia elata extract accounts for 85wt%, and the walnut peptides account for 15wt%, and the concentration of the composition is 1000 mug/mL.
2. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable excipient; the pharmaceutical composition is tablets, capsules, granules, injections, powder injections, dropping pills, powder, extracts, soft extracts, oral liquid preparations and other formulations acceptable in the pharmaceutical field.
3. Use of the composition of claim 1 or the pharmaceutical composition of claim 2 for the manufacture of a medicament for preventing, ameliorating or treating mycophenolate mofetil-induced central nerve injury.
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