A kind of betel nut extract and its preparation method and application

Abstract

The present invention relates to the technical field of plant effective component extraction, and specifically discloses a betel nut extract and a preparation method and application thereof. The preparation method of the betel nut extract provided by the present invention adopts a simulated saliva solution as an extraction solvent under a specific alkaline condition (pH 11.5-12.5), an auxiliary ultrasonic extraction, and a condition and process of simulating a person chewing betel nut, so that the active components of the betel nut are fully released, while ensuring that the integrity of the active component structure and the biological activity do not change, thereby significantly improving the extraction rate of alkaloids, polyphenols, flavonoids and total sugars in the betel nut, and the total extraction rate of the betel nut can reach 21.4%. The extracted betel nut extract has high anti-inflammatory, anti-oxidation and learning and memory function improvement effects, can be widely used in various betel nut products, and is of great significance for promoting and expanding the sustainable development of the betel nut industry.

Description

Technical Field

[0001] The invention relates to the technical field of plant effective component extraction, in particular to a betel nut extract and a preparation method and application thereof. Background Art

[0002] Betel nut is the mature fruit of Areca atechu L., a plant of the genus Areca in the palm family. It is one of the four major southern medicinal herbs in my country and has the effects of strengthening teeth and killing bacteria, eliminating accumulation and digestion, eliminating athlete's foot and expelling insects. The composition of betel nut is extremely complex. Studies have shown that at least dozens of ingredients have been detected in it. Betel nut is rich in alkaloids, polyphenols, coumaric acid, flavonoids, polysaccharides, proteins, amino acids, fats and other functional active ingredients. In recent years, the output of betel nut has jumped to the second place among tropical cash crops in Hainan Province, second only to rubber. It is a good fruit for residents of the southeastern coastal provinces of China to welcome guests and entertain relatives and friends.

[0003] The Compendium of Materia Medica records that betel nut is warm and spicy, bitter and non-toxic. Modern scientific research shows that betel nut has a protective effect on the nervous system, endocrine system and digestive system. Therefore, betel nut has great development prospects as a traditional Chinese medicine. However, most betel nuts have not entered the market of medicinal materials or health products, but are eaten fresh or simply processed into dried betel nuts for chewing. Because the fiber of betel nut is rough, regular chewing of betel nut will rub the oral mucosa, which is easy to cause repeated trauma to the mucosa and form local chronic damage, thereby causing oral diseases and even oral cancer. Therefore, the safety of traditional betel nut consumption has received widespread attention in recent years. How to make full use of betel nut resources, tap its medicinal, edible and health value, and solve the problem of comprehensive utilization of betel nut fine processing and deep processing is of great significance to promoting the development of agricultural economy in tropical regions and leading the sustainable development of the betel nut industry.

[0004] At present, the methods for extracting the effective components of betel nut mainly include solvent extraction, microwave-assisted extraction, supercritical CO2 fluid extraction, enzymatic extraction, etc., which have the problems of high operating conditions, high production costs, and low effective component extraction rate. Therefore, it is urgent to develop a method for extracting the effective components of betel nut with high extraction rate, low cost, and simple operation. Summary of the invention

[0005] In view of the problems that the existing method for extracting betel nut has complex operation, high extraction cost and low extraction rate, the present invention provides a betel nut extract and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for preparing betel nut extract comprises the following steps:

[0008] The betel nut is crushed, a simulated saliva solution is added to the betel nut powder, the mixture is mixed evenly, the pH value is adjusted to 11.5-12.5, extraction is performed under ultrasonic conditions, solid-liquid separation is performed, concentration is performed, and drying is performed to obtain a betel nut extract;

[0009] Wherein, the simulated saliva solution comprises sodium chloride, potassium chloride, disodium ethylenediaminetetraacetic acid, edible gelatin, calcium chloride and magnesium chloride.

[0010] Compared with the prior art, the preparation method of the betel nut extract provided by the present invention adopts the conditions and process of human chewing betel nut to simulate the conditions and process of human chewing betel nut under specific alkaline conditions, with simulated saliva solution as the extraction solvent, so as to fully release the active ingredients of the betel nut, while ensuring that the integrity of the structure of the active ingredients and the biological activity do not change, thereby significantly improving the extraction rate of alkaloids, polyphenols, flavonoids and total sugars in the betel nut, and the total extraction rate of the betel nut can reach 21.4%. The extracted betel nut extract has high anti-inflammatory, antioxidant and learning and memory function improvement effects, can be widely used in various betel nut products, and is of great significance for promoting and expanding the sustainable development of the betel nut industry.

[0011] The preparation method of the betel nut extract provided by the invention is simple and easy to operate, has low cost, and adopts green and environmentally friendly solvent for extraction, which not only significantly improves the extraction rate of betel nut, but also effectively ensures the safety of the extract. The extracted betel nut extract can be directly used in the preparation of medicines, is environmentally friendly, and will not cause environmental pollution. It provides a new process for the green and efficient preparation of betel nut extract, and has high promotion and application value.

[0012] Preferably, each 1000 mL of the simulated saliva solution comprises: 550-650 mg of sodium chloride, 700-800 mg of potassium chloride, 250-350 mg of disodium edetate, 40-60 mg of edible gelatin, 180-250 mg of calcium chloride and 80-120 mg of magnesium chloride.

[0013] Adding gelatin to the simulated saliva solution can simulate the process of chewing betel nut in the human mouth, improve the dissolution of various active components in the betel nut, and improve the stability of the extracted active components; adding disodium ethylenediaminetetraacetic acid to the simulated saliva solution can reduce the oxidation of the extract during the extraction process and further improve the stability of the extract, thereby ensuring that the structural integrity and biological activity of the extract do not change.

[0014] It should be noted that the solvent of the simulated saliva solution is distilled water.

[0015] Preferably, the volume-to-mass ratio of the simulated saliva solution to betel nut powder is 8-12:1, wherein the unit of volume is milliliter and the unit of mass is gram.

[0016] Preferably, calcium hydroxide and sodium carbonate are used to adjust the pH to 11.5-12.5.

[0017] Preferably, the temperature of ultrasonic extraction is 35-40°C, the power of ultrasonic extraction is 100-120W, and the time of ultrasonic extraction is 10-15min.

[0018] Preferably, the number of ultrasonic extractions is 4-6 times.

[0019] The preferred extraction conditions can improve the extraction rate of multiple active components in betel nut and improve the extraction efficiency. The extracted betel nut extract has no unpleasant odor, has the unique flavor of betel nut, and can be widely used in various betel nut products.

[0020] The present invention also provides a betel nut extract, which is prepared by any of the above-mentioned methods for preparing the betel nut extract.

[0021] The present invention also provides the use of the betel nut extract in preparing a medicine for assisting in improving learning and memory functions.

[0022] The betel nut extract provided by the present invention can improve cognitive disorders such as learning and memory of aging mice induced by D-Gal, has a certain repair effect on liver and brain damage, can improve the impairment of spatial memory and working memory, and promote the activity of antioxidant enzymes in the body, inhibit the production of malondialdehyde and accelerate its clearance, and regulate the release of cytokines (IL-1β, IL-6 and TNF-α), and has broad application prospects in the field of medicine.

[0023] The invention also provides a medicine for assisting in improving learning and memory functions, comprising the betel nut extract.

[0024] Exemplarily, the betel nut extract and a pharmaceutically acceptable carrier can be made into a drug in the form of granules, capsules, tablets, pills, pellets, powders, pellets, decoctions, syrups, mixtures, decoctions or extracts.

[0025] The preparation method of the betel nut extract provided by the present invention is conducive to fully utilizing the clinical pharmacological value and health-care effects of betel nut, is of great significance for reducing or eliminating the negative effects of betel nut and improving its positive effects, and also provides a new idea for the comprehensive utilization of betel nut processing by-products and the development of high value-added products, which is of great significance to the development of betel nut products and has high promotion and application value. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Example 1

[0028] A preparation method of betel nut extract:

[0029] Step 1: Wash the betel nut, crush it with a grinder, and pass it through a 40-mesh sieve to obtain betel nut powder;

[0030] Step 2: Take 100 g of betel nut powder, add it to 1000 mL of simulated saliva solution, mix well, adjust the pH to 12.0 with calcium hydroxide and sodium carbonate, perform ultrasonic extraction at 37° C. and 110 W for 10 min, extract 5 times, combine the extracts, concentrate, and dry to obtain a betel nut extract;

[0031] The simulated saliva solution includes: 500 mg sodium chloride, 750 mg potassium chloride, 300 mg disodium ethylenediaminetetraacetic acid, 50 mg edible gelatin, 200 mg calcium chloride, 100 mg magnesium chloride and 1000 mL distilled water.

[0032] It was measured that the extraction rate of betel nut under this method was 22.5%.

[0033] Extraction rate = (mass of betel nut extract / 100) x 100%.

[0034] Example 2

[0035] A preparation method of betel nut extract:

[0036] Step 1: Wash the betel nut, crush it with a grinder, and pass it through a 40-mesh sieve to obtain betel nut powder;

[0037] Step 2: Take 100 g of betel nut powder, add it to 800 mL of simulated saliva solution, mix well, adjust the pH to 12.5 with calcium hydroxide and sodium carbonate, perform ultrasonic extraction at 38° C. and 120 W for 12 min, extract 6 times, combine the extracts, concentrate, and dry to obtain a betel nut extract;

[0038] The simulated saliva solution includes: 550 mg sodium chloride, 800 mg potassium chloride, 250 mg disodium ethylenediaminetetraacetic acid, 40 mg edible gelatin, 180 mg calcium chloride, 120 mg magnesium chloride and 1000 mL distilled water.

[0039] It was measured that the extraction rate of betel nut under this method was 20.3%.

[0040] Extraction rate = (mass of betel nut extract / 100) x 100%.

[0041] Example 3

[0042] A preparation method of betel nut extract:

[0043] Step 1: Wash the betel nut, crush it with a grinder, and pass it through a 40-mesh sieve to obtain betel nut powder;

[0044] Step 2: Take 100 g of betel nut powder, add it to 1200 mL of simulated saliva solution, mix well, adjust the pH to 11.5 with calcium hydroxide and sodium carbonate, perform ultrasonic extraction at 40° C. and 100 W for 15 min, extract 4 times, combine the extracts, concentrate, and dry to obtain a betel nut extract;

[0045] The simulated saliva solution includes: 650 mg sodium chloride, 700 mg potassium chloride, 350 mg disodium ethylenediaminetetraacetic acid, 60 mg edible gelatin, 250 mg calcium chloride, 80 mg magnesium chloride and 1000 mL distilled water.

[0046] It was measured that the extraction rate of betel nut under this method was 21.4%.

[0047] Extraction rate = (mass of betel nut extract / 100) x 100%.

[0048] Comparative Example 1

[0049] This comparative example provides a method for preparing a betel nut extract, and the steps are exactly the same as those in Example 1, except that the gelatin in the simulated saliva solution is replaced with an equal amount of pectin.

[0050] Comparative Example 2

[0051] This comparative example provides a method for preparing a betel nut extract, and the steps are exactly the same as those in Example 1, except that the disodium ethylenediaminetetraacetic acid in the simulated saliva solution is replaced by an equal amount of sodium citrate.

[0052] Comparative Example 3

[0053] This comparative example provides a method for preparing a betel nut extract, and the steps are exactly the same as those in Example 1, except that gelatin is not added to the simulated saliva solution.

[0054] Comparative Example 4

[0055] This comparative example provides a method for preparing a betel nut extract, and the steps are exactly the same as those in Example 1, except that the pH adjusted with calcium hydroxide and sodium carbonate in step 2 is changed to 8.5.

[0056] Analysis of components of betel nut extract

[0057] 1.1 Determination of total alkaloid content

[0058] 1.1.1 Preparation of Standards

[0059] Weigh 2.5 mg of arecoline hydrobromide reference substance, place it in a 50 mL volumetric flask, add pH 5.0 acetic acid-sodium acetate buffer to dissolve and dilute to the scale, shake well to obtain the arecoline hydrobromide stock solution.

[0060] 1.1.2 Establishment of standard curve

[0061] Take 0.4mL, 0.7mL, 1.0mL, 1.5mL, 2.0mL, and 2.5mL of the prepared arecoline hydrobromide stock solution respectively, place them in separatory funnels respectively, add 4mL of acetic acid-sodium acetate buffer solution of pH 5.0 and 2mL of 0.05% bromocresol green solution in sequence, shake gently, add 5mL of chloroform respectively, extract twice, shake for 2min each time, let stand for 30min, take the chloroform layer, place it in a dry volumetric flask with 0.2g of anhydrous sodium sulfate in advance, shake, let stand, draw 5mL of supernatant, add 1mL of 0.01mol / L potassium hydroxide anhydrous ethanol solution, shake well, measure the absorbance at 618nm, measure three times for each concentration, calculate the average value, and establish the regression equation of the standard curve according to the relationship between concentration and absorbance, Y=0.0077X-0.0144.

[0062] 1.1.3 Determination of total alkaloid content of test samples

[0063] Weigh 1.0 g of the prepared betel nut extract, add 10 mL of distilled water, dissolve with ultrasound assistance, add 40 mL of 90% ethanol solution and precipitate for 12 h, filter with suction, and dry the filtrate to obtain the test sample.

[0064] Weigh 25.0 mg of the test sample, place it in a 10 mL volumetric flask, add pH 5.0 acetic acid-sodium acetate buffer to dissolve and dilute to the scale to obtain the test solution. Use a pipette to measure 1 mL of the prepared test solution and place it in a separatory funnel, add pH 5.0 acetic acid-sodium acetate buffer 4 mL and 0.05% bromocresol green solution 2 mL, shake gently, add 5 mL of chloroform to extract twice, shake for 2 minutes each time, let stand for 30 minutes, separate the chloroform layer, place it in a dry volumetric flask with 0.2 g of anhydrous sodium sulfate in advance, shake, let stand, absorb 5 mL of the supernatant, add 1 mL of 0.01 mol / L potassium hydroxide anhydrous ethanol solution, shake well, measure the absorbance at 618 nm, and then calculate the total alkaloid content of the test sample according to the regression equation, and calculate the total alkaloid content in the sample accordingly. The results are shown in Table 1.

[0065] 1.2 Determination of total polyphenol content

[0066] 1.2.1 Preparation of Standards

[0067] Weigh 20.0 mg of gallic acid standard, place in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the scale, shake well to obtain gallic acid stock solution.

[0068] 1.2.2 Establishment of standard curve

[0069] Measure different volumes of the above gallic acid stock solution, place in a 10mL volumetric flask, add distilled water to dissolve and dilute to the scale, shake well, and obtain gallic acid standard solutions with concentrations of 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL, and 120μg / mL, respectively. Pipette 0.5mL of the gallic acid standard solution into a graduated test tube, add 2.5mL of 0.25mol / L Folin phenol test solution, shake well, react for 3-8min, add 2.0mL of 7.5% Na2CO3 solution, shake well, place at room temperature for 60min, and measure the absorbance at 765nm with a spectrophotometer. Each concentration is measured three times in parallel, and the average value is calculated. Use purified water instead of the gallic acid standard solution, prepare a blank solution according to the above method, and adjust to zero with the blank solution. According to the relationship between different concentrations and absorbance, the regression equation of the standard curve is established, Y=0.0123X+0.0746.

[0070] 1.2.3 Determination of total polyphenol content of test samples

[0071] Weigh 1.0 g of the prepared betel nut extract, add 10 mL of distilled water, dissolve with ultrasound assistance, add 40 mL of 90% ethanol by volume and precipitate for 12 h, filter with suction, and dry the filtrate to obtain the test sample.

[0072] Weigh 10.0 mg of the above test sample, place it in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the scale, shake well, and the test solution is obtained. Measure 0.5 mL of the test solution into a graduated test tube, add 2.5 mL of 0.25 mol / L Folin phenol test solution, shake well, react for 3 to 8 minutes, add 2.0 mL of 7.5% Na2CO3 solution, shake well, place at room temperature for 60 minutes, measure the absorbance at 765 nm with a spectrophotometer, and then calculate the total polyphenol content of the test sample according to the regression equation, and calculate the total polyphenol content in the sample accordingly. The results are shown in Table 1.

[0073] 1.3 Determination of total flavonoids content

[0074] 1.3.1 Preparation of Standards

[0075] Weigh 20.0 mg of rutin standard, place in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the scale, shake well to obtain rutin stock solution.

[0076] 1.3.2 Establishment of standard curve

[0077] Measure the above-mentioned rutin stock solution of different volumes, put in a 10mL volumetric flask, add water to the scale, shake up, and obtain the rutin standard solution with a concentration of 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL, and 120μg / mL. Pipette 2.5mL of rutin standard solution in a graduated test tube, add 0.3mL5%NaNO2 test solution, shake up, let stand for reaction for 7min, add 0.3mL 10%Al(NO3)3 test solution, shake up, let stand for reaction for 6min, then add 4mL 4%NaOH solution, shake up, let stand for reaction for 15min, measure absorbance with an ultraviolet spectrophotometer at 510nm, and each concentration is measured in parallel three times to calculate mean value. Replace the rutin standard solution with distilled water, prepare a blank solution as described above, and adjust to zero with the blank solution. The regression equation of the standard curve was established based on the relationship between different concentrations and absorbance, Y=0.0030X-0.0008.

[0078] 1.3.3 Determination of total flavonoid content of test samples

[0079] Weigh 1.0 g of the prepared betel nut extract, add 10 mL of distilled water, dissolve with ultrasound assistance, add 40 mL of 90% ethanol by volume and precipitate for 12 h, filter with suction, and dry the filtrate to obtain the test sample.

[0080] Weigh 10 mg of the above test sample, place it in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the scale, shake well, and the test solution is obtained. Measure 2.5 mL of the above test sample solution into a graduated test tube, add 0.3 mL of 5% NaNO2 test solution, shake well, let it stand for 7 minutes, add 0.3 mL of 10% Al(NO3)3 test solution, shake well, let it stand for 6 minutes, then add 4 mL of 4% NaOH solution, shake well, let it stand for 15 minutes, measure the absorbance at 510 nm with an ultraviolet spectrophotometer, and then calculate the total flavonoid content in the betel nut extract according to the regression equation. The results are shown in Table 1.

[0081] 1.4 Determination of total soluble sugar content

[0082] 1.4.1 Preparation of Standards

[0083] Weigh 25.0 mg of D-glucose reference substance, dissolve it in water and make up to the volume in a 25 mL volumetric flask to obtain a glucose stock solution with a concentration of 1.0 mg / mL.

[0084] 1.4.2 Establishment of standard curve

[0085] The above glucose stock solution was used, and water was used as diluent to prepare standard solutions with concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL. 2 mL of each of the above standard solutions was pipetted, and 0.5 mL of 6% phenol and 2.5 mL of 98% concentrated sulfuric acid were added, respectively, and the mixture was shaken and allowed to stand for 30 min. The absorbance was measured at 490 nm, and the regression equation of the standard curve was established, Y=15.6730X-0.0153.

[0086] 1.4.3 Determination of total sugar content of test sample

[0087] Weigh 1.0 g of the prepared betel nut extract, add 10 mL of distilled water, dissolve with ultrasound assistance, add 40 mL of 90% ethanol solution and precipitate for 12 h, filter with suction, and dry the residue to obtain the test sample.

[0088] Take 20.0 mg of the above test sample, dissolve it in water and transfer it to a 100 mL volumetric flask, add water to the mark, and shake well. Measure 5 mL of the above test sample solution and place it in a 100 mL volumetric flask, add water to the mark, and shake well to obtain the test sample solution. Prepare the test sample solution into a 0.04 mg / mL aqueous solution, transfer 2 mL, add 0.5 mL of 6% phenol and 2.5 mL of 98% concentrated sulfuric acid, shake well, let stand for 30 minutes, measure the absorbance at 490 nm, and then calculate the total sugar content in the betel nut extract according to the regression equation. The results are shown in Table 1.

[0089] 1.2.5 Determination of total ash

[0090] In this experiment, the total ash content of betel nut extract was studied by thermogravimetric analysis (TGA). The thermal stability analysis was performed at 20-800℃ with a heating rate of 10℃ per minute under N2 protective gas. The results are shown in Table 1.

[0091] Table 1 Main components and contents of betel nut extract prepared in Example 1

[0092] Element content(%) Total alkaloids 1.1±0.3 Total polyphenols 8.3±0.5 Total flavonoids 19.6±0.7 Total Sugar 22.5±0.6 Total ash 40.6±0.7

[0093] As can be seen from the above table, in the component analysis of betel nut extract, the total alkaloid content determined by acid dye colorimetry was (1.1±0.3)%, the total polyphenol content determined by Folin phenol method was (8.3±0.5)%, the total flavonoid content determined by nitrite-aluminum nitrate colorimetry was (19.6±0.7)%, the total sugar content determined by phenol-sulfuric acid method was (22.5±0.6)%, and the total ash content determined by differential scanning thermogravimetry was (40.6±0.7)%.

[0094] The contents of the main components in the betel nut extract prepared in Example 2-3 are substantially the same as those in Example 1.

[0095] Evaluation of Areca Extract in Improving Learning and Memory Function

[0096] 2.1 Antioxidant effect of betel nut extract in vitro

[0097] 2.1.1 Determination of DPPH free radical scavenging ability

[0098] Accurately weigh 0.16g of the prepared betel nut extract, add it to a 100mL volumetric flask, dissolve it in distilled water and dilute it to the mark to prepare an aqueous solution of betel nut extract with a concentration of 1.6mg / mL. Take the above 1.6mg / mL test solution as the reference solution, use a pipette to measure 5mL, 2.5mL, 1.25mL, 0.625mL, 0.3125mL, and 0.15625mL of the prepared 1.6mg / mL reference solution to be tested, add it to a 10mL volumetric flask, dilute it to the mark with distilled water, and dilute it step by step to 0.8mg / mL, 0.4mg / mL, 0.2mg / mL, 0.1mg / mL, 0.05mg / mL, and 0.025mg / mL of the betel nut extract aqueous solution, accurately pipette 2mL of the above concentration samples into a test tube, and add 1mL of 0.16mmol·L -1 Shake the DPPH solution and place it at room temperature for 30 minutes. Use a UV-visible spectrophotometer to measure the absorbance at 517nm and record the value as A1. Perform three parallel measurements for each sample concentration and calculate the average value. Replace 2mL of the sample in the system with 2mL of distilled water, and keep other solutions unchanged. Measure the absorbance under the same conditions to get A0. Replace 1mL of DPPH in the system with 1mL of anhydrous ethanol, and keep other solutions unchanged. Measure the absorbance under the same conditions to get A2. Repeat the different sample concentrations and tests 3 times in parallel.

[0099] Positive control group: accurately weigh 0.16g of Vc, add it to a 100mL volumetric flask, dissolve it in distilled water and dilute it to the scale to prepare a 1.6mg / mL Vc aqueous solution, and then dilute it step by step to 0.8mg / mL, 0.4mg / mL, 0.2mg / mL, 0.1mg / mL, 0.05mg / mL, 0.025mg / mL Vc aqueous solution. Test A1, A0, and A2 in the same way as above, and calculate the DPPH free radical scavenging rate. The results are shown in Table 2. The DPPH free radical scavenging rate formula is as follows:

[0100]

[0101] K—clearance rate, %

[0102] A0—Absorbance of blank group

[0103] A1—Absorbance of sample group

[0104] A2—Absorbance of sample control group

[0105] Table 2 DPPH free radical scavenging rate

[0106]

[0107]

[0108] The results showed that betel nut extract had the activity of scavenging DPPH free radicals, and the activity first increased with the increase of concentration and then tended to be stable. 50 The smaller the value, the better the scavenging activity. The concentration of betel nut extract is 0.8 mg·mL -1 The clearance rate was the best when the concentration of each group was 0.8 mg·mL -1 IC 50 After calculation, the IC of the betel nut extract of Example 1 is 50 The IC value of positive control Vc is 0.01mg / mL. 50 is 0.008 mg / mL, and the IC 50 is 0.015 mg / mL, and the IC of the betel nut extract of Comparative Example 2 is 50 The IC value of the betel nut extract of Comparative Example 3 is 0.017 mg / mL. 50 The IC value of the betel nut extract of Comparative Example 4 is 0.022 mg / mL. 50 It is 0.034 mg / mL, indicating that the betel nut extract prepared in Example 1 has good DPPH free radical scavenging activity in the concentration range of 0.025-0.8 mg / mL, and is better than Comparative Examples 1-4.

[0109] 2.1.2 Hydroxyl radical scavenging experiment

[0110] Weigh 0.4 g of the prepared betel nut extract, add appropriate amount of distilled water to dissolve it, and finally make up the volume in a 50 mL volumetric flask to obtain a betel nut extract solution with a concentration of 8 mg / mL. Taking the above 8 mg / mL test solution as the reference solution, use a pipette to measure 5 mL, 2.5 mL, 1.25 mL, 0.625 mL, and 0.3125 mL of the prepared 8 mg / mL test reference solution, add it to a 10 mL volumetric flask and dilute to the scale with distilled water to prepare extract sample solutions with concentrations of 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. Pipette 2 mL of the extract samples of the above concentrations into test tubes, add 0.5 mL of 9 mmol / L FeSO4 solution and 0.5 mL of 8.8 mmol / L H2O2 solution, shake and mix, and let it stand at room temperature for 10 min. Then add 0.5 mL of 9 mmol / L salicylic acid solution, shake and mix, and let it stand at room temperature for 10 min. Use a UV-visible spectrophotometer to measure the absorbance at 510 nm, record the value, which is A1, and measure each sample concentration in parallel three times to calculate the average value. Replace 2 mL of sample in the system with 2 mL of distilled water, keep other solutions unchanged, and measure the absorbance value under the same conditions, which is A0. Replace 0.5 mL of H2O2 in the system with 0.5 mL of distilled water, keep other solutions unchanged, and measure the absorbance value under the same conditions, which is A2.

[0111] Positive control group: accurately weigh 0.4g of Vc, add it to a 50mL volumetric flask, dissolve it in distilled water and dilute it to the scale to prepare an 8mg / mL Vc aqueous solution, which is gradually diluted to 4mg / mL, 2mg / mL, 1mg / mL, 0.5mg / mL, and 0.25mg / mL Vc aqueous solutions. A1, A0, and A2 were tested in the same manner as above, and the hydroxyl scavenging rate was calculated using the following formula. The results are shown in Table 3.

[0112]

[0113] K—clearance rate, %

[0114] A0—Absorbance of blank reference

[0115] A1—Absorbance of the sample

[0116] A2—Background absorbance

[0117] Table 3 Hydroxyl removal rate

[0118]

[0119]

[0120] The results showed that betel nut extract had the activity of scavenging hydroxyl free radicals in a concentration-dependent manner. 50 The smaller the value, the better the scavenging activity. After calculation, the IC 50 is 0.039 mg / mL, while the IC of the betel nut extract prepared in Example 1 is 50 The IC value of the betel nut extract of Comparative Example 1 is 3.62 mg / mL. 50 The IC value of the betel nut extract of Comparative Example 2 is 4.25 mg / mL. 50 The IC value of the betel nut extract of Comparative Example 3 is 4.43 mg / mL. 50 The IC value of the betel nut extract of Comparative Example 4 is 4.83 mg / mL. 50 It is 5.99 mg / mL, indicating that the betel nut extract prepared in Example 1 has a certain hydroxyl radical scavenging activity at 0.25-8 mg / mL, and is better than Comparative Examples 1-4.

[0121] 2.2 Anti-inflammatory effect of betel nut extract in vitro

[0122] 2.2.1 Phagocytic ability

[0123] 5×10 RAW264.7 cells were added 5 100 μL of betel nut extract aqueous solution (1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL) and LPS (1 μg / mL) as a positive control were added to each well to treat the cells for 24 hours. The supernatant was discarded, 100 μL of 0.1% neutral red solution was added to each well, and incubated in a 37°C incubator for 1 hour, then the supernatant was discarded, and then washed three times with PBS (0.01M, pH 7.4). 100 μL of cell lysis solution (a mixed solution of anhydrous ethanol and acetic acid in a volume ratio of 1:1) was added to each well and kept at room temperature for 2 hours, and the absorbance was measured at a wavelength of 540 nm using an enzyme reader. The phagocytic rate was calculated according to the following formula, and the results are shown in Table 4.

[0124]

[0125] Drug group: Aqueous solutions of betel nut extract with different concentrations (1μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, 250μg / mL, 500μg / mL).

[0126] Blank group: basic culture medium.

[0127] Blank control group: RAW264.7 cells + basic culture medium.

[0128] Table 4 Phagocytosis rate

[0129]

[0130]

[0131] Note: Compared with the positive control group, *P<0.05, **P<0.01.

[0132] The results show that the betel nut extract prepared in Example 1 improves the phagocytic ability of macrophages RAW264.7 in the range of 1-100 μg / mL, and can significantly improve the phagocytic ability of macrophages RAW264.7 at 250-500 μg / mL (P<0.01), and the effects are better than those of Comparative Examples 1-4.

[0133] 2.2.2 Determination of reactive oxygen species (NO)

[0134] Macrophages RAW264.7 (2×10 5 Cells were inoculated in 96-well plates for 24 h, and LPS (1 μg / mL) and 100 μL of different concentrations of betel nut extract (1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL) were added to each well to treat the cells for 24 h. After incubation, the supernatant was collected into a sterile centrifuge tube. The nitrite level was determined by the Griess test. The specific method was as follows: NaNO2 was used as the standard, a NaNO2 stock solution with a concentration of 2 mmol / L was prepared, and then dilute it with distilled water to (0, 10, 20, 30, 40, 50 μmol / L) NaNO2 solution, where the concentration of NaNO2 solution was equivalent to the concentration of NO, 100 μL of NaNO2 solution of different concentrations was added, 100 μL of Griess reagent was added in the dark, and then reacted at room temperature for 10 min. The absorbance was evaluated at 540 nm by an enzyme marker. A standard curve was drawn with the measured absorbance as the ordinate and the NO concentration as the abscissa. The NO content in the positive control and different concentrations of betel nut extracts was determined in the same manner as the NaNO2 standard solution. The results are shown in Table 5.

[0135] Griess reagent: 0.1% naphthylethylenediamine hydrochloride solution and 1.0% p-aminobenzenesulfonamide solution were mixed in a volume ratio of 1:1.

[0136] Table 5 NO concentration

[0137]

[0138]

[0139] Note: Compared with the LPS group, *P<0.05, **P<0.01.

[0140] The results show that within the concentration range of 1-250 μg / mL, the release of NO in the cell supernatant decreases as the concentration of betel nut extract increases. Compared with the LPS group, the betel nut extract prepared in Example 1 can significantly inhibit the NO concentration in the cell supernatant at 100 μg / mL (P<0.05), and can significantly inhibit the NO concentration in the cell supernatant at 250-500 μg / mL (P<0.01), indicating that the betel nut extract has a certain anti-inflammatory effect at high concentrations, and the effect is better than that of comparative examples 1-4.

[0141] 2.3 Effects of Areca Extract on D-galactose-induced Aging Model Mice

[0142] 2.3.1 Establishment of D-Gal-induced aging mouse model

[0143] 162 Kunming male mice (20±2g, 6-8 weeks old) were placed in an animal room at a temperature of 23±2°C, a humidity of 60±5%, and a light intensity of 12h / d. They were allowed to adapt to this environmental condition for 3-5 days.

[0144] After the mice were adapted, they were randomly divided into 9 groups (18 mice / group), including 1 normal control (NC) group, 1 model control (MC) group, 1 positive control (PC) group, 2 Example 1 dose groups (ACE-L and ACE-H) and 4 comparative groups.

[0145] The NC group was intraperitoneally injected with normal saline; the other groups were intraperitoneally injected with D-Gal (200 mg / kg / d), with a dosing volume of 0.15 mL / 10 g. At the same time, the NC group and the MC group were gavaged with distilled water, the PC group was given huperzine A (0.2 mg / kg / d), the ACE-L group was gavaged with a low dose of the betel nut extract of Example 1 (250 mg / kg / d), the ACE-H group was gavaged with a high dose of the betel nut extract of Example 1 (500 mg / kg / d), and the comparative examples 1-4 groups were gavaged with a high dose of the betel nut extract prepared in comparative examples 1-4 (500 mg / kg / d). All were administered in the morning every day for 5 consecutive weeks, with a dosing volume of 0.25 mL / 10 g.

[0146] 2.3.2 General Observations

[0147] The daily status of mice in each group was observed, including water drinking, food intake, feces, activity, fur gloss changes, etc., and inter-group comparisons were made. The results are shown in Table 6.

[0148] Table 6 Observation table of general status of mice (n=18)

[0149]

[0150]

[0151] The results show that the mice in the model control group showed a series of aging phenomena, indicating that the modeling was successful. The feces of the mice in the example group were not formed in the early stage of the experiment, which may be because the short-term administration of betel nut extract had no obvious therapeutic effect on aging mice. As the administration time increased, the mice in the example group gradually recovered normal defecation.

[0152] 2.3.3 Body weight and food intake

[0153] The mice were weighed before and at the end of the experiment. They were weighed once every 7 days before feeding, and then the weight gain, weight gain rate and feed utilization rate of the mice were calculated. The results are shown in Tables 7 and 8.

[0154] Weight gain rate (%) = weight gain (g) / body weight before test (g) × 100%

[0155] Feed utilization rate (%) = weight gain (g) / feed consumption (g) × 100%

[0156] Table 7 Weight gain rate and feed utilization rate of mice

[0157]

[0158] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0159] The results show that the weight of the 9 groups of mice showed an upward trend over time. Compared with the normal control group, the weight gain rate of the model group mice was significantly reduced (P < 0.01). Compared with the model group, the weight gain rate of the high-dose group of the embodiment increased significantly (P < 0.05), and the weight gain rate of the mice in the high-dose groups of the comparative examples 1-4 increased significantly compared with the model group (P < 0.05). The weight gain rates were all higher than the low-dose group of the embodiment but lower than the high-dose of the embodiment. The results show that betel nut extract can improve the weight gain rate and feed utilization rate of mice, and the high-dose group of the embodiment is the optimal solution, and the weight gain effect and feed utilization rate are the best.

[0160] 2.3.4 Organ Index

[0161] After the experiment, all mice were fasted overnight and weighed. The eyeballs were immediately removed to collect blood. After centrifugation (3500r / min, 15 min), the supernatant was collected for later use. After blood collection, the mice were killed and dissected. The liver and brain were immediately removed and washed in 0.9% saline. The surface moisture was then dried with filter paper, weighed and recorded. The results are shown in Table 8.

[0162] Organ index (g / g) = organ mass / body mass

[0163] Table 8 Organ Index

[0164]

[0165] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0166] The results show that the liver and brain indexes of the model group are significantly lower than those of the normal control group (P < 0.01), indicating that the liver and brain tissues of the mice in the model group may have pathological damage. Compared with the model group, the liver and brain indexes of the mice in the low-dose group of the embodiment are significantly increased (P < 0.001), and the liver and brain indexes of the mice in the high-dose group of the embodiment are significantly increased (P < 0.05), proving that the betel nut extract can alleviate pathological damage in the liver and brain. Compared with the model group, the liver and brain indexes of the mice in the high-dose groups of comparative examples 1-4 are significantly increased (P < 0.05), indicating that the betel nut extract can alleviate pathological damage in the liver and brain. However, the liver and brain indexes of the mice are lower than those in the embodiment group (P < 0.05). And the liver and brain indexes of the mice in the high-dose group of the embodiment are lower than those in the low-dose group of the embodiment. The results show that a certain concentration of betel nut extract can alleviate pathological damage in the liver and brain, among which the low-dose group of the embodiment has the best effect.

[0167] 2.3.5 Behavioral evaluation

[0168] Behavioral experiments were started after 35 days of continuous drug administration, and drug administration continued during the behavioral period.

[0169] (1) New object recognition experiment

[0170] The experiment is divided into a familiarization period and a test period. First, the familiarization period is carried out. The four corners of the square mine are divided into four square areas with a side length of 10 cm, recorded as areas 1, 2, 3, and 4. Then, two identical objects are placed in areas 1 and 2 in the open field. The two objects are 20 cm apart, and the distance between the two objects and the side wall is 10 cm. That is, they are placed at the corners of areas 1 and 2 and recorded as objects A and B. The square areas 1 and 2 surrounded by the two objects and the side walls are recorded as the old space, and the square areas 3 and 4 are recorded as the new space. The time the mice are active in areas 1 and 2 is recorded as the time of exploring the old space, and the time they are active in areas 3 and 4 is recorded as the time of exploring the new space. Then, each group of mice is placed in turn from the middle of the non-object area of ​​the open field against the wall, and they are taken out after being allowed to explore freely for 10 minutes. After resting for 30 minutes, the test period experiment is carried out.

[0171] First, to test the spatial recognition ability, one of the objects in the open field needs to be taken out and replaced in the position of the 3rd or 4th division area in the field to prevent the mouse from remembering the position of objects A and B. The 3rd and 4th division areas are recorded as new spaces. Then the mouse is put in from the same position of the box, and the mouse explores freely for 10 minutes and records the time the mouse explores the new space and the old space. After that, to test the ability to recognize new objects, one of the objects in the open field needs to be taken out during the test period and replaced with a new object of the same material but different color and size. This object is the new object. Then the mouse is put in from the same position of the box and freely explored for 10 minutes. The time the mouse smells, sniffs, and circles around the new object is recorded as the time of exploring the new object, and the time the mouse smells, sniffs, and circles around the old object is recorded as the time of exploring the old object. After each mouse experiment, wipe the box and objects with 75% alcohol to prevent the residual smell from affecting the next mouse. The camera on the top of the device tracked and collected the mouse's trajectory, analyzed the time the mouse spent exploring new objects and old objects, and calculated the novel space recognition index (NSI) and novel object recognition index (NOI) of each group of mice. The results are shown in Table 9.

[0172] NSI = time to explore new space / (time to explore new space+time to explore old space)×100%

[0173] NOI = time spent exploring new objects / (time spent exploring new objects + time spent exploring old objects) × 100%

[0174] Table 9 New object and new space recognition index

[0175]

[0176] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0177] The results showed that the NSI of the model group mice was reduced, indicating that the spatial memory of the mice was impaired. The NSI values ​​of the mice in the positive control group and the low-dose group of the example were significantly improved (P < 0.05), and the high-dose group of the example was able to significantly improve the spatial memory impairment of aging mice induced by D-Gal (P < 0.01).

[0178] Compared with the normal control group, the NOI of the model group mice was significantly reduced (P < 0.05), indicating that the working memory of the mice was impaired. The positive control group and the high-dose group of the embodiment can significantly improve the working memory impairment of aging mice induced by D-Gal (P < 0.01), the low-dose group of the embodiment can significantly improve the working memory of mice recognizing new objects (P < 0.05), and the NSI and NOI values ​​of the high-dose groups of the comparative examples 1-4 are significantly improved compared with the model group (P < 0.01). It can significantly improve the spatial memory impairment and working memory impairment of aging mice induced by D-Gal (P < 0.01). Compared with the embodiment, the new object and new space recognition index are higher than the low-dose group of the embodiment but lower than the high-dose group of the embodiment, among which the high-dose group of the comparative example 4 has the worst improvement effect. It can be seen that oral administration of a certain dose of betel nut extract can effectively improve the spatial memory and working memory impairment of aging mice induced by D-Gal. Among them, the high-dose group of the embodiment has the best effect.

[0179] (2) Y-maze test

[0180] The Y-maze body consists of three identical gray closed arms of 30cm×15cm×7cm, with the middle joint forming a triangle, and the angle between the three arms is 120°. A camera is installed in the center of the body to track and collect the mouse trajectory. During the experiment, each group of mice was placed in the intersection of the three arms in turn and allowed to explore freely for 8 minutes. The mouse entered the arm when its limbs were completely inside the arm. If the third entry into the arm was different from the previous two times, it was considered to be a correct entry into the arm. The order in which the mice entered the arm was recorded, and the spontaneous alternation accuracy of the mice in the Y-maze was calculated according to the following formula. The results are shown in Table 10.

[0181] Spontaneous alternation accuracy rate = [number of entries into the correct arm / (total number of entries into the arm - 2)] × 100%

[0182] Table 10 Spontaneous alternation accuracy

[0183]

[0184]

[0185] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0186] The results showed that the accuracy of spontaneous alternation in the model group was lower than that in the normal control group, indicating that the spatial working memory of the model group mice was impaired. Compared with the model group, the accuracy of spontaneous alternation in the positive group and the high-dose group of the embodiment mice was significantly increased (P < 0.05). It can be seen that the difference in the accuracy of spontaneous alternation among the 9 groups may be caused by impaired spatial memory.

[0187] 2.3.6 Antioxidant enzyme activity

[0188] After all behavioral experiments on mice were completed, all mice were fasted overnight and weighed, and their eyes were immediately removed to collect blood. The mice were then killed and dissected. Whole blood, liver and brain tissues were collected from the mice, and the collected tissues were washed and weighed in 0.9% saline. Then, they were placed in a 10mL centrifuge tube and stored at -80°C for later use. When the activity of antioxidant enzymes was determined, the mouse tissues were removed, thawed at room temperature, and the liver or brain tissues of each group of mice were added with 0.9% saline at a ratio of 1:9 (m:V), that is, 9mL 0.9% saline was added to 1g of tissue and organ, and tissue homogenate was prepared using a homogenizer, and then centrifuged at 7000r / min using a high-speed centrifuge model TGL-15B for 5 minutes. The supernatant was aspirated with a pipette to obtain the tissue sample to be tested. Serum preparation method: After the whole blood was collected and placed in a refrigerator at 4°C for 20 minutes, it was centrifuged at 3500r using a high-speed centrifuge model TGL-15B for 10 minutes, and the supernatant was taken twice as serum. The changes of SOD, GSH-Px and MDA in serum, liver and brain tissue were measured. The results are shown in Table 11.

[0189] Note: For the specific determination methods of SOD, GSH-Px and MDA, please refer to the instructions of Nanjing Jiancheng SOD, MDA and GSH-PX test kits.

[0190] Table 11 Antioxidant activity

[0191]

[0192]

[0193]

[0194] Note: Compared with the normal control group, the model group *P<0.05, **P<0.01, ***P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0195] The results showed that the SOD inhibition rate, SOD and GSH-Px activities in the serum, liver and brain tissues of the model group mice were lower than those in the normal control group, and the MDA content was higher than that in the normal control group, indicating that the model was successfully established. The SOD inhibition rate, SOD and GSH-Px activities in the serum, liver and brain tissues of the example group mice were higher than those in the model group, and the MDA content was lower than that in the model group, and all of them were statistically significant (P < 0.05).

[0196] It can be seen that the betel nut extract prepared in the example can promote the activity of the body's antioxidant enzymes (SOD and GSH-Px), inhibit the production of MDA and accelerate its clearance, thereby reducing peroxidation damage, showing good antioxidant activity on D-Gal-induced aging mice, and improving the cognitive dysfunction of aging mice. The high-dose group of the example is better than the low-dose group of the example, and is better than comparative examples 1-4.

[0197] 2.3.7 Content of inflammatory factors

[0198] After all behavioral experiments on mice were completed, all mice were fasted overnight and weighed, and their eyeballs were immediately removed to collect blood. The mice were then killed and dissected immediately. Whole blood, liver and brain tissues were collected from the mice, and the tissues were washed and weighed in 0.9% saline. They were then placed in a 10mL centrifuge tube and stored at -80°C for later use. During the measurement, the mouse tissues were removed and thawed at room temperature. The preparation method of the tissue samples to be tested was the same as in 2.3.6. Serum preparation method: After the whole blood was collected and placed in a refrigerator at 4°C for 20 minutes, it was centrifuged at 3500r / min in a high-speed centrifuge model TGL-15B for 10 minutes, and the supernatant was collected twice as serum. The changes in the levels of IL-1β, IL-6 and TNF-α in the serum, liver and brain tissues of each group of mice were measured. The results are shown in Table 12

[0199] Table 12 Inflammatory factors

[0200]

[0201]

[0202]

[0203] Note: Compared with the normal control group, the model group *P<0.05, **P<0.01, ***P<0.001; compared with the model group,# P<0.05, ## P<0.01, ### P<0.001.

[0204] The results showed that the levels of IL-1β, IL-6 and TNF-α in the serum, liver and brain homogenates of the mice in the model group were higher than those in the normal control group, indicating that the model was successfully established. The levels of IL-1β, IL-6 and TNF-α in the serum, liver and brain homogenates of the mice in the example group were lower than those in the model group, and were statistically significant (P < 0.05).

[0205] It can be seen that the betel nut extract prepared in the embodiment of the present invention can effectively alleviate the aging reaction caused by D-Gal by regulating the content of cytokines (IL-1β, IL-6 and TNF-α) in mice, improve cognitive dysfunction such as memory in aging mice, and the effect of the high-dose group of the embodiment is better than that of the low-dose group of the embodiment, and the embodiment groups are better than the comparative examples 1-4.

[0206] The betel nut extracts prepared in Examples 2-3 can achieve technical effects substantially equivalent to those in Example 1.

[0207] In summary, the betel nut extract provided by the present invention has a good in vitro antioxidant effect, can significantly improve the proliferation ability and phagocytic ability of macrophages RAW264.7 within a certain concentration range, and extremely significantly reduce the NO concentration in the supernatant of macrophages RAW264.7, has a certain in vitro anti-inflammatory effect, can improve cognitive disorders such as learning and memory in D-Gal-induced aging mice, has a certain repair effect on liver and brain damage, can improve its spatial memory and working memory impairment, and at the same time, can also promote the body's antioxidant enzymes (SOD and GSH-Px) activity, inhibit MDA production and accelerate its clearance, and regulate the release of cytokines (IL-1β, IL-6 and TNF-α), and the preparation method of the betel nut extract provided by the present invention is simple, green, environmentally friendly and efficient, and can be industrially produced.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing betel nut extract, characterized in that: The following steps are involved: The betel nut is crushed, a simulated saliva solution is added to the betel nut powder, the mixture is mixed evenly, the pH value is adjusted to 11.5-12.5 with calcium hydroxide and sodium carbonate, extraction is performed under ultrasonic conditions, solid-liquid separation is performed, concentration is performed, and drying is performed to obtain a betel nut extract; Wherein, each 1000 mL of the simulated saliva solution comprises: 550-650 mg of sodium chloride, 700-800 mg of potassium chloride, 250-350 mg of disodium ethylenediaminetetraacetic acid, 40-60 mg of edible gelatin, 180-250 mg of calcium chloride and 80-120 mg of magnesium chloride.

2. The method for preparing the betel nut extract according to claim 1, wherein The volume-to-mass ratio of the simulated saliva solution to betel nut powder is 8-12:1, wherein the unit of volume is milliliter and the unit of mass is gram.

3. The method for preparing the betel nut extract according to claim 1, wherein The temperature of ultrasonic extraction is 35-40°C, the power of ultrasonic extraction is 100-120W, and the time of ultrasonic extraction is 10-15min.

4. The method for preparing the betel nut extract according to claim 1 or 3, characterized in that: The number of ultrasonic extractions was 4-6 times.

5. A betel nut extract, characterized in that The betel nut extract is prepared by the preparation method of the betel nut extract according to any one of claims 1 to 4.

6. Use of the betel nut extract according to claim 5 in the preparation of a drug for assisting in improving learning and memory functions.

7. A drug for assisting in improving learning and memory function, characterized in that: Prepared from the betel nut extract according to claim 5.

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