CN114425062A - Organic extract of negundo chastetree fruit and preparation method and application thereof - Google Patents
Organic extract of negundo chastetree fruit and preparation method and application thereof Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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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
- A61K2236/30—Extraction of the material
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a negundo chastetree fruit organic extract and a preparation method and application thereof. The invention provides a preparation method of a negundo chastetree fruit organic extract, and the negundo chastetree fruit organic extract achieves the treatment effect on Alzheimer disease by improving learning ability and memory ability and reducing the level of beta amyloid 1-42 in brain tissue.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a negundo chastetree fruit organic extract and a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is the most common type of dementia in the elderly, accounting for 65% of all cases of dementia. AD is clinically manifested primarily as progressive memory and cognitive impairment, with its major pathological features including selective neuronal cell loss, neuronal fibrillar tangles, and formation of extra-neuronal senile plaques, among others. The increase in global life expectancy has led to an increase in the frequency of numerous age-related diseases, including AD. Currently, the total number of AD patients worldwide has exceeded 5000 million, and is expected to exceed 1.315 million by year 2050, which is consistent with the trend of aging of the population. The burden of AD affects both the individual patient as well as the family, and it is estimated that around 1 trillion dollars per year are used globally for anti-AD therapy. However, the pathogenesis and mechanism of AD are not clear, and at present, drug development is based on various hypothesis theories, including beta-amyloid deposition, neurofibrillary tangle, neuroinflammation, craniocerebral trauma, etc., and the success rate of new drug development for AD is only 2.7% at present, which is far lower than the average success rate in the field of new drug development. Although some existing treatments can ameliorate the symptoms of AD, they are far from reaching the goal of curing AD in the field. Therefore, further search for effective drugs for the treatment of AD remains an endeavor of pharmacological workers today.
Fructus Viticis negundo is the fruit of Vitex negundo L.of Vitex of Verbenaceae, and is widely distributed in Yangtze river basin and southern provinces of China. As a traditional Chinese medicine rich in medicinal activity, negundo chastetree fruit is applied to treat various diseases, such as common cold, cough, asthma, stomachache, acid regurgitation, dyspepsia, indigestion, diarrhea, cholecystitis, gallstone, hernia and the like. However, no research report that negundo chastetree fruit is taken as a main component and applied to prevention and treatment of Alzheimer disease exists at present.
Disclosure of Invention
The invention aims to provide a negundo chastetree fruit organic extract, a preparation method and an application thereof, wherein the negundo chastetree fruit organic extract can effectively improve learning ability and memory ability and reduce the level of beta amyloid 1-42 in brain tissues, and has good prevention and treatment effects on Alzheimer's disease.
In order to achieve the above object, the present invention provides a method for preparing the organic extract of negundo chastetree fruit, comprising the steps of: extracting the negundo chastetree fruit powder with an organic solvent under heating; concentrating the extracting solution to obtain the negundo chastetree fruit organic extract.
Preferably, the organic solvent comprises absolute ethanol.
Preferably, the heat extraction comprises a first heat extraction, a second heat extraction and a third heat extraction; and carrying out second heat extraction on the filter residue after the first heat extraction, carrying out third heat extraction on the filter residue after the second heat extraction, and combining the extracting solutions.
Preferably, the first heat extraction is carried out at a mass-to-volume ratio of the negundo chastetree fruit powder to the organic solvent of 1 g: 5-15 mL; performing the second heat extraction and the third heat extraction with a mass to volume ratio of independently 1 g: 5-15 mL.
Preferably, the time for 3 times of heat extraction is 2 h.
The invention also provides application of the negundo chastetree fruit organic extract in preparing preparations for improving learning ability and memory ability and reducing beta amyloid 1-42 content in brain tissues.
The invention provides application of an organic extract of negundo chastetree fruit in preparing a medicament for preventing and treating Alzheimer disease.
Preferably, the fructus viticis organic extract comprises an alcohol extract of fructus viticis negundo.
Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the medicine comprises tablets, injections, capsules, granules, pills, powder, decoction, syrup, mixture, soft extract or extract.
The invention also provides a medicament for preventing and treating Alzheimer disease, and the effective components of the medicament comprise the negundo chastetree fruit organic extract extracted by the preparation method.
Has the beneficial effects that:
the invention provides a preparation method of a negundo chastetree fruit organic extract, and the negundo chastetree fruit organic extract prepared by the invention can improve learning ability and memory ability and reduce the content of beta amyloid 1-42 in brain tissues, thereby achieving the treatment effect on Alzheimer's disease. The mouse animal model experiment shows that the negundo chastetree fruit organic extract obviously shortens the evasion latency period of a mouse in a Morris water maze, increases the time percentage of the mouse in a quadrant where a platform is located, the action effect of the high-dose negundo chastetree fruit organic extract is better than that of the low-dose negundo chastetree fruit organic extract, and the negundo chastetree fruit organic extract can reduce the level of beta amyloid protein 1-42 in brain tissues.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIG. 1 is the effect of the organic extract of Vitex negundo L in example 1 on the latency of the localized sailing experiment in experimental mice;
FIG. 2 is a graph showing the effect of the organic extract of Vitex negundo L in example 1 on the percentage of time spent in positioning navigation experiment target quadrant of experimental mice;
FIG. 3 is a graph of the effect of organic extract of Vitex negundo L in example 1 on the percentage of time in the spatial exploration experiment target quadrant of experimental mice;
FIG. 4 is a graph showing the effect of the organic extract of Vitex negundo L in example 1 on the number of times that the experimental mouse crosses the target platform in the space exploration experiment;
FIG. 5 is the effect of organic extract of Vitex negundo L in example 1 on the MDA content in brain tissue of experimental mice;
FIG. 6 shows the effect of the organic extract of Vitex negundo L in example 1 on the activity of T-SOD in experimental mouse brain tissue;
FIG. 7 shows the comparison of the organic extract of Vitex negundo L in example 1 on the brain tissue Abeta of experimental mice1-42The effect of the level.
Detailed Description
The invention provides a preparation method of an organic extract of negundo chastetree fruit, which comprises the following steps: extracting the negundo chastetree fruit powder with an organic solvent under heating; concentrating the extracting solution to obtain the negundo chastetree fruit organic extract.
The fructus viticis organic extract provided by the invention preferably comprises an alcohol extract of fructus viticis, and more preferably comprises an ethanol extract of fructus viticis.
The invention preferably uses a multifunctional grinder to grind the negundo chastetree fruit to obtain the negundo chastetree fruit powder. The source, model and use mode of the multifunctional crusher are not particularly limited, and the multifunctional crusher and use mode in the prior art can be selected, such as a high-speed mode in the embodiment of the invention. The invention has no special limit on the particle size of the negundo chastetree fruit powder, and the particle size of the traditional Chinese medicine powder in the field can be adopted.
After the negundo chastetree fruit powder is obtained, the invention preferably uses an organic solvent to carry out first heat extraction on the negundo chastetree fruit powder, and filtration is carried out to obtain a first negundo chastetree fruit extracting solution and a first filter residue. The mass volume ratio of the negundo chastetree fruit powder to the organic solvent is preferably 1 g: 5-15 mL, more preferably 1 g: 10 mL. The time of the first heat extraction is preferably 1-2 h, and more preferably 2 h.
After the first filter residue is obtained, the first filter residue is preferably subjected to second heat extraction by absolute ethyl alcohol, and is filtered to obtain a second negundo chastetree fruit extracting solution and a second filter residue. The mass-volume ratio of the first filter residue to the absolute ethyl alcohol is preferably 1 g: 5-15 mL, more preferably 1 g: 10 mL. The time of the second heat extraction is preferably 1-2 h, and more preferably 2 h.
After the second filter residue is obtained, the second filter residue is preferably subjected to third heat extraction by absolute ethyl alcohol, and is filtered to obtain a third negundo chastetree fruit extracting solution and a third filter residue. The mass-volume ratio of the second filter residue to the absolute ethyl alcohol is preferably 1 g: 5-15 mL, more preferably 1 g: 10 mL. The time of the third heat extraction is preferably 1-2 h, and more preferably 2 h.
The heat extraction according to the present invention preferably comprises reflux extraction. The judgment standards for finishing the first heat extraction, the second heat extraction and the third heat extraction are the same, the solutions are in a micro-boiling state, and fine bubbles can be continuously formed on the liquid level from the bottle body to be broken when the solution is in the micro-boiling state. The method for filtering is not particularly limited, and the conventional filtering mode in the field can be adopted.
Preferably, the first fructus viticis negundo extract, the second fructus viticis negundo extract and the third fructus viticis negundo extract are combined to obtain the fructus viticis negundo extract.
After the fructus viticis negundo extract is obtained, the invention preferably recovers the solvent from the fructus viticis negundo extract in vacuum to obtain the fructus viticis negundo concentrate. The invention preferably adopts a rotary evaporator matched with a circulating water type vacuum pump to carry out the vacuum solvent recovery. The specific process and steps for recovering the solvent under vacuum are not particularly limited in the invention, and the conventional steps and processes in the field can be adopted.
After the fructus viticis negundo concentrate is obtained, the fructus viticis negundo concentrate is preferably dried by natural volatilization to obtain the fructus viticis negundo organic extract. The present invention preferably performs the natural evaporation in an evaporation dish. The natural volatilized organic extract of the negundo chastetree fruit is not sticky and can be suspended in sterile physiological saline, and the level of the extract for animal experiment intragastric administration can be reached.
The invention also provides application of the negundo chastetree fruit organic extract in preparing preparations for improving learning ability and memory ability and reducing beta amyloid 1-42 level in brain tissues. The negundo chastetree fruit organic extract achieves the effect of treating the Alzheimer disease by improving the learning ability and the memory ability and reducing the beta amyloid 1-42 in brain tissues.
The invention provides application of an organic extract of negundo chastetree fruit in preparing a medicament for preventing and treating Alzheimer disease.
The application of the present invention is preferably the same as described above and will not be described further herein.
The medicament also preferably comprises pharmaceutically acceptable auxiliary materials. The dosage form of the medicine provided by the invention preferably comprises tablets, injections, capsules, granules, pills, powder, decoction, syrup, mixture, soft extract or extract; the pill preferably comprises a pellet or a drop pill.
The invention also provides a medicament for preventing and treating Alzheimer disease, and the effective component of the medicament comprises the negundo chastetree fruit organic extract extracted by the preparation method.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of the negundo chastetree fruit organic extract comprises the following steps:
taking a proper amount of negundo chastetree fruit, putting the negundo chastetree fruit into a high-speed multifunctional grinder for grinding, and then mixing the ground medicinal powder according to the weight ratio of 1 g: adding 10mL of absolute ethyl alcohol into the feed liquid according to the mass-volume ratio for reflux extraction, and performing reflux extraction for 3 times, 2 hours each time. The filtrates from the 3 extractions were combined and the solvent was recovered in vacuo to give a concentrate. Putting the concentrate into an evaporating dish and naturally volatilizing to obtain the negundo chastetree fruit organic extract.
Example 2
Experimental study of fructus Viticis negundo organic extract for resisting Alzheimer disease:
experimental materials:
fructus Viticis negundo is purchased from Ziboyou, commerce and trade company; amyloid beta 1-42 was purchased from Shanghai peptide Biotech, Inc.; a protein quantitative kit, a Malondialdehyde (MDA) kit and a total superoxide dismutase (T-SOD) kit are purchased from Nanjing to build a bioengineering research institute; mouse Abeta1-42The ELISA kit was purchased from Wuhan Huamei bioengineering GmbH.
Animal experiments:
taking 24 male KM mice of 25-30g, injecting Abeta into lateral ventricle1-42And (5) constructing an AD mouse model. After one week of feeding, the mice were randomly divided into a control group (physiological saline), a low dose group (50mg/kg/d) and a high dose group (100mg/kg/d) of the organic extract of negundo chastetree prepared in example 1. The administration is performed by gavage for 1 time every day and for 30 days.
The evaluation method comprises the following steps:
1) preparation of oligomeric Abeta1-42
Treating Abeta with sterile physiological saline1-42Diluting to 2g/L solution, incubating in a thermostat at 37 deg.C for 120h to obtain condensed Abeta1-42。
2) Construction of AD model mice
First, 4.5% chloral hydrate (0.1ml/10g) was intraperitoneally injected for anesthesia. After the mice are anesthetized successfully, the mice are fixed on a brain stereotaxic instrument, a 0.8cm incision is cut on the skin of the head of the mice along the midline in the longitudinal direction by taking the connecting line of the two eyes of the mice as the center, the periosteum is stripped, and the bregma is exposed. Quickly find fontanel, vertically hang the microsampler on fontanel, position it with needle tip, and record its position. Using this position as the starting position, the plate was moved 1.8mm to the right and 2.3mm to the lower. The skull is drilled by a dental drill, and then a micro-syringe is inserted vertically for 2.0mm from the surface of the brain. At a sample introduction speed of 0.8ul/minCondensed state of Abeta with a degree of 2ul1-42The injection is slowed. After the injection is finished, the needle is left for 5min to allow Abeta1-42And (4) fully dispersing. Slowly withdrawing the needle, coating penicillin G sodium powder at the opening for diminishing inflammation before suturing, and then suturing the wound by using an operation thread. Each mouse was post-operatively given an intramuscular injection of penicillin G sodium injection (4 ten thousand units/mouse) into the leg for 3 consecutive days.
3) AD model mouse spatial learning and memory ability detection
The Morris water maze system is widely applied to scientific researches of a plurality of subjects such as learning memory, senile dementia, neurobiology, behavioral biology and the like, and is mainly used for detecting the spatial localization learning memory ability of experimental animals. The system mainly comprises a water maze device, an automatic water maze image acquisition system and a software analysis system. The water maze device is composed of a round water pool with the diameter of 1.2m, and the water pool is artificially defined as NE-I (first quadrant in the northeast), NW-II (second quadrant in the northwest), SW-III (third quadrant in the southwest) and SE-IV (fourth quadrant in the southeast) according to the four directions of the east, the south and the north. Before the experiment is started, the Morris water maze experiment can be carried out by putting water to a preset water level (namely the position that the water surface is 1.5cm higher than the platform) and maintaining the water temperature at 21 +/-1 ℃. The experimental procedure is divided into two parts of a positioning navigation experiment and a space exploration experiment.
Positioning navigation experiment: and (3) carrying out continuous 6-day water maze hidden platform experiments on mice of each experimental group after adapting to the environment so as to explore the space learning capacity and the space memory capacity of the mice. The hidden platform experiment requires that a platform with the diameter of 8cm is placed at a position which is about 15cm away from the center of a SW-III quadrant, the platform is hidden at a position which is 1.5cm below the liquid level in a water maze, and the position of the platform is kept unchanged within 6 days. Each mouse was placed gently into the water with each mouse facing the pool wall according to the four quadrants, and the time required for the mouse to find and stay on the hidden platform for 10s was recorded as the latency period. Each mouse was subjected to a round of experiments in four quadrants each day, with the interval between entry into the water in each quadrant being 15-20min to ensure adequate rest time for the mice.
Space exploration experiment: and carrying out a mouse space exploration experiment 24h after carrying out the last positioning navigation experiment of the hidden platform. And (4) keeping the external conditions such as contrast, water depth, illumination, temperature, humidity and the like unchanged, and only removing the water maze platform. Mice were gently placed into the water facing the NE-i water maze pool wall. Recording the swimming track of the mouse in water within 60s, the time percentage and the route percentage in the SW-III quadrant, the times of crossing the platform position and the like, and later using the result for analyzing the space positioning capacity and the space exploration process of the mouse.
4) Pathological index detection of AD model mouse
After the water maze experiment is finished, the mouse is deeply anesthetized by chloral hydrate and then is killed, brain tissue is rapidly taken, and brain tissue homogenate is prepared by adopting a conventional method. And performing an experiment according to the kit steps, measuring the protein concentration of the sample, and further calculating the MDA content and the T-SOD activity of the sample according to the protein concentration. Simultaneous detection of AD model mouse Abeta1-42And (4) horizontal.
5) Analysis of Experimental data
Data obtained from the experiment were analyzed by using the software GraphPad Prism 8.0.2 for both biological statistics and graphic design, and data in the group were presented in the standard deviation manner. Comparison of data between groups was analyzed using Multiple tests-one per row. P <0.05 is statistically significant. P <0.05, P <0.01, P < 0.001.
The experimental results are as follows:
1. the influence of the organic extract of the negundo chastetree fruit on the spatial learning and memory ability of the experimental mice:
1.1 FIG. 1 shows the 6-day latency of four groups of mice in a positional voyage experiment: the escape latency time of the control mice during the 6-day experiment was significantly higher than that of the 2 groups of mice to which the organic extract of negundo chastetree fruit of example 1 was added. The escape latency time of each group of mice is obviously reduced along with the increase of the training days, but the mice of the 2 groups which are administrated show obvious reduction in the first 3 days of the experiment, the mice of the control group do not show obvious reduction until the fourth day, and the latency time of the subsequent 3 days is stable without obvious reduction. In general, the time to escape from the incubation period is shorter as the concentration of the organic extract of negundo chastetree fruit is increased. The same results are also shown in the target quadrant time percentage of fig. 2, and at the beginning of the experiment, the 2 groups of mice administered showed strong targeting with a rapid increase in the time spent in the target quadrant. The final percent time in target quadrant for stability was significantly higher than the control. The vitex negundo fruit organic extract provided by the invention obviously improves the learning and memory abilities of experimental mice.
1.2 can be seen from FIG. 3: the time percentage of target quadrant of mice in the low-dose and high-dose administration groups is significantly higher than that of the control group, and the difference is more obvious as the administration dose is increased. The results of the number of platform crossings in FIG. 4 also confirm the above conclusions. The vitex negundo fruit organic extract provided by the invention obviously improves the spatial memory capacity of experimental mice.
2. Influence of fructus Viticis negundo organic extract on brain tissue oxidative damage of experimental mice
The MDA content is an important parameter of the antioxidant potential capability of a reactor, can reflect the lipid peroxidation rate and strength of the reactor, and can also indirectly reflect the tissue peroxidation damage degree. As can be seen from FIG. 5, the MDA content in the brain tissue of the mouse is significantly reduced after the drying of the organic extract of fructus Viticis negundo.
T-SOD is a kind of metalloenzyme widely distributed in tissue cells, is used for catalyzing superoxide anion free radical to carry out disproportionation reaction, and plays an important role in balancing oxidation and oxidation resistance of organisms and avoiding damage of free radicals. As can be seen from FIG. 6, there was no significant change in T-SOD activity in the three groups of mice after administration. Therefore, the fructus viticis negundo organic extract described in example 1 has a certain effect of alleviating tissue oxidative damage.
3. Vitex negundo organic extract for experimental mouse brain tissue Abeta1-42Influence of level
Misfolding of a β plays a very important role in the onset and progression of alzheimer's disease, such as: a β is the major component of amyloid neuritic plaques, produced by the metabolism of Amyloid Precursor Protein (APP), from which a β is altered by the following mechanisms: APP is a type I transmembrane glycoprotein, and APP is cleaved near the membrane by the extracellular protease of alpha-secretase, releasing the soluble extracellular fragment sAPP alpha, and is also cleaved by the aspartyl protease of beta-secretase 1, releasing the soluble extracellular fragment APP beta and the membrane-bound fragment (C99). C99 is cleaved by the gamma-secretase complex within the membrane, releasing the a β protein and intracellular peptides. The A beta protein is secreted and released into interstitial fluid under the environment of enhanced neuron activity, and is aggregated to form oligomers and fibrils, and finally plaques are formed. A β can cause formation of senile plaques in the brain and apoptosis of nerve cells, and is an important factor causing alzheimer's disease.
As shown in FIG. 7, the two doses of the organic extract of fructus Viticis negundo can significantly reduce the A beta of the mouse brain tissue1-42And (4) horizontal.
The experimental results show that the fructus viticis organic extract provided by the invention can effectively improve the space learning and space memory abilities of an AD model mouse, obviously shorten the latency avoidance time of the experimental mouse in the Morris water maze, increase the time percentage of the experimental mouse in the quadrant where the platform is located, and the action effect of the fructus viticis with high dose is better than that of the fructus viticis with low dose. And the negundo chastetree fruit organic extract can reduce the level of beta amyloid 1-42 in brain tissues and has good prevention and treatment effects on AD.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. The preparation method of the negundo chastetree fruit organic extract is characterized by comprising the following steps:
extracting fructus Viticis negundo powder with organic solvent under heating; concentrating the extracting solution to obtain the negundo chastetree fruit organic extract.
2. The method of claim 1, wherein the organic solvent comprises absolute ethanol.
3. The method of manufacturing of claim 1, wherein the heat extraction comprises a first heat extraction, a second heat extraction, and a third heat extraction; and carrying out second heat extraction on the filter residue after the first heat extraction, carrying out third heat extraction on the filter residue after the second heat extraction, and combining the extracting solutions.
4. The method according to claim 3, wherein the first heat extraction is carried out in a mass-to-volume ratio of the negundo chastetree fruit powder to the organic solvent of 1 g: 5-15 mL; and when the second heat extraction and the third heat extraction are carried out, the mass-to-volume ratio of the feed liquid is independently 1 g: 5-15 mL.
5. The method of claim 4, wherein the time for each of the 3 heat extractions is 2 hours.
6. Use of the organic extract of negundo chastetree fruit extracted by the method of any one of claims 1 to 5 in the preparation of a preparation for improving learning ability and memory ability and reducing the level of amyloid-beta 1-42 in brain tissue.
7. Use of the fructus Viticis negundo organic extract obtained by the preparation method according to any one of claims 1-5 in the preparation of a medicament for preventing and treating Alzheimer's disease.
8. The use of claim 7, wherein the medicament further comprises a pharmaceutically acceptable excipient.
9. The use according to claim 7, wherein the medicament is in the form of tablets, injections, capsules, granules, pills, powders, decoctions, syrups, mixtures, soft extracts or extracts.
10. A medicine for preventing and treating Alzheimer disease, which is characterized in that the effective component of the medicine comprises the organic extract of negundo chastetree fruit extracted by the preparation method of any one of claims 1 to 5.
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| Title |
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| ABHINAV KANWAL等: "Anti-Amnesic Activity of Vitex Negundo in Scopolamine Induced Amnesia in Rats", 《PHARMACOLOGY & PHARMACY》 * |
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