CN114246934B - Active substance composition for synergistically protecting nerves and application thereof - Google Patents

Active substance composition for synergistically protecting nerves and application thereof Download PDF

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CN114246934B
CN114246934B CN202111549058.8A CN202111549058A CN114246934B CN 114246934 B CN114246934 B CN 114246934B CN 202111549058 A CN202111549058 A CN 202111549058A CN 114246934 B CN114246934 B CN 114246934B
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ginseng
water
walnut
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powder
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CN114246934A (en
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闵伟红
宋晓兵
刘春雷
傅俊曦
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Jilin Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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

An active substance combination for synergistically protecting nerves and application thereof in preparing medicaments for protecting nerves and improving memory belong to the technical field of pharmaceutical compositions. The walnut protein peptide is prepared by adopting alkaline protease and leucine aminopeptidase for complex enzymolysis, and the molecular weight of the polypeptide is 300-5000 Da. The ginseng water decoction is prepared by adopting a water decoction method, water-soluble active substances are completely leached, substances such as saponin, polypeptide, polysaccharide, organic acid, amino acid and the like are contained, and the multi-component of the composition determines the characteristics of synergism, multi-point fine tuning, multi-target point, multi-way and the like of pharmacological action mechanisms of the composition, so that the memory activity is improved to be far higher than that of single action, the development of brain nerve cells can be promoted, the cognitive dysfunction can be improved, and the learning and memory capacity can be improved. Has the characteristics of simple whole process, less investment, high return rate, no environmental pollution, mild operation, low carbon and high efficiency.

Description

Active substance composition for synergistically protecting nerves and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical compositions, and particularly relates to an active substance composition for synergistically protecting nerves and application of the active substance composition in preparation of a medicament for protecting nerves and improving memory.
Background
With the increasing global population aging, the incidence of a series of neurodegenerative diseases such as parkinson's disease, alzheimer's disease, etc., which are closely related to memory impairment, increases year by year. To date, there is no particularly effective therapeutic agent, namely, a chemically synthesized drug such as Donepezil hydrochloride (Donepezil) and Piracetam (Piracetam), which can improve spatial learning and memory cognition of patients, but can also produce adverse effects such as mental disorders such as hallucinations, irritability and aggression. Therefore, development of active substances of natural origin having neuroprotective function is particularly important.
The walnut (Juglans mandshurica Maxim.) protein content is up to 20%, and the walnut-derived oligopeptide prepared by using the enzyme-controlled hydrolysis technology has advantages in various aspects such as safety, absorbability, processing characteristics, antigenicity reduction and the like, and particularly has a high-activity neuroprotection function. For example, the walnut peptide has remarkable improvement effect on learning skills, memory, anxiety, movement and movement coordination of AD-tg mice; aβ -induced neurotoxicity can be inhibited by improving active oxygen, mitochondrial membrane potential and adenosine triphosphate content; has regulatory effect on cholinergic system.
The ginseng (panaxgineng C.A.Meyer) has extremely high medicinal value, the active ingredients are diversified, the central nervous system of a human body can be regulated and controlled, for example, ginsenoside Rg1, rb1, rd, rg2, re, rh1 and the like can promote the development of brain nerve cells, meanwhile, acetylcholinesterase can be inhibited, tau protein hyperphosphorylation and Abeta deposition can be inhibited, and the cognitive function of an animal model of dysmnesia can be improved by mobilizing an endogenous protection mechanism of the organism. Meanwhile, the ginseng non-saponin substances such as protein, polysaccharide, organic acid, polypeptide and the like have the activities of resisting neuroinflammation, oxidative stress, nerve cell apoptosis and the like. Ginseng is therefore an important raw material for developing neuroprotective substances.
Chinese patent publication No. 201910211839.2 discloses a ginseng small-molecule peptide beverage and a preparation method thereof, wherein main materials are ginseng, walnut kernel, red date, medlar, quinoa, cyperus esculentus, bone collagen peptide and fish collagen peptide, and the raw material components are complex; meanwhile, complex enzyme systems (alpha-amylase, cellulase, neutral papain and trypsin) are added, so that the cost is high, the enzymolysis products are undefined and uncontrollable, and the optimal enzyme reaction conditions are difficult to unify; the process is complicated, macroporous resin purification and ethanol elution are needed in the preparation process of ginseng pulp, and the ginseng pulp is easy to pollute; the invention mainly aims at invigorating primordial qi, promoting fluid production, tonifying kidney, supplementing calcium, improving body immunity, preventing osteoporosis and the like. The invention discloses ginseng and walnut oligopeptide powder and a preparation method thereof, wherein main raw materials of the ginseng and walnut oligopeptide powder are ginseng and walnut peptide powder, DHA algae oil, medlar powder, semen euryales, myotonin, taurine, phospholipid, maltodextrin, food-grade preservative and the like are supplemented, and the technical scheme of the invention is mainly to extract walnut source and ginseng source polypeptide.
Disclosure of Invention
One of the purposes of the invention is to combine walnut protein peptide and ginseng decoction to form a new formula to obtain an active substance composition for synergistically protecting nerves, wherein the compound mass ratio of the walnut protein peptide to the ginseng decoction is 1:0.2 to 5.
The walnut protein peptide is prepared by the following steps: preparing a solution with mass fraction of 2-7% of walnut defatted protein powder (prepared by an alkali dissolution and acid precipitation method, functional Properties of the Protein Isolate and Major Fractions of Pine Nut Proteins Prepared from the Changbai Mountain in China, dan Wu and the like) and distilled water, and carrying out constant-temperature water bath for 10-20 min at 90-100 ℃; then cooling to 40-60 ℃, and regulating the pH value of the solution to 7.5-9.5 by using NaOH with the concentration of 1.0 mol/L; keeping the pH value constant, adding alkaline protease Alcalase 2.4L and leucine aminopeptidase with the enzyme amount of 1000-9000U/g respectively, performing enzymolysis for 1-5 h at 40-60 ℃, and inactivating the enzyme of the solution at 90 ℃ for 10-20 min; finally cooling to room temperature, regulating the pH value to be neutral, centrifuging at 5000-8000 rpm for 10-20 min, and freeze-drying the supernatant to obtain the walnut protein peptide.
The ginseng water decoction is prepared by the following method: cleaning ginseng, drying, crushing into powder, and sieving with a 40-80 mesh sieve to obtain ginseng powder; adding 8-35 times of distilled water of the volume of the ginseng powder, fully stirring, and carrying out ultrasonic treatment at the temperature of 50-80 ℃ and the pressure of 320-640W for 20-60 min; decocting the solid-liquid mixture obtained by ultrasonic treatment at 100 ℃ for 1-2.5 h, cooling the prepared ginseng liquid to room temperature, centrifuging at 4000-6000 r/min for 15-30 min, and taking supernatant; adding 8-35 times of distilled water into the obtained precipitate, repeatedly boiling, cooling to room temperature, centrifuging, and collecting supernatant; mixing the supernatants, recovering distilled water under reduced pressure, vacuum freeze drying to obtain Ginseng radix water decoction, and preserving at-20deg.C.
Compared with the prior art, the invention has the advantages that:
the invention discloses an active substance composition for synergistically protecting nerves, a preparation method and application thereof for the first time. Wherein, the preparation of the walnut protein peptide adopts the compound enzymolysis of alkaline protease and leucine aminopeptidase, and the molecular weight of the polypeptide ranges from 300 Da to 5000Da. The ginseng water decoction is prepared by adopting a water decoction method, water-soluble active substances are completely leached, substances such as saponin, polypeptide, polysaccharide, organic acid, amino acid and the like are contained, and the multi-component of the composition determines the characteristics of synergism, multi-point fine tuning, multi-target point, multi-way and the like of pharmacological action mechanisms of the composition, so that the memory activity is improved to be far higher than that of single action, the development of brain nerve cells can be promoted, the cognitive dysfunction is improved, and the learning and memory capacity is improved.
The invention has simple whole process and does not use equipment with higher cost, thereby meeting the control requirement of industrial mass production on cost and promoting the smooth conversion of experimental process to production process. The method has the advantages of low process investment, high return rate, no environmental pollution, mild operation, low carbon and high efficiency, and is suitable for industrial production.
Drawings
Fig. 1: space exploration test path diagrams of mice in a blank control group;
fig. 2: a test path diagram is explored in the space of the mice in the memory damage model group;
fig. 3: space exploration test path diagram of positive control group (piracetam) mice;
fig. 4: space exploration test path diagram of ginseng powder group mice;
fig. 5: space exploration test path diagram of ginseng peptide group mice;
fig. 6: a test path diagram is explored in the space of a ginsenoside group (Rg 1) mouse;
fig. 7: a walnut peptide group mouse space exploration test path diagram;
fig. 8: composition group mice spatially explored the experimental path graph;
placing a platform with the diameter of 65mm into the SW quadrant, wherein the height of the platform is 1cm away from the water surface, and simultaneously lightly placing the mouse facing the pool wall into the NE quadrant, and recording the swimming track of the mouse within 120s by using an image acquisition system. If the mouse can find the platform in the SW quadrant within 120s, the using time is recorded as the incubation period(s), meanwhile, the mouse stays on the platform for 10s, and then the water is wiped and put back into the mouse cage, so that the mouse is prevented from hypothermia and occurrence of diseases; if the mice were unable to find the platform spontaneously within 120s, they were guided to the platform using the baffle assist, and were allowed to rest on the platform for 10s as well, and latency was recorded as 120s.
After the positioning experiment is finished, the underwater platform is taken out. The mice were again gently placed into the NE quadrant facing the pool wall and the swim track of the mice was recorded using the image acquisition system.
Detailed Description
The invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the invention more apparent, but the invention is not limited to these examples. It should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below. In the present invention, unless otherwise specified, all parts and percentages are mass units, and the equipment, raw materials, etc. used are commercially available or are commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1:
(1) Preparation of walnut protein peptide
Dissolving 5g of walnut defatted protein powder in 100mL of distilled water to prepare a solution with the mass fraction of 5%, and carrying out water bath at 90 ℃ for 10min; then cooling to 40 ℃, and regulating the pH value of the solution to 8 by using 1.0mol/L NaOH; keeping the pH value constant, adding alkaline protease Alcalase 2.4L and leucine aminopeptidase, respectively adding 7000U/g enzyme, performing enzymolysis at 40deg.C for 3 hr, and inactivating the solution at 90deg.C for 10min; and finally cooling to 40 ℃, centrifuging at 5000rpm for 10min, and freeze-drying the supernatant to obtain the walnut protein peptide powder.
(2) Preparation of ginseng decoction
Cleaning ginseng, drying, crushing into powder, and sieving with a 80-mesh sieve to obtain ginseng powder; adding distilled water 10 times of the volume of the ginseng powder, fully stirring, and performing ultrasonic treatment at 74 ℃ and 600W for 40min; decocting the solid-liquid mixture obtained by ultrasonic treatment at 100deg.C for 2.5 hr, cooling the decoction to room temperature, centrifuging at 5000r/min for 20min, and collecting supernatant; adding distilled water with volume of 10 times to the obtained precipitate, repeatedly boiling, cooling to room temperature, centrifuging, and collecting supernatant; mixing the supernatants, recovering distilled water under reduced pressure, vacuum freeze drying to obtain Ginseng radix water decoction, and preserving at-20deg.C.
(3) The walnut protein peptide and ginseng decoction are compounded
The obtained walnut protein peptide and ginseng water decoction are compounded (namely, the mixture is simply mixed), and the mass ratio of the compound is 1: and 0.5, preparing the active substance composition for synergistically protecting nerves.
Example 2: the invention has the effect of improving the cognitive function of mice
(1) Test animal
Male C57BL/6 mice, 5-6 weeks old, 80 available from Liaoning long life Biotechnology Co.
(2) Experimental grouping
Male C57BL/6 mice were conditioned for one week in an environment with a temperature of 20.+ -. 2 ℃ and a relative humidity of 55.+ -. 5% and a 12/12 hour light/dark cycle. The randomization was divided into 8 groups (n=10). The composition comprises a blank control group, a memory injury model group, a positive control group, a ginseng powder group, a ginseng peptide group, a ginsenoside group, a walnut peptide group and a composition group. Starting at week 2, mice in each group were weighed, recorded and stomach-filled at 8 am each day. 250 mg/kg/bw of ginseng powder group (ginseng powder solution), 600 mg/kg/bw of ginseng peptide group (ginseng peptide is the product of enzymolysis of ginseng powder by cellulase and beta-amylase), 5 mg/kg/bw of ginsenoside group (Rg 1 solution), 600 mg/kg/bw of walnut peptide group (walnut peptide solution) and 600 mg/kg/bw of composition group (composition solution). Meanwhile, the blank control group, the memory damage model group and the positive control group are filled with physiological saline with the same volume as the stomach. Starting from week 6, the positive control group was perfused with a piracetam solution (400 mg/kg. Bw); meanwhile, except for a blank control group, each group of mice is subjected to scopolamine intraperitoneal injection, and the dosage of the intraperitoneal injection is 1mg/kg.bw (scopolamine is a non-selective cholinergic receptor and can block cholinergic signal paths and inhibit acetylcholine transmission).
(3) Morris water maze behavioural test
A circular platform with a diameter of 65mm was fixed in the NW quadrant, 1cm under water, 6d total. Each mouse was tested 4 times daily, each time the mice were gently placed into water facing the pool wall. The first and fourth times water is admitted from the quadrant (SE) opposite the target quadrant, and the second and third times water is admitted randomly from the SW and NE quadrants. The monitoring time of the swimming of the mice is 120s, and the computer acquisition system automatically records the data and the process from the water entering of the mice to the finding of the hidden platform. The time from the entry of water into the mice until the platform was found was noted as latency. After the mice find the platform, the mice stay on the platform for 10s, the mice with failed platform seeking are guided to the platform to stay for 10s by using the partition board, the latency period is recorded as 120s, each time training is finished, and the mice are wiped by a dry towel so as to prevent stress caused by hypothermia.
The spatial exploration test in the test method for evaluating spatial memory capacity of mice is an authoritative test method (Vouros, a., gehring, t.v., szydlowska, K.A generalised framework for detailed classification of swimming paths inside the Morris Water maze.scientific Report,2018,8 (1), 15089). The circular platform was removed from the pool and each mouse was tested 1 time a day, each time with the mouse gently placed into the water facing the pool wall. The monitoring time of the swimming of the mice is 120s, and the computer acquisition system automatically records the times of the mice passing through the original platform. The space memory capacity of the mice to the original platform position is detected by removing the platform hidden under water.
The results in table 1 show that the blank control group has good learning and memory ability, so that the travel path is relatively regular, and the retention time of the escape platform, the entering times of the escape platform, the movement time of the effective area and the distance are all higher. Compared with a blank control group, the wandering path of the scopolamine model injury group mice is irregular, mainly moves at the edge and has randomness, and the retention time of an escape platform, the entering times of the escape platform and the movement time and distance of an effective area are obviously reduced, so that the scopolamine injection causes the test mice to lose memory of the original platform. The mice exploration strategy of the ginseng water decoction and walnut peptide compound group mainly adopts trend, has remarkable repairing effect on the mice memory injury, remarkably improves the retention time of an escape platform, the entering times of the escape platform and the movement time and distance of an effective area compared with the model injury group, has better effect than the independent effect of ginseng powder, ginseng peptide, ginsenoside and walnut peptide, and proves that the composition is a high-efficiency neuroprotection composition.
Table 1: time for staying on mouse escape platform, times for entering escape platform, effective area movement time and effective area movement distance
Note that: different letters represent significant differences.
The foregoing examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the foregoing examples, and it will be apparent to those skilled in the art that modifications and color rendering can be made without departing from the principles of the present invention, and the modifications and color rendering should also be regarded as the protection scope of the present invention.

Claims (2)

1. A synergistically nerve-protecting active substance composition, characterized in that: the composition is prepared by compounding walnut protein peptide and ginseng decoction, wherein the compounding mass ratio is 1: 0.2-5;
wherein, the walnut protein peptide is prepared by the following method,
preparing a solution with the mass fraction of 2-7% of the walnut defatted protein powder with distilled water, and carrying out constant-temperature water bath for 10-20 min at the temperature of 90-100 ℃; then cooling to 40-60 ℃, and regulating the pH of the solution to 7.5-9.5 by using 1.0mol/L NaOH; keeping the pH value constant, adding alkaline protease Alcalase 2.4L and leucine aminopeptidase with the enzyme amount of 1000-9000U/g respectively, performing enzymolysis for 1-5 h at 40-60 ℃, and inactivating the solution at 90 ℃ for 10-20 min; cooling to room temperature, regulating the pH value to be neutral, centrifuging at 5000-8000 rpm for 10-20 min, and freeze-drying the supernatant to obtain the walnut protein peptide;
the ginseng water decoction is prepared by the following method,
cleaning ginseng, drying, crushing into powder, and sieving with a 40-80 mesh sieve to obtain ginseng powder; adding distilled water with the volume of 8-35 times of that of the ginseng powder, fully stirring, and carrying out ultrasonic treatment at the temperature of 50-80 ℃ and the pressure of 320-640W for 20-60 min; boiling the solid-liquid mixture obtained by ultrasonic treatment at 100 ℃ for 1-2.5 h, cooling the boiled ginseng liquid to room temperature, centrifuging at 4000-6000 r/min for 15-30 min, and taking supernatant; adding 8-35 times of distilled water into the obtained precipitate, repeatedly boiling, cooling to room temperature, centrifuging, and collecting supernatant; mixing the supernatants, recovering distilled water under reduced pressure, and vacuum freeze drying to obtain Ginseng radix water decoction.
2. Use of a synergistically nerve-protecting active substance combination according to claim 1 for the preparation of a medicament for neuroprotection and memory improvement.
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