CN110169987B - Acer truncatum extract with high-efficiency inhibitory activity on acetylcholinesterase and preparation method thereof - Google Patents
Acer truncatum extract with high-efficiency inhibitory activity on acetylcholinesterase and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase and a preparation method thereof, wherein the method comprises the following steps: firstly, crushing acer truncatum seeds, and carrying out vacuum freeze drying on the obtained powder; sequentially carrying out ultrasonic extraction, centrifugation and filtration by using normal hexane as an extraction solvent, collecting filter residues, and drying the filter residues; extracting the filter residue with methanol solution as extractant, stirring, ultrasonic extracting, centrifuging, filtering, collecting supernatant, and drying the supernatant to obtain Acer Truncatum Bunge extract with high inhibitory activity on acetylcholinesterase. The method can guide the reutilization of the acer truncatum seed residues, and solve the possible pollution problem. The extraction solvent has the characteristics of small pollution, low price, suitability for mass production, low requirements on instruments and equipment and easy operation. Vacuum freeze drying and normal temperature nitrogen flow drying are adopted to avoid the influence of temperature on the components and activity of the extract.
Description
Technical Field
The invention belongs to the technical field of development and utilization of acer truncatum resources, and particularly relates to an acer truncatum extract with high-efficiency inhibitory activity on acetylcholinesterase and a preparation method thereof.
Background
Acer truncatum Bunge is a perennial deciduous tree of Acer of Aceraceae (now incorporated into Sapindaceae), and naturally distributed in the north of China; it is not only a unique tree species in China, but also an important resource plant with various values of edible, medicinal, ornamental and industrial utilization. The acer truncatum leaves are raw materials of ancient precious tea products in China, namely maple dew tea, and are processed and developed into various instant tea and health-care tea in modern times. The Acer Truncatum Bunge leaf has effects of dispelling pathogenic wind and removing dampness, and can be used for treating traumatic injury, arthralgia, fracture, etc.; the acer truncatum leaves are also used for treating coronary arteriosclerosis, cardiovascular and cerebrovascular diseases, angina and other diseases in China folks. Modern pharmacological research shows that the acer truncatum leaf extract also has the activities of resisting oxidation, resisting bacteria, resisting tumors and inhibiting the activity of fatty acid synthetase. The acer truncatum leaf contains various chemical components such as flavonoids, polyphenols, organic acids, tannin, polysaccharide and the like, wherein the flavonoids, the polyphenols and chlorogenic acid are main active components.
The mature fruits (seeds) of acer truncatum have the habit of directly stir-frying the fruits for eating in a plurality of areas in the north of China. The acer truncatum seeds not only have high oil content (42.6-46.6%), but also are high-quality woody plant oil. The oil contains 92% of unsaturated fatty acids, mainly comprising oleic acid (23.8-26.0%), linoleic acid (33.9-37.3%) and high-content nervonic acid (5.2-6.2%). Nervonic acid is the core component of brain nerve cells, and can promote intelligence development of newborn infants, delay nerve cell aging and repair damage of nerve cells. The acer truncatum buge oil also contains abundant vitamin E (2352.0-2654.3 mu mol/kg) and phytosterol (1961.9-2402.8 mu mol/kg), and the content of the vitamin E is even higher than that of common edible oil, such as soybean oil, sesame oil, rapeseed oil and peanut oil. In addition, the acer truncatum buge oil also has the pharmacological effects of resisting oxidation, bacteria and tumors, relieving senile dementia and the like.
Pathological studies find that there is a significant reduction in acetylcholine in the brain of alzheimer's patients, and acetylcholinesterase is a key enzyme catalyzing the hydrolysis of acetylcholine; inhibiting the activity of the enzyme can effectively reduce the hydrolysis of acetylcholine in brain, thereby achieving the purpose of improving the content of acetylcholine in brain. The inhibition of the activity of acetylcholinesterase is a path for effectively treating the Alzheimer disease internationally acknowledged at present, and the key point is that a strong acetylcholinesterase activity inhibitor is found.
Disclosure of Invention
The invention aims to provide a preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase.
The preparation method of the acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase comprises the following steps: s101: firstly, putting naturally dried acer truncatum seeds into an ultramicro jet mill for grinding, and carrying out vacuum freeze drying on the obtained powder; s102: taking the powder treated in the step S101, sequentially carrying out ultrasonic extraction, centrifugation and filtration by using normal hexane as an extraction solvent, collecting filter residues, repeatedly extracting for 2-3 times, and then drying the filter residues in a protective gas atmosphere; s103: and (2) extracting the filter residue obtained in the step (S102) by using a methanol solution as an extracting agent, sequentially stirring, ultrasonically extracting, centrifuging, filtering, collecting supernatant, repeatedly extracting for 2-3 times, and drying the supernatant in a protective gas atmosphere to obtain the acer truncatum bunge extract with efficient inhibitory activity on acetylcholinesterase.
The invention relates to a preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase, which comprises the steps of crushing seeds, freeze-drying at low temperature, extracting for removing oil, ultrasonically extracting residues, and drying in nitrogen to obtain the extract; biological activity experiments show that the extract has extremely strong acetylcholinesterase inhibition activity and can be used for developing medicaments for treating the Alzheimer disease. The extract disclosed by the invention has the advantages of natural and easily-obtained raw materials, simplicity and easiness in preparation, and extremely strong biological activity, can reduce the pollution of waste residues in the Acer truncatum oil obtaining process, guides the comprehensive development and utilization of Acer truncatum resources, and has a great market prospect in developing medicaments and health care products for treating the Alzheimer disease.
The invention relates to a preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase, wherein acer truncatum bunge seed residues are waste left after oil is obtained from acer truncatum bunge kernels. The extraction solvent is 80% methanol, is a common extraction solvent, has the characteristics of small pollution, low price and suitability for large-scale production, has low requirements on instruments and equipment, is easy to obtain equipment, has low purchase cost and is easy to operate. Ultrasonic extraction is adopted, so that the method is economic and environment-friendly; vacuum freeze drying and normal temperature nitrogen flow drying are adopted to avoid the influence of temperature on the components and activity of the extract.
In addition, the preparation method of the acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase can also have the following additional technical characteristics:
further, in the step S103, the mass concentration of the methanol solution is 78% to 82%.
Further, in the step S101, the temperature of the vacuum freeze drying is-78 ℃ to-82 ℃, the vacuum pressure is less than 13.3Pa, and the drying time is 45h to 50 h.
Further, in the step S102, the ratio of the mass of the powder processed in the step S101 to the volume of the n-hexane is 1 g: (18-22) mL.
Further, in the step S102, the time of ultrasonic extraction is 18min to 22 min; the centrifugation time is 5min to 8min, and the rotation speed during centrifugation is 3000rpm to 4000 rpm; the filter membrane adopted during filtration is a polytetrafluoroethylene membrane with the aperture of 0.4-0.5 mu m.
Further, in the step S103, the ratio of the mass of the filter residue obtained in the step S102 to the volume of the methanol solution is 1 g: (18 mL-22 mL); stirring for 3-8 min, ultrasonic extracting for 15-20 min, centrifuging for 5-8 min at 3000-4000 rpm, and filtering with polytetrafluoroethylene membrane of 0.4-0.5 micron pore size.
The invention also aims to provide the acer truncatum bunge extract which is prepared by the preparation method and has high-efficiency inhibitory activity to acetylcholinesterase.
The invention also aims to provide the application of the acer truncatum extract with high-efficiency inhibitory activity to acetylcholinesterase in preparing the medicines and health products for treating the Alzheimer disease.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and not to be construed as limiting the invention.
The invention provides a preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase, which comprises the following steps:
(1) firstly, putting naturally dried acer truncatum seeds into an ultramicro jet mill for grinding, and carrying out vacuum freeze drying on the obtained powder. Wherein the temperature of the vacuum freeze drying is-78 ℃ to-82 ℃, the vacuum pressure is less than 13.3Pa, and the drying time is 45h to 50 h.
(2) And (2) taking the powder treated in the step (1), sequentially carrying out ultrasonic extraction, centrifugation and filtration by using n-hexane as an extraction solvent, collecting filter residues, repeatedly extracting for 2-3 times, and drying the filter residues under the atmosphere of protective gas. Wherein the volume ratio of the mass of the powder treated in the step (1) to the n-hexane is 1 g: (18-22) mL. The ultrasonic extraction time is 18 min-22 min; the centrifugation time is 5min to 8min, and the rotation speed during centrifugation is 3000rpm to 4000 rpm; the filter membrane adopted during filtration is a polytetrafluoroethylene membrane with the aperture of 0.4-0.5 mu m.
(3) And (3) extracting the filter residue obtained in the step (2) by using a methanol solution with the mass concentration of 78-82% as an extracting agent, sequentially stirring, ultrasonically extracting, centrifuging, filtering, collecting supernatant, repeatedly extracting for 2-3 times, and drying the supernatant in a protective gas atmosphere to obtain the acer truncatum bunge extract with high-efficiency inhibitory activity on acetylcholinesterase. Wherein the volume ratio of the mass of the filter residue obtained in the step (2) to the methanol solution is 1 g: (18 mL-22 mL); stirring for 3-8 min, ultrasonic extracting for 15-20 min, centrifuging for 5-8 min at 3000-4000 rpm, and filtering with polytetrafluoroethylene membrane of 0.4-0.5 micron pore size.
The invention is described in detail below by means of specific examples:
example 1
Example 1 provides a method for preparing acer truncatum extract having high inhibitory activity against acetylcholinesterase, comprising the steps of:
(1) crushing and drying: naturally drying collected mature acer truncatum seeds, then crushing the seeds by using an ultramicro airflow crusher, and further drying the obtained powder in a vacuum freeze dryer under the following drying conditions: -80 ℃ and vacuum pressure <0.133mBar, 48 h. Wherein, the mature acer truncatum seeds are collected from the great sulcus protection area of Tongliao city of autonomous region of inner Mongolia.
(2) Oil removal treatment: placing 0.5g of dry powder in a 20mL glass vial with a silicone resin diaphragm cover, rapidly adding 10mL of n-hexane into the vial, screwing the vial cover, and performing ultrasonic extraction for 20 min; after the sonication was completed, centrifugation was carried out for 5min (3500rpm), and then the supernatant was transferred to a clean vial. Then, to the vial from which the supernatant had been transferred, 10mL of n-hexane was added, and the above extraction step was repeated. After the third repeated extraction, the supernatant was filtered through a polytetrafluoroethylene membrane (0.45 μm) and transferred out of the extraction flask, and then the extraction flask containing the remaining seed residue was dried under a nitrogen stream at room temperature for 24 hours to obtain a dried seed residue.
(3) Extracting and drying: to the extraction flask containing the dried seed residue in step (2) was added 10mL of 80% methanol solution, stirred with shaking for 5min, followed by sonication for 15min, followed by centrifugation for 5min (3500 rpm). The supernatant from the extraction flask was transferred to a 20mL clean glass vial, and then 10mL of 80% methanol solution was added to the sample flask and the extraction procedure was repeated. Mixing the supernatants, filtering with polytetrafluoroethylene membrane (0.45 μm), drying the filtrate at room temperature under nitrogen flow for 48 hr to obtain dry seed residue extract, i.e. Acer Truncatum Bunge extract, and storing at-20 deg.C.
Example 2
The seeds of example 2 were collected from the city of mahogany in the inner Mongolia autonomous region, and the preparation method and parameters were the same as those of example 1.
Example 3
The seed of example 3 was collected from the park of the purple bamboo institute of Hai lake district, Beijing, and the preparation method and parameters were the same as those of example 1.
Example 4
The seeds of example 4 were obtained from the Fenghuang mountain scenic spot of the Hai lake area of Beijing, and the preparation method and parameters were the same as those of example 1.
Example 5
The seeds of example 5 were collected from Acer truncatum seeds from Fufeng county, Baoji City, Shaanxi province, and the preparation method and parameters were the same as those of example 1.
Experiment of inhibitory effect of acer truncatum seed residue extract on acetylcholinesterase activity:
the experimental method mainly refers to the Ellman method which is most commonly used in academia at present, and the simple description after the adjustment according to the characteristics of the extract is as follows:
(1) phosphate buffer (0.1M Na per 100mL phosphate buffer)2HPO494.7mL of solution; 0.1M NaH2PO45.3mL of solution, adjusting pH to 8.0) to dilute acetylcholinesterase (AChE) into 0.1U/mL of working solution;
(2) preparing 6.25mM solution (working solution) of thioacetyl choline iodide and DTNB (color development agent) by using phosphate buffer solution;
(3) the extract was diluted with DMSO and water to 1mM working solution, ensuring that DMSO concentrations in different concentrations of extract solutions were the same (2% each), DMSO concentration in the final reaction system was 0.1%, and the extract concentration was 20. mu.g/mL. The positive control is tacrine (TA, AChE inhibitor clinical drug) with a final concentration of 0.333 μ M; negative control (NC group) was 2% DMSO solvent control;
(4) the reaction was performed in a 96-well plate, to which the following reagents (the volume of the added reagents was one reaction volume) were sequentially added, in triplicate for each sample; as shown in table 1:
TABLE 1
(5) The absorbance at 405nm was measured every 30 seconds for 1 hour after addition of the developer and substrate.
(6) The absorbance of the extract at about 1 in the average of the absorbance values of the NC group was selected, the average of the absorbance values of the extract (extract measurement value-background value) was calculated, and the AChE inhibition ratio of the extract was calculated as (NC-average of absorbance values of the extract)/NC × 100%.
The results of the experimental examples are shown in tables 2 and 3:
TABLE 2 inhibitory Effect of extract at a concentration of 20. mu.g/mL on acetylcholinesterase (unit:%)
Note: the concentration of tacrine is 0.333 mu M
It is generally accepted in academia that when the inhibition rate of acetylcholinesterase by a plant extract at a concentration of 100. mu.g/mL is greater than 50%, it indicates that the extract has acetylcholinesterase inhibitory activity. The results of the above preliminary screening experiments of the present invention show that, at a concentration of 20 μ g/mL, the acetylcholinesterase inhibition rates of examples 1-5 are all close to 90%, which is much lower than the standard of 100 μ g/mL concentration, thus indicating that the acer truncatum seed residue extract has significant acetylcholinesterase inhibition activity.
Further, the extracts of the examples were subjected to a series of concentration gradients (0.01, 0.05, 0.125, 0.25, 2.5 and 10. mu.g/mL), and the other steps and reagent concentrations were the same as those described above, and the extracts of the examples were subjected to a double-screening test to determine the half inhibitory activity against acetylcholinesterase, i.e., IC50The value is obtained. The results are shown in Table 3.
TABLE 3 IC inhibition of acetylcholinesterase activity by extracts of the examples50Measurement (Unit: μ g/mL)
| Sample (I) | Tacrine (D) | Extract 1 | Extract 2 | Extract 3 | Extract 4 | Extract 5 |
| IC50 | 0.07 | 0.124 | 0.170 | 0.162 | 0.133 | 0.232 |
| Standard deviation of | 0.004 | 0.012 | 0.013 | 0.013 | 0.004 | 0.013 |
The results in Table 3 show that the examples all have extremely low IC50A value, and only slightly greater than the IC of tacrine, an acetylcholinesterase inhibitor for clinical use50The value further proves that the extract has obvious and high-efficiency acetylcholinesterase inhibitory activity and has great development and application prospects.
In conclusion, the preparation method of the acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase comprises the steps of crushing seeds, freeze-drying at low temperature, extracting for removing oil, ultrasonically extracting residues, and drying in nitrogen to obtain the extract; biological activity experiments show that the extract has extremely strong acetylcholinesterase inhibition activity and can be used for developing medicaments for treating the Alzheimer disease. The extract disclosed by the invention has the advantages of natural and easily-obtained raw materials, simplicity and easiness in preparation, and extremely strong biological activity, can reduce the pollution of waste residues in the Acer truncatum oil obtaining process, guides the comprehensive development and utilization of Acer truncatum resources, and has a great market prospect in developing medicaments and health care products for treating the Alzheimer disease.
The invention relates to a preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase, wherein acer truncatum bunge seed residues are waste left after oil is obtained from acer truncatum bunge kernels. The extraction solvent is 80% methanol, is a common extraction solvent, has the characteristics of small pollution, low price and suitability for large-scale production, has low requirements on instruments and equipment, is easy to obtain equipment, has low purchase cost and is easy to operate. Ultrasonic extraction is adopted, so that the method is economic and environment-friendly; vacuum freeze drying and normal temperature nitrogen flow drying are adopted to avoid the influence of temperature on the components and activity of the extract.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (3)
1. A preparation method of acer truncatum bunge extract with high-efficiency inhibitory activity to acetylcholinesterase is characterized by comprising the following steps:
s101: firstly, putting naturally dried acer truncatum seeds into an ultramicro jet mill for grinding, and carrying out vacuum freeze drying on the obtained powder;
s102: taking the powder treated in the step S101, sequentially carrying out ultrasonic extraction, centrifugation and filtration by using normal hexane as an extraction solvent, collecting filter residues, repeatedly extracting for 2-3 times, and then drying the filter residues in a protective gas atmosphere;
s103: extracting the filter residue obtained in the step S102 by using a methanol solution as an extracting agent, sequentially stirring, ultrasonically extracting, centrifuging, filtering, collecting supernatant, repeatedly extracting for 2-3 times, and drying the supernatant in a protective gas atmosphere to obtain the acer truncatum bunge extract with efficient inhibitory activity to acetylcholinesterase;
in the step S103, the mass concentration of the methanol solution is 78% to 82%;
in the step S101, the temperature of vacuum freeze drying is-78 ℃ to-82 ℃, the vacuum pressure is less than 13.3Pa, and the drying time is 45h to 50 h;
in the step S102, the ratio of the mass of the powder processed in the step S101 to the volume of the n-hexane is 1 g: 18-22 mL;
in the step S102, the ultrasonic extraction time is 18 min-22 min; the centrifugation time is 5min to 8min, and the rotation speed during centrifugation is 3000rpm to 4000 rpm; the filter membrane adopted during the filtration is a polytetrafluoroethylene membrane with the aperture of 0.4-0.5 mu m;
in step S103, the ratio of the mass of the residue obtained in step S102 to the volume of the methanol solution is 1 g: 18-22 mL; stirring for 3-8 min, ultrasonic extracting for 15-20 min, centrifuging for 5-8 min at 3000-4000 rpm, and filtering with a filter membrane of 0.4-0.5 micron size.
2. Acer truncatum bunge extract with high inhibitory activity on acetylcholinesterase, which is obtained by the preparation method of claim 1.
3. Use of Acer truncatum extract having potent acetylcholinesterase inhibitory activity according to any one of claims 1-2 for the manufacture of a medicament for the treatment of Alzheimer's disease.
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| 元宝枫种仁70%乙醇提取物的化学成分分析;张玉伟等;《食品科学》;20171231;第38卷(第24期);第137-143页，尤其是第138页右栏第4段以及第139页左栏第2段 * |
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