Seabuckthorn seed meal extract containing tryptamine alkaloids, uses and detection methods thereof

Abstract

The present application provides an extract based on seabuckthorn seed meal and having the effects of improving sleep and memory. The present application also provides the use and detection method of the extract.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant extracts and their medical uses and detection. BACKGROUND

[0002] Sleep is an important physiological process, which is an important factor for maintaining physical and mental health and life status of human body. The repair of human body damage, the establishment and consolidation of memory, etc. cannot be separated from the regulation of sleep activity. In modern society, the phenomenon of sleep deprivation is extremely common, which is a major hidden danger affecting human health. Sleep deprivation not only affects the mental state and the ability of work and study of human, but also increases the risk of various diseases, including cancer, heart disease, diabetes, obesity, Alzheimer's disease, etc.

[0003] Sufficient sleep is the basis of memory formation, and good sleep prepares the brain for the formation of new memory. Sleep deprivation has been widely proven to cause memory impairment, which is manifested as memory decline, memory loss, memory misconstruction, etc. Previous studies have pointed out that sleep deprivation affects the three processes of memory coding, consolidation and extraction. Complete sleep deprivation will continuously impair the coding process of many declarative memories, which is related to the functional defects of neural networks supporting memory and related processes such as executive function, and sleep deprivation will lead to a decrease in the coding speed of memory. Non-rapid eye movement sleep deprivation has been shown to impair memory consolidation and extraction, i.e. if non-rapid eye movement sleep deprivation is performed during the consolidation phase after memory formation, the formed memory will also be significantly reduced.

[0004] The damage of sleep deprivation to memory involves complex mechanisms, mainly including synaptic plasticity and neuroinflammation, etc. At the same time, the damage of memory involves multiple brain regions, including hippocampus, medial prefrontal cortex, amygdala, etc., among which the hippocampus is more susceptible to sleep. SUMMARY

[0005] Hippophae rhamnoides Linn. is a perennial shrub or tree of Hippophae in Elaeagnaceae, which is an ecological and economic forest plant resource widely planted in northwest China, and its resource amount is extremely rich in China. As a food and medicine dual-purpose berry plant, Hippophae rhamnoides Linn. has a wide application in national medicine, and it can also be used to produce juice, fruit wine, fruit powder, seed oil and other processed products.

[0006] Hippophae rhamnoides seed meal is the residue remaining after the extraction of Hippophae rhamnoides seed oil, which contains rich chemical components such as flavonoids, proanthocyanidins, polyphenols, proteins, alkaloids, etc., and has high research and utilization value. Hippophae rhamnoides seed meal has been rarely paid attention to by people, which causes great waste of resources, and its active ingredients and treatment potential need to be developed.

[0007] The present application provides a kind of based on seabuckthorn seed meal, and has the efficacy of improving sleep, memory etc.Extract.The present application research finds that seabuckthorn seed meal crude extract has no obvious improvement on memory disorder caused by sleep loss etc.;However, when seabuckthorn seed meal crude extract is purified by appropriate method, the improvement on memory etc.is significantly improved.

[0008] Based on the above findings, the present application actually provides a kind of seabuckthorn seed meal extract for improving memory, which is prepared by the following method: (1) seabuckthorn seed meal, acetic acid water, extraction, alcohol precipitation, after removing the solvent of filtrate to obtain crude extract;

[0009] (2) crude extract on macroporous adsorption resin, eluted with water and 10%v / v ethanol in turn, collect ethanol elution part, remove solvent to obtain seabuckthorn seed meal extract.

[0010] In step (1), the concentration of acetic acid water is 2.5%; add ethanol to the alcohol content of 80%v / v for alcohol precipitation.

[0011] In step (2), AB-8 macroporous adsorption resin is selected.

[0012] In the present application, acetic acid water is used to heat extract tryptamine alkaloids in seabuckthorn seed meal to obtain alkaloid crude extract. Through the selection of resin material, AB-8 macroporous adsorption resin is selected as the enrichment material. Combined with single factor experiment, three factors of sample loading amount, sample solution pH and eluent ethanol concentration are selected for process optimization. Based on the principle of Box-Behnken Design, Design Expert 8.0 software is used to design variables as sample loading amount (A), sample solution pH (B) and eluent ethanol concentration (C), and response value as product tryptamine alkaloid content (Y) to make three-factor and three-level response surface experiment, and the enrichment process of seabuckthorn seed meal tryptamine alkaloids is optimized.

[0013] The model is extremely significant (p<0.01) as a whole, and the non-significant term is not significant (p>0.05), which indicates that the model is accurate and reliable, and can be used for the optimization of the current process. Among them, A 2 reach a significant level (p<0.05), A and B 2 reach an extremely significant level (p<0.01).

[0014] The optimal enrichment process conditions predicted by the model are as follows: sample loading amount 0.34BV, sample solution pH 6.83, and eluent ethanol concentration 8.3%, under which the predicted tryptamine alkaloid content can reach 29.30%. According to the actual situation, the optimized conditions are adjusted as follows: sample loading amount 0.34BV, sample solution pH 7, and eluent ethanol concentration 8%. Under these conditions, the experimental verification of the predicted results is carried out, and the tryptamine alkaloid content in the obtained enrichment product is 28.99%, which is close to the predicted value.

[0015] On this basis, the enrichment process is scaled up according to the optimal process conditions, and when the extraction raw material is scaled up to 3.2 kg, the content of the tryptamine alkaloid can reach 25.71%.

[0016] The application also provides use of the seabuckthorn seed meal extract in preparation of a product for improving sleep and memory, increasing the content of melatonin, and improving hippocampal neuron injury.

[0017] The application also provides use of the seabuckthorn seed meal extract in preparation of a product for increasing the level of monoamine neurotransmitters and glutamic acid in brain tissue.

[0018] Further, the monoamine neurotransmitters are 5-hydroxytryptamine and dopamine.

[0019] In the research of the application, a sleep-deprived mouse model is established by intraperitoneal injection of p-chlorophenylalanine (PCPA) to investigate the memory improvement effect of the seabuckthorn seed meal tryptamine alkaloid enrichment product. The results of behavior experiments such as Morris water maze and Y maze show that the enrichment product can significantly improve the spatial memory ability of mice, the H&E and Nissl staining results of the hippocampus show that it can improve the injury of hippocampal neurons and increase the number of Nissl bodies of neurons. The results of biochemical index determination show that it can increase the MT level in the serum of mice and the levels of 5-HT, NA, DA and glutamic acid in the hippocampus, and inhibit the activity of MAO. In summary, the seabuckthorn seed meal tryptamine alkaloid can improve the memory and rhythm of sleep-deprived mice and regulate the level of neurotransmitters and their metabolic capacity at a certain dose.

[0020] The application also provides a detection method of the seabuckthorn seed meal extract, which simultaneously detects 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harman alkaline and N-acetyl-5-hydroxytryptamine by using HPLC, and includes the following detection contents:

[0021] (1) taking the crude extract prepared in claim 1 or the seabuckthorn seed meal extract to prepare a test sample solution;

[0022] (2) taking 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harman alkaline and N-acetyl-5-hydroxytryptamine to prepare a control sample solution;

[0023] (3) qualitatively or quantitatively detecting the test sample solution and the control sample solution by HPLC, and the chromatographic conditions are as follows:

[0024] Column: C18

[0025] Mobile phase: methanol as A, 0.1-0.3% trifluoroacetic acid aqueous solution as B, gradient elution procedure: 0-15 min, 5%-7% A; 15-20 min, 7% A; 20-50 min, 7%-35% A; 50-70 min, 35%-95% A; 70-80 min, 95%-5% A.

[0026] Further, the concentration of the trifluoroacetic acid aqueous solution is 0.15%.

[0027] Further, in the HPLC, the detection column temperature is 30℃, and the detection wavelength is 280 nm.

[0028] Further, the chromatographic column is selected from an Xcharge C18 chromatographic column.

[0029] A method for simultaneously determining six kinds of tryptamine alkaloids (5-hydroxytryptamine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, shepherdine, L-tryptophan, N-acetyl-5-hydroxytryptamine and harmine) is established in the application, and the established analysis method is verified for methodology, including linear relationship investigation, precision, repeatability, 12h stability and sample recovery rate investigation. The results show that the analysis method has good precision and repeatability, and good stability within 12h, and the linear ranges of the six kinds of tryptamine alkaloids are 6.280-251.4, 5.710-228.6, 1.140-457.1, 0.8000-32.00, 0.3100-12.60 and 0.4600-18.30 μg·mL -1 (r 2 >0.9993) respectively. Four kinds of tryptamine alkaloids are detected in the test sample, which are 5-hydroxytryptamine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, shepherdine and L-tryptophan, and the average sample recovery rates are 93.63%, 89.86%, 91.58% and 90.91% respectively, and the RSD values are all less than 3.0%. The established method is applied to the content determination of tryptamine alkaloids in seabuckthorn samples, and it is found that the tryptamine alkaloids are mainly concentrated in seabuckthorn seeds and seabuckthorn seed meal, and the content of tryptamine alkaloids in seabuckthorn seed meal is more than 0.4%. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Screening of resin types

[0031] Figure 2 Effect of sample loading solution pH on content of tryptamine alkaloids

[0032] Figure 3 Effect of eluent ethanol concentration on content of tryptamine alkaloids

[0033] Figure 4 Effect of sample loading amount on content of tryptamine alkaloids

[0034] Figure 5 Chromatogram of reference substance

[0035] Figure 6 Chromatogram of test sample of seabuckthorn seed meal extract

[0036] Figure 7 Chromatogram under chromatographic condition 1

[0037] Figure 8 Chromatogram under chromatographic condition 2

[0038] Figure 9 Chromatogram under chromatographic condition 3

[0039] Figure 10 Chromatogram under chromatographic condition 4

[0040] Figure 11 Chromatogram under chromatographic condition 5

[0041] Figure 12 Variation of average latency per day in each group in place navigation experiment

[0042] Figure 13 Movement trajectory and exploration results of mice in each group in spatial exploration experiment

[0043] Figure 14 Spontaneous alternation rate of mice in each group in Y maze

[0044] Figure 15 H&E stained tissue section of hippocampus of mice

[0045] Figure 16 Nissl stained tissue section of hippocampus of mice

[0046] Figure 17 Melatonin level in serum of mice

[0047] Figure 18 Levels of various neurotransmitters in hippocampus tissue of mice

[0048] Figure 19 Activity level of monoamine oxidase in brain of mice DETAILED DESCRIPTION

[0049] Example 1

[0050] 1 Instruments and reagents

[0051] 1.1 Material reagents

[0052] Hippophae rhamnoides L. seed meal sample (purchased from Qinghai Kangfu Biological Technology Co., Ltd., which is the residue of Hippophae rhamnoides L. seed after supercritical CO2 extraction of Hippophae rhamnoides L. seed oil). Ethanol, water, acetic acid, and NaOH are all analytical pure.

[0053] 1.2 Instrument equipment

[0054] Agilent 1260 type high performance liquid chromatograph (USA Agilent); DK-98-IIA type electric heating constant temperature water bath (Tianjin Test Instrument Co., Ltd.); JP100-S type ultrasonic cleaner (Shenzhen Jielian Cleaning Equipment Co., Ltd.). AB-8 macroporous adsorption resin, DM301 macroporous adsorption resin, D101 macroporous adsorption resin.

[0055] 2 Experimental method

[0056] 2.1 Extraction of tryptamine alkaloids in Hippophae rhamnoides L. seed meal

[0057] The Hippophae rhamnoides L. seed meal raw material was extracted with 2.5% acetic acid water: Hippophae rhamnoides L. seed meal = 20:1 (L: kg). After the extract was concentrated, ethanol was added to 80% to alcoholize the macromolecules. Then the filtrate of alcoholization was concentrated, the ethanol in the filtrate was evaporated, and the concentrated liquid was made up to 500 mL per 3.2 kg of Hippophae rhamnoides L. seed meal raw material to serve as the sample for the next enrichment treatment. A portion was dried and detected according to the established analysis method. The content of tryptamine alkaloids in the crude extract was 3.07%.

[0058] 2.2 Preliminary optimization of tryptamine alkaloid enrichment conditions in Hippophae rhamnoides L. seed meal

[0059] (1) Screening of tryptamine alkaloid enrichment materials in Hippophae rhamnoides L. seed meal

[0060] In order to investigate the enrichment effect of different enrichment materials on tryptamine alkaloids, a total of 3 kinds of macroporous adsorption resins, D101, AB-8 and DM301, were selected as screening materials. The volume of the resin was 100 mL, and the loading amount of the concentrated liquid was 10 mL (the concentrated liquid pH 6). The adsorption treatment was carried out respectively. Then 5 BV of water and 10% ethanol were used for elution respectively, and the eluate was collected. The 10% ethanol part was dried, and the content of tryptamine alkaloids was calculated.

[0061] (2) Effect of pH of extract concentrate on enrichment effect

[0062] The concentrated solution initially contains a high concentration of acetic acid, and the pH of the concentrated solution is adjusted by adding dry NaOH. The high concentration of acetic acid buffers the dramatic effect of NaOH on pH, allowing the pH to be adjusted stably in the near-neutral range. The pH of the concentrated solution is detected at any time using precision pH test paper, and the pH of the concentrated solution is adjusted to 2, 3, 4, 5, 6, 7, 8, 10, and 12, respectively. The volume of AB-8 macroporous resin is 100 mL, and the volume of the extraction concentrated solution is 10 mL for each adsorption. Each experiment is repeated three times. Then, 5 BV of water and 10% ethanol are used for elution, and the eluate is collected. The 10% ethanol fraction is dried, and the content of tryptamine alkaloids is determined.

[0063] (3) Effect of eluent ethanol concentration on enrichment

[0064] After studying the effect of pH of the concentrated solution on enrichment, pH 7 is determined as the sample concentration solution, and the effect of different ethanol concentrations on the enrichment of tryptamine alkaloids is investigated. The volume of AB-8 macroporous resin is 100 mL, and the volume of the extraction concentrated solution is 10 mL at pH 7. After adsorption of the concentrated solution, 5 BV of water is used for flushing, and then 5%, 10%, 15%, 20%, and 25% ethanol is used as eluent for elution, with a volume of 5 BV. The eluate is collected, dried, and the content of tryptamine alkaloids is determined.

[0065] (4) Effect of sample size on enrichment

[0066] Prepare 10, 20, 30, 40, and 50 mL of concentrated solution at pH 7, and the volume of resin is 100 mL. The concentrated solution is loaded, i.e., the sample size is 0.1, 0.2, 0.3, 0.4, and 0.5 BV, respectively. After adsorption, 5 BV of water and 10% ethanol are used for flushing, and the content of tryptamine alkaloids in the 10% ethanol fraction is determined after drying.

[0067] 2.3 Response surface experiment design

[0068] Based on the results of the above single-factor experiment, the sample size, sample solution pH, and eluent ethanol concentration are selected for process optimization. Using Design Expert 8.0 software, based on the principles of Box-Behnken Design, the variables are sample size (A), sample solution pH (B), and eluent ethanol concentration (C), and the response value is the content of product tryptamine alkaloids (Y). A three-factor, three-level response surface experiment is conducted to optimize the enrichment process of seabuckthorn seed meal tryptamine alkaloids, and the factors and levels are shown in Table 1.

[0069] Table 1 Factors and levels of response surface

[0070]

[0071] Regression analysis was performed on the measured experimental values to fit a second-order polynomial model. The optimal point within the experimental condition range was predicted by the model, and the optimized process was obtained. The results were verified according to the optimized conditions, and the actual content was compared with the predicted value to determine whether they were close.

[0072] 2.4 Scale-up experiment of the enrichment process of tryptamine alkaloids in seabuckthorn seed meal

[0073] According to the results of the response surface experiment, the preparation conditions were determined as follows: sample amount 0.34 BV, sample solution pH 7, and eluent ethanol concentration 10%. AB-8 macroporous adsorption resin was used to purify 500 mL of the concentrated solution prepared from 3.2 kg of seabuckthorn seed meal according to the method in item 2.1. The column filling volume was 2 L, the pH was adjusted to 7, and 5 BV of water and 10% ethanol were used for elution, respectively. The 10% ethanol fraction was collected, concentrated, and dried to obtain a dry powder, and the content of tryptamine alkaloids in the enriched product was detected.

[0074] 3 Experimental results

[0075] 3.1 Optimization of the enrichment conditions of tryptamine alkaloids in seabuckthorn seed meal

[0076] (1) Effect of different types of resins on the enrichment of tryptamine alkaloids

[0077] D101, AB-8, and DM301 macroporous resins were used to enrich the extract, respectively, as shown in Figure 1 . The content of tryptamine alkaloids in the D101 enriched product was the lowest, while the contents of tryptamine alkaloids in the AB-8 and DM301 enriched products were similar. Based on the universality and cost-effectiveness of AB-8 macroporous resin, AB-8 macroporous adsorption resin was finally selected as the material for further optimization and subsequent related experimental research.

[0078] (2) Effect of pH of the extract concentrate on the enrichment of tryptamine alkaloids

[0079] The effect of pH on the enrichment of tryptamine alkaloids is shown in Figure 2 . Within the pH range of 2-7, the product content gradually increased with the increase of pH, reached a maximum at a pH close to 7, and then decreased, showing high pH sensitivity. The reason may be that most tryptamine alkaloids have amphoteric groups, and excessive acid or alkali can easily ionize the molecules, leading to a decrease in lipophilicity and affecting the adsorption retention in the column.

[0080] (3) Effect of ethanol concentration of the eluent on the enrichment of tryptamine alkaloids

[0081] Because the lipophilicity and hydrogen bonding of tryptophan alkaloids vary significantly with pH, ​​their adsorption strength and elution also change. Therefore, preliminary studies on eluents were conducted. Figure 3 As shown, within the ethanol concentration range of 5% to 25%, the alkaloid content in the enriched product exhibits a trend of first increasing and then decreasing, with an inflection point between 5% and 15%. The content of tryptophan alkaloids reaches its highest level under elution with a 10% ethanol concentration.

[0082] (4) Effect of sample loading amount on enrichment effect of tryptamine alkaloids

[0083] from Figure 4 As can be seen, the loading amount has a relatively small effect on the content of tryptophan alkaloids in the enriched product. An inflection point appears between the loading amount of 0.2 and 0.4 BV, but the variation is slightly smaller. This may be due to the product content changes caused by the competition of various components of the extract for adsorption sites.

[0084] 3.2 Response surface optimization enrichment conditions and process scale-up

[0085] Response surface methodology experiments were conducted based on the preliminary optimization results, as shown in Table 2. Design Expert 8.0 was used to analyze and fit the results in Table 2, yielding the response surface model equation:

[0086] Y=28.67+2.53A-0.74B-0.88C+0.83AB-0.82AC+0.44BC-3.74A 2 -5.23B 2 -1.79C 2 .

[0087] Table 2 Results of Box-Behnken response surface methodology experiment

[0088]

[0089]

[0090] The results of the analysis of variance for this model are shown in Table 3.3. The model is highly significant overall (p<0.01), while the lack-of-fit term is not significant (p>0.05), indicating that the model is accurate and reliable and can be used for the optimization of the current process. A 2 The significance level was reached (p < 0.05), A and B 2 It reached a highly significant level (p<0.01).

[0091] Table 3. Results of Analysis of Variance

[0092]

[0093] The optimal enrichment process conditions predicted by the model are as follows: the loading amount is 0.34 BV, the loading solution pH is 6.83, and the eluent ethanol concentration is 8.3%. Under these conditions, the predicted content of tryptamine alkaloids can reach 29.30%. According to the actual situation, the optimized conditions are adjusted as follows: the loading amount is 0.34 BV, the loading solution pH is 7, and the eluent ethanol concentration is 8%. The experimental verification of the predicted results is carried out under these conditions, and the content of tryptamine alkaloids in the obtained enriched product is 28.99%, which is close to the predicted value.

[0094] 3.3 Scale-up experiment of the enrichment process of tryptamine alkaloids in seabuckthorn seed meal

[0095] Subsequently, the process was scaled up. In order to ensure the convenience of the process, appropriate adjustments were made in actual preparation. The final dry powder obtained by the preparation process in step 3.2 contains 25.71% of tryptamine alkaloids, and the yield is 2.31 g / kg. That is, about 2.31 g of enriched product with a content of 25.71% can be obtained from each kg of seabuckthorn seed meal. The obtained dry powder will be used for subsequent research on improving memory.

[0096] Example 2

[0097] 1. Instruments and materials

[0098] 1.1 Instruments

[0099] Agilent 1260 type high performance liquid chromatograph including four-element gradient pump, online vacuum degassing machine, DAD detector and automatic sampler (Agilent, USA), electronic analytical balance (Mettler-Toledo Instruments Co., Ltd.), pharmaceutical low-temperature ultrafine grinder (Jinan Tianfang Machinery Co., Ltd.), ultrapure water production system (Chengdu Youpu Technology Co., Ltd.), constant-temperature water bath (Shanghai Ailang Instruments Co., Ltd.), centrifuge (Shanghai Anting Scientific Instruments Factory).

[0100] 1.2 Materials

[0101] Seabuckthorn seed meal produced after supercritical CO2 fluid extraction of oil from seabuckthorn seeds was purchased from Qinghai Kangpu Biological Technology Co., Ltd. After drying, the seabuckthorn seed meal was ground for 15 min using a pharmaceutical low-temperature ultrafine grinder, and the powder was sieved through a 60-mesh sieve. The material was stored in a 4°C refrigerator for standby use.

[0102] 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harmine, N-acetyl-5-hydroxytryptamine were self-made in the laboratory. UP water was self-made in the laboratory, methanol (Hubei Fudon Science and Technology Co., Ltd., HPLC), formic acid (Chengdu Kolon Chemicals Co., Ltd., AR), pure water (Wahaha Group Co., Ltd.), trifluoroacetic acid (Shanghai McLean Biochemical Technology Co., Ltd., AR)

[0103] 2. Method results

[0104] 2.1 Chromatographic conditions

[0105] An Xcharge C18 chromatographic column (250 mm x 4.6 mm, 5 μm) was used, and the mobile phase was methanol (A)-0.15% trifluoroacetic acid aqueous solution (B) with gradient elution. Elution program (0-15 min, 5%-7% A; 15-20 min, 7% A; 20-50 min, 7%-35% A; 50-70 min, 35%-95% A; 70-80 min, 95%-5% A); column temperature 30 ℃, detection wavelength 280 nm, flow rate 1.0 mL / min.

[0106] 2.2 Preparation of solutions

[0107] 2.2.2 Mixed control solution one

[0108] An appropriate amount of 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harmine, N-acetyl-5-hydroxytryptamine reference substance was accurately weighed, diluted with 0.2% formic acid water to constant volume, and the concentrations were 0.0628, 0.0571, 0.0114, 0.0080, 0.0031, 0.0046 mg / mL, respectively. The reference substance chromatogram is shown in Figure 5 .

[0109] 2.2.3 Test solution

[0110] 1.000 g of seabuckthorn seed meal sample was accurately weighed into a conical flask, 20 mL of 2.5% acetic acid water was accurately added, heated at 75 ℃ water bath for 1 h, shaken uniformly, cooled, filtered, and diluted to 25 mL to obtain the filtrate, and then filtered through a 0.45 μm filter membrane to obtain the test solution. The test solution chromatogram is shown in Figure 6 .

[0111] 2.3 Linear relationship investigation

[0112] Take 2.2.1 in the mixed reference solution 1, 5, 10, 20, 40 μL, as in 2.1 chromatographic conditions for sampling, with the mass of the reference contained in the sample as the abscissa, the peak area as the ordinate, draw the standard curve, with the signal-to-noise ratio of 3:1 to calculate the detection limit, the signal-to-noise ratio of 10:1 to calculate the limit of quantification.

[0113] Table 4 of the method of establishing 6 kinds of tryptamine alkaloids analysis

[0114]

[0115] x: mg / mL y: peak area

[0116] 2.4 Precision test

[0117] Take 2.2.2 in the mixed reference solution two according to 2.1 chromatographic conditions for continuous sampling 6 times, 5 μL each time, finally calculated 5-hydroxytryptamine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, shepherdine, L-tryptophan, N-acetyl-5-hydroxytryptamine, harmine peak area RSD were 0.38%, 1.31%, 1.08%, 1.09%, 0.40%, 0.68%, indicating that the instrument precision is good.

[0118] 2.5 Stability test

[0119] Take the test solution at 0, 2, 4, 6, 8, 12, 16, 24, 48 h according to 2.1 chromatographic conditions, 10 μL of sample, finally calculated 5-hydroxytryptamine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, shepherdine, L-tryptophan, N-acetyl-5-hydroxytryptamine, harmine peak area RSD were 0.84%, 4.29%, 1.70%, 2.42%, 1.24%, 0.93%, indicating that the test solution is stable within 48 h.

[0120] 2.6 Reproducibility test

[0121] Take the same batch of sea buckthorn seed meal sample according to the method in item 2.2.3 to prepare 6 test sample solutions in parallel, and determine by injecting sample according to the chromatographic conditions in 2.1, with a sample injection amount of 10 μL. The peak area RSDs of 5-hydroxytryptamine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, shepherdine, L-tryptophan, N-acetyl-5-hydroxytryptamine, and harmane are 1.83%, 2.98%, 2.47%, 1.55%, 2.45%, and 1.28%, respectively.

[0122] 2.7 Sample injection recovery rate test

[0123] Precisely take 1 mL of the test sample solution with a known content, and precisely add 1 mL of 5-hydroxytryptamine (0.22 mg / mL), 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid (0.032 mg / mL), shepherdine (0.080 mg / mL), L-tryptophan (0.056 mg / mL), N-acetyl-5-hydroxytryptamine (0.021 mg / mL), and harmane (0.032 mg / mL) reference substance solutions, respectively. Inject sample according to the chromatographic conditions in 2.1, with a sample injection amount of 20 μL, and calculate the recovery rate. The average sample injection recovery rates of the respective compounds are 96.81%, 103.12%, 92.50%, 103.57%, 106.09%, and 112.5%, with RSDs of 1.54%, 1.78%, 2.56%, 1.21%, 2.03%, and 1.02%, respectively.

[0124] 2.8 Content determination of the sample

[0125] Take the sea buckthorn seed meal sample and prepare test sample solutions according to the method in item 2.2.3, and determine by injecting sample according to the chromatographic conditions in 2.1, with a sample injection amount of 10 μL. Calculate the content of the tryptamine alkaloids in the sample.

[0126] Table 5 Determination results of the content of tryptamine alkaloids in the sea buckthorn seed meal sample (mg / g, n=3)

[0127]

[0128]

[0129] In this study, HPLC-DAD method was used to simultaneously detect the contents of 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harman alkaline and N-acetyl-5-hydroxytryptamine for the first time. The preparation of sample solution was simple, and the determination results were accurate and reproducible, which could provide certain reference for the analysis and detection of tryptophan alkaloids in Hippophae rhamnoides L.

[0130] Screening of detection chromatographic conditions for comparative examples

[0131] The following are the liquid chromatograms of the sample from the reconfigured mixed standard under different conditions and different chromatographic conditions:

[0132] Mixed control solution two: accurately weigh 5-hydroxytryptamine, L-tryptophan, shepherdine, 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid, harman alkaline and N-acetyl-5-hydroxytryptamine control substances, dilute with 0.2% formic acid water to constant volume, and obtain the concentrations of 0.0376, 0.0552, 0.0353, 0.0382, 0.0259, 0.01765, and 0.01294 mg / mL, respectively.

[0133] Chromatographic condition 1: Xcharge C18 column, methanol A-water B: 0-15-20-50-70-80 min, 5%-7%-7%-35%-95%-5% A. See the chromatogram at Figure 7 As can be seen from the figure, only 2 components can be separated under this condition, and the detection of 6 tryptophan alkaloids cannot be realized.

[0134] Chromatographic condition 2: Kromosil C18 column, mobile phase methanol A-0.15 formic acid aqueous solution B: 0-15-20-50-70-80 min, 5%-7%-7%-35%-95%-5% A. See the chromatogram at Figure 8 As can be seen from the figure, only 4 components can be separated under this condition, and there are many detection interference peaks.

[0135] Chromatographic condition 3: Kromosil C18 column, mobile phase methanol A-0.15 formic acid aqueous solution B: 0-15-20-50-70-80 min, 5%-7%-7%-35%-95%-5% A. See the chromatogram at Figure 9 As can be seen from the figure, only 5 components can be separated under this condition.

[0136] Chromatography condition 4: Chromatography column Kromosil C18, mobile phase methanol A-0.15% trifluoroacetic acid aqueous solution B: 0-15-20-50-70-80 min, 5%-7%-7%-35%-95%-5% A. From Figure 10 It can be seen that only 5 components can be separated under this condition.

[0137] Chromatography condition 5: Chromatography column Xcharge C18, mobile phase methanol A-0.15% trifluoroacetic acid aqueous solution B: 0-15-40-45-50 min, 5%-20%-95%-95%-5% A. From Figure 11 It can be seen that the separation degree is not good under this condition.

[0138] Example 3

[0139] 1. Experimental materials, reagents and instruments

[0140] 1.1 Experimental reagents

[0141] The seabuckthorn seed meal tryptamine alkaloid enrichment part (HTA) was prepared in the third chapter, and the tryptamine alkaloid content was 25.71%; the seabuckthorn seed meal tryptamine alkaloid crude extract (HE) had a tryptamine alkaloid content of 3.07%; DL-4-chlorophenylalanine (purity 98%, Shanghai Maikelin Biochemical Technology Co., Ltd.); Estazolam tablets (Huazhong Pharmaceutical Co., Ltd.); Tween-80, NaHCO3 (National Pharmaceutical Group Chemical Reagent Co., Ltd.); carboxymethylcellulose sodium (Shanghai Yuanye Biological Technology Co., Ltd.); physiological saline and PBS buffer, Nissl staining kit and H&E staining kit (Wuhan Sivier Biological Technology Co., Ltd.); melatonin enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Biological Technology Co., Ltd.); 5-HT, DA, NA enzyme-linked immunosorbent assay kit, glutamate colorimetric assay kit and monoamine oxidase colorimetric assay kit (Hubei Elabscience Biotechnology Co., Ltd.).

[0142] 1.2 Experimental instruments

[0143] High-speed refrigerated centrifuge (Eppendorf 5430R, Eppendorf); multifunctional enzyme label (Epoch 2, Times Atlas Biological Technology Co., Ltd.); vortex mixer (Mixer 4K, Shengwo Biological Engineering (Shanghai) Co., Ltd.); microplate incubation oscillator (EB-9010, Jiangsu Haimen Qisilber Instrument Manufacturing Co., Ltd.); automatic snowflake ice maker (IMS-20, Changshu Xueke Electrical Appliance Co., Ltd.); pure water machine (Master-S15 UVF, Shanghai Hetai Instrument Co., Ltd.).

[0144] 2. Experimental method

[0145] 2.1 Establishment of animal model and drug administration

[0146] 72 SPF KM mice (half male and half female) (8 weeks old, average weight 30-40 g) were purchased from Beijing Sbiif Biological Technology Co., Ltd. Experimental Animal Co., Ltd. The mice were adaptively fed in the animal room for 1 week, 12 h light / 12 h dark, room temperature 23℃, humidity 60%±10%. All animals were free to eat. The normal group, model control group, positive drug group (estazolam, 3x10 －4 g / kg), HTA group (low dose group HTA-L, 100 mg / kg, high dose group HTA-H, 300 mg / kg), extract administration group (HE, 100 mg / kg), 12 in each group, half male and half female.

[0147] Modeling: Intraperitoneal injection of PCPA 450 mg / kg was used to induce sleep deprivation mouse model every day, and 1% Tween-80 was intraperitoneally injected in the normal group, a total of 10 days. At the end of modeling, it was observed that the model mice had dull fur, restlessness, irritability, and circadian rhythm regression, while the normal mice had bright fur, stable temperament, and obvious circadian rhythm.

[0148] Drug treatment: Drug treatment started the next day after modeling ended, and the administration method was gavage administration, which lasted for 8 days.

[0149] 2.2 Behavioral experiment

[0150] (1) Water maze

[0151] Place navigation experiment: It started from the 3rd day of administration and lasted for 5 days. First, the mice were put into the pool (without platform) to swim freely for 2 min to familiarize themselves with the maze environment. Each day was scheduled at a fixed time period, and each time period trained 4 times. At the beginning of training, the platform was placed in the corresponding quadrant, and the mouse was put into the pool from any of the four starting points on the pool wall, facing the pool wall, and the time for the mouse to find the platform (escape latency) was recorded. Each mouse was trained 4 times. The mouse was put into the water from the four different quadrants of the pool, respectively. When the mouse found the platform or failed to find the platform within 120 s (latency was recorded as 120 s), the experimenter took it onto the platform, and after resting on the platform for 15 s, the next test was performed. The average value of the latency of the mouse in 4 training times each day was taken as the learning score of the mouse on that day.

[0152] Spatial exploration experiment: It started the next day after the place navigation experiment and lasted for 1 day. The original platform was removed, and the mouse was put into the water at a random entry point, and all mice had to be the same entry point. Each mouse explored for 2 min, and the exploration activities of each mouse were recorded using a free video recording system. The target distance (movement distance in the quadrant where the original platform was located), target time (time spent in the quadrant where the original platform was located), total distance, average speed, and platform crossing times of each mouse were recorded.

[0153] (2) Y maze alternation behavior test

[0154] Y maze alternation behavior test was performed the day after the water maze test for 1 day. The Y maze apparatus was made of three opaque plastic arms (labeled I, II, and III, respectively) with a 120° angle between the two arms. The mouse was gently placed in the center of the maze and allowed to explore freely for 8 min. The criterion for entry into each arm was complete entry of all four limbs. A sequence of three different arms (e.g., I II III, I III II, II III I, II I III, III I II, III II I) was an alternation. The calculation of alternation score was the total number of alternations divided by the total number of choices minus 2 multiplied by 100, as shown in the equation: Spontaneous alternation rate (%) = [(number of spontaneous alternations) / (total number of arm entries - 2)] x 100. The maze was wiped with 75% alcohol and allowed to evaporate completely between the two mice to ensure that the smell did not interfere with the experiment.

[0155] 2.3 Animal sampling

[0156] The day after the behavioral test, 4 mice from each group were randomly selected, half male and half female, anesthetized, and each mouse was perfused with 50 mL of pre-cooled physiological saline through the left ventricle. At this time, the mouse organs gradually lost their color as the perfusion progressed, the lungs turned white, and the liver tissue turned yellow. Then, 4% paraformaldehyde solution was perfused, and the whole brain was obtained by decapitation. The whole brain was immersed in 4% paraformaldehyde solution and stored in a 4°C refrigerator for future use.

[0157] The remaining mice were sacrificed by decapitation after blood was taken from the orbital plexus, and then the whole brain was obtained by decapitation. The hippocampal tissue was quickly stripped on ice, and the hippocampal tissue and the rest of the brain were collected separately and stored in a -80°C refrigerator for future use. The blood samples were left to stand at room temperature for 2 h, and then centrifuged at 3000 rpm for 20 min at room temperature. The supernatant was stored at -80°C.

[0158] 2.4 Histopathology experiment

[0159] Slicing: The whole brain tissue in a good fixed state was taken out of the fixative and trimmed with a scalpel in a fume hood to make the target tissue flat. The trimmed tissue was dehydrated with gradient alcohol and immersed in wax. The wax- immersed tissue was then embedded in an embedding machine. The embedded tissue was cut into sections on a paraffin sectioning machine, with a thickness of 4 μm.

[0160] HE staining: The paraffin section was dewaxed to water, and then treated with high-definition constant staining reagent for 1 min, stained with hematoxylin solution for 3-5 min, washed with water, differentiated with differentiation solution, washed with water again, and returned to blue. After washing with water again, dehydrated with 95% alcohol for 1 min, and stained with eosin solution for 15 s. Finally, after gradient dehydration, the section was mounted with neutral balsam, observed under an optical microscope, and images were collected.

[0161] Nissl staining: The paraffin section was dewaxed to water, and then colored with staining solution for 2-5 min, washed with water, and terminated by washing with 0.1% glacial acetic acid after differentiation, and the degree of differentiation was controlled under a microscope. After washing with water, the section was dried and then transparentized in clean xylene for 10 min, mounted with neutral balsam, observed under an optical microscope, and images were collected.

[0162] 2.5 Detection of biochemical indicators

[0163] (1) Melatonin (MT) level in serum

[0164] The content of MT in serum was detected by ELISA (competitive method), and the operation steps were as follows

[0165] The kit was equilibrated at room temperature for more than 30 min. Standard solutions of different concentrations were prepared. Blank wells (only color reagent and termination solution were added, and the rest was not added), zero wells (50 μL of standard diluent was added, and no sample or standard was added, and the rest was the same as the standard well and the sample well), standard wells, and sample wells were set. 50 μL of the corresponding standard diluent, standard solution, and serum sample was added to each well of the zero well, standard well, and sample well, respectively, and then 50 μL of biotin-labeled recognition antigen was added to each well, and incubated at 37°C for 30 min. Discard the liquid in the wells, wash the plate 5 times to remove the unbound biotin antigen, and then add avidin-HRP 50 μL to each well, and incubate at 37°C for 30 min. Discard the liquid in the wells, wash the plate 5 times, and dry. After adding color reagent to all wells including blank wells for color development for 10 min, add termination solution to terminate the reaction, mix well, set the zero well with the blank well, and measure the absorbance value of each well at 450 nm. The corresponding concentration of the sample was calculated by the standard curve.

[0166] (2) Neurotransmitter level in hippocampus

[0167] The monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NA), and dopamine (DA) in the hippocampus were determined by ELISA, and glutamate was determined by colorimetry.

[0168] a. Tissue homogenate: After the hippocampus tissue was taken out from the freezer, it was balanced at 4°C. Then the hippocampus tissue was weighed and mixed with PBS (1 g:9 mL) at the corresponding volume. The mixture was homogenized on ice. The homogenate was centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was used for analysis.

[0169] b. The process of detecting monoamine neurotransmitters by ELISA (competitive method) was the same, and the operation was as follows:

[0170] The kit was pre-equilibrated to room temperature. Standard solutions of different concentrations were prepared. Standard wells, blank wells (50 μL of standard & sample diluent was added, no sample or standard was added, and the remaining steps were the same as other wells), and sample wells were set up, and the corresponding standard solution, standard & sample diluent, and sample (the supernatant of hippocampus tissue homogenate was diluted by the diluent, 5-HT and NA were diluted 1:1, and DA was not diluted) were added. Immediately, 50 μL of biotin-labeled monoclonal antibody was added. The film was incubated at 37°C for 45 min. The liquid in the wells was discarded, and the plate was washed 3 times and dried. 100 μL of avidin-HRP enzyme was added to each well, and the film was incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plate was washed 5 times and dried. 90 μL of color developing substrate solution was added to each well, and the incubation was continued for 15 min. 50 μL of stop solution was added, and the absorbance value of each well was measured immediately at 450 nm. The content of the sample was calculated by the standard curve.

[0171] c. The level of glutamate (GLU) in the hippocampus tissue was determined by colorimetry, and the steps were as follows:

[0172] Before detection, all reagents were equilibrated to room temperature. Standard solutions of different concentrations and reaction working solutions (a mixture containing enzymes, substrates, and color developing agents) were prepared. Standard wells and sample determination wells were set up, and 30 μL of the corresponding standard solution and sample solution (supernatant of hippocampus tissue homogenate) were added to each well. 60 μL of reaction working solution was added to each well. Incubation was performed at 37°C for 20 min, and the absorbance value of each well was measured at 450 nm. The content of the sample was calculated according to the standard curve.

[0173] (3) Colorimetric determination of monoamine oxidase (MAO) activity in brain tissue

[0174] a. Sample processing

[0175] Animal tissue sample: 0.1-0.5g sample was added to reagent one working solution according to the ratio of weight (g): volume (mL) = 1:9 for homogenization. Centrifugation at 1000xg for 10min at 4℃, and the supernatant was taken. The supernatant was centrifuged at 10000xg for 30min at 4℃, and the supernatant was discarded. 1mL reagent two was added, mixed well, and centrifuged at 16000xg for 40min at 4℃, and the supernatant was discarded. 1mL reagent three working solution was added, mixed well, and stored in an ice box for use. (Convert the unit of centrifugal force to rpm, and the unit of centrifugation in the full text should be uniform)

[0176] b. Determination of total protein concentration of sample by BCA method

[0177] The supernatant after homogenization of reagent one working solution was determined for total protein concentration according to the BCA protein concentration kit instruction.

[0178] c. The final sample obtained in step a was used as the determination sample, and the monoamine oxidase (MAO) activity of each sample was detected according to the kit instruction. The enzyme activity was defined as the amount of enzyme required to catalyze the production of 1nmol of p-dimethylaminobenzaldehyde (generated from the reaction substrate 4-dimethylaminobenzylamine under the catalysis of MAO) per minute per gram of tissue protein at 37℃ as an activity unit (U / gprot).

[0179] 3. Results and analysis

[0180] 3.1 Effect of tryptamine alkaloids in seabuckthorn seed meal on the positioning and navigation memory behavior of mice

[0181] From the behavior trajectory of mice every day Figure 12 and the related analysis results, it can be seen that during the 5-day training period, the latency of all mice was shortened to find the hidden platform. Compared with the normal group, the latency of mice in the model group was significantly prolonged, with significant or extremely significant difference (p<0.05 or p<0.01), indicating that the learning and memory ability of mice was reduced. After treatment, the latency of mice in the high-dose group and the extract control group was significantly different from that in the model group (p<0.05), and the difference between the low-dose group and the positive drug group and the model group was extremely significant (p<0.01). (The identification of each group in the picture is normal group Con, model group Mod, positive drug group Pos, extract HE, low-dose tryptamine group HTAL, and high-dose tryptamine group HTAH, in the order of normal group, model group, positive drug group, extract group, low-dose serotonin group and high-dose serotonin group).

[0182] 3.2 Effect of tryptamine alkaloids in seabuckthorn seed meal on the spatial exploration memory behavior of mice

[0183] As Figure 13The figures show the trajectory maps, heatmaps, and corresponding statistics of the number of platform crossings and the distance to the target quadrant / total distance for each group of mice in the spatial exploration experiment. In the spatial exploration experiment, the number of platform crossings by the model group mice was significantly different from that of the normal group (p < 0.05), while there was no significant difference between the crude extraction group and the model control group. The high-dose group of tryptamine alkaloids was significantly higher than that of the model control group (p < 0.05), and the positive control group and the low-dose group of tryptamine alkaloids were significantly higher than those of the model group (p < 0.01).

[0184] Analysis using the target distance / total distance ratio showed that the model control group was significantly lower than the normal group (p<0.01), the crude extract group had no significant difference compared with the model group, the high-dose tryptamine alkaloid group was significantly higher than the model control group (p<0.05), and the positive drug group and the low-dose tryptamine alkaloid group were significantly different from the model group (p<0.01).

[0185] 3.3 Effects of tryptophan alkaloids in sea buckthorn seed meal on memory exploration behavior in mice during Y-maze alternation.

[0186] like Figure 14 As shown, in the spontaneous alternation behavior experiment in the Y-maze, the spontaneous alternation rate of the model group mice was significantly different from that of the control group (p<0.05). The extract group and the positive control group were significantly higher than the model control group (p<0.05), and the high-dose and low-dose tryptamine alkaloid groups were significantly higher than the model control group (p<0.01). These findings indicate that tryptamine alkaloids can significantly improve the exploratory memory ability of mice in the Y-maze.

[0187] 3.4 Effects of tryptophan alkaloids in sea buckthorn seed meal on cell morphology in mouse hippocampus

[0188] (1) H&E staining

[0189] H&E staining results are as follows Figure 15 As shown, in the normal group, a small number of neuronal shrinkages (black arrows) were observed in the CA3 region of the hippocampus, with no obvious inflammatory cell infiltration. In the model group, a large number of neuronal shrinkages (black arrows) were observed in the CA1, CA3, CA4, and DG regions. After treatment with the extract and high-dose tryptamine alkaloids, a large number of neuronal shrinkages (black arrows) were still observed in the CA1, CA3, CA4, and DG regions. After intervention with the positive control drug and low-dose tryptamine alkaloids, the neuronal shrinkages (black arrows) were significantly reduced. The effect of the low-dose group was comparable to that of the positive control drug and was superior to that of the high-dose group.

[0190] (2) Nissl staining

[0191] Nissl staining results as follows Figure 16In the model group, the number of Nissl bodies and neurons in the hippocampus were significantly less than those in the normal group. Furthermore, the neurons were disorganized, with enlarged intercellular spaces and numerous shrunken, irregularly shaped cell bodies (black arrows), indicating damage to both neuronal morphology and structure. After treatment with positive control drugs and tryptophan alkaloids, the number of Nissl bodies and neurons significantly increased to varying degrees. The cell bodies were fuller and regularly round or oval, suggesting some improvement in neuronal damage and a certain degree of repair effect on the damaged neuronal morphology and structure.

[0192] 3.5 Effects of tryptophan alkaloids in sea buckthorn seed meal on melatonin levels in mouse serum

[0193] like Figure 17 As shown, compared with the normal group, the serum melatonin level in the model group mice was significantly lower (p<0.01). The extract group had a slightly higher level than the model control group, but the difference was not statistically significant (p>0.01). The melatonin levels in the low-dose tryptamine alkaloid group and the positive control group were significantly higher than those in the model control group (p<0.05), while the melatonin level in the high-dose tryptamine alkaloid group was extremely significantly higher than that in the model control group (p<0.01). These results indicate that tryptamine alkaloid treatment can restore melatonin levels in mice to a certain extent.

[0194] 3.6 Effects of tryptophan alkaloids in sea buckthorn seed meal on neurotransmitters in the hippocampus of mice

[0195] like Figure 18 As shown, compared with the normal group, the 5-HT content in the hippocampus of the model group mice was significantly different (p<0.01). Although the 5-HT level in the hippocampus of the extract group mice was increased to some extent, it was not statistically significant. The positive drug group was significantly higher than the model control group (p<0.05), and the two dose groups of tryptophan alkaloids were significantly higher than the model control group (p<0.01).

[0196] Compared with the normal group, the DA level in the hippocampus of the model group mice was significantly decreased (p<0.01), while the DA level in the extract group and the positive hippocampus was increased to some extent, but not statistically significant. The levels of tryptophan alkaloids in both dosage groups were significantly higher than those in the model group (p<0.01).

[0197] Compared with the normal group, the NA level in the hippocampus of mice in the model group was significantly increased (p<0.05). The NA level in the hippocampus of mice in the extract group and the positive drug group was increased to some extent, but not statistically significant. The levels of tryptophan alkaloids in both dosage groups were significantly higher than those in the model group (p<0.01).

[0198] Compared with the normal group, the GLU level in the hippocampus of mice in the model group was significantly lower than that in the normal group (p<0.05). The GLU level in the hippocampus of mice in the extract, positive control drug, and low-dose tryptamine alkaloid groups was significantly increased (p<0.01) and comparable to that in the normal group. However, the GLU level in the hippocampus of mice in the high-dose tryptamine alkaloid group showed a decreasing trend, and the difference was significant compared with the model group (p<0.05).

[0199] 3.7 Effects of tryptophan alkaloids in sea buckthorn seed meal on monoamine oxidase (MAO) in brain tissue

[0200] like Figure 19 As shown, compared with the normal group, the MAO level in the mouse brain tissue increased, but the difference was not statistically significant (p>0.05). The extract, positive control group, and high-dose tryptophan alkaloid group showed some reduction compared with the model group, but the difference was not statistically significant. The low-dose treatment group showed a significant reduction compared with the model group (p<0.05).

[0201] 4. Analysis and Discussion

[0202] Serotonin (5-HT) is an important neurotransmitter in animals, highly correlated with sleep and memory. In the central nervous system, HT synthesis mainly occurs within neurons, using tryptophan as a precursor. Tryptophan is converted to 5-hydroxytryptophan under the catalysis of tryptophanase, and further decarboxylation yields HT. In this process, tryptophan hydroxylase acts as the rate-limiting enzyme. p-Chlorophenylalanine (PCPA) is a HT synthesis inhibitor that irreversibly inhibits tryptophan hydroxylase, thus blocking HT synthesis. Ultimately, the body experiences insomnia due to HT depletion. Therefore, PCPA is often used to establish mouse sleep deprivation models.

[0203] 4.1 Improvement of memory behavior in sleep-deprived mice by tryptophan alkaloids in sea buckthorn seed meal

[0204] The experimental results show that, compared with the extract group, tryptophan alkaloids significantly improved memory, indicating that increasing the content of tryptophan alkaloids can provide a better memory improvement effect. Meanwhile, during the experiment, mice in the high-dose group showed a decrease in motor tendency compared to other groups, which was more pronounced in the water maze test.

[0205] 4.2 Effects of tryptophan alkaloids in sea buckthorn seed meal on hippocampal cell morphology

[0206] From the H&E staining and Nissl staining results of each group, sleep deprivation caused damage to the neurons, which was more obvious in the changes in cell morphology, and the number of Nissl bodies decreased. In the normal group, the cell body was full and the cell morphology was normal. In the drug administration groups, the cell morphology and the number of Nissl bodies in the low-dose administration group and the eszopiclone positive control group recovered most obviously, while the recovery of neurons in the high-dose administration group and the extract administration control group was slightly worse. In summary, within a certain dose range, the enriched product showed a good promoting effect on the recovery of neurons, but too high a dose might have a negative effect on the neuronal tissue.

[0207] 4.3 Effect of tryptamine alkaloids in seabuckthorn seed meal on melatonin in mouse plasma

[0208] Melatonin is closely related to biological rhythms, and sleep deprivation in mice leads to rhythm disorders, so the melatonin content in the model group mice is significantly lower than that in the normal mice. After drug intervention, the positive drug and tryptamine alkaloids increase the content of melatonin in the hippocampus of mice, but the extract drug does not show a significant increase. This shows that tryptamine alkaloids in seabuckthorn can increase the content of melatonin in sleep-deprived mice to some extent and improve the sleep rhythm of mice.

[0209] 4.4 Effect of tryptamine alkaloids in seabuckthorn seed meal on the level of neurotransmitters in the hippocampus of mice

[0210] (1) Monoamine neurotransmitters

[0211] The 5-HT level in the hippocampus of the model group mice was significantly lower than that in the normal group, and after drug intervention, the 5-HT levels in the hippocampus of the positive drug and tryptamine alkaloid groups were significantly improved. Moreover, the 5-HT levels in the positive control group and the low-dose administration group were similar to those in the normal group, while the 5-HT level in the high-dose administration group was even much higher than that in the other groups. The results of DA level were similar to those of 5-HT, the DA level of the model group was significantly lower. Although there was no significant difference in the positive control group, it was slightly higher than that in the model group, and the effect of tryptamine alkaloids on the DA level of mice was significantly greater than that of the positive drug eszopiclone. The DA levels of the two dose groups were significantly improved, and the DA level of the high-dose group was significantly higher than that of any other group. On the effect of NA level, the mice showed different results, and the NA content of the model group mice was higher than that of the normal mice.

[0212] From the results, it can be seen that sleep deprivation with PCPA affects the level of monoamine neurotransmitters in mice, and then affects the memory ability of mice. Estazolam can increase the level of 5-HT and DA in mice, but has no obvious effect on the level of NA. The effect of tryptamine alkaloids in sea buckthorn seed meal on the level of monoamine neurotransmitters in the hippocampus of mice is extremely significant. It can greatly increase the level of monoamine neurotransmitters including 5-HT, DA and NA, and its effect on improving memory may be related to this. However, it is also worth noting that excessive medication may lead to excessive increase of the level of monoamine neurotransmitters, and choosing the right dose is a key problem.

[0213] (2) Glutamate

[0214] The level of glutamate in the hippocampus of model mice is significantly lower than that of normal mice. Except for the high-dose group, the glutamate level of other drug groups shows a reversal. This may prove that positive drugs such as estazolam and tryptamine alkaloids can increase the level of glutamate in mice to a certain extent, and then affect memory. The high-dose group shows a completely opposite trend to the low-dose group. Glutamate is an excitatory neurotransmitter in the brain of living organisms, responsible for regulating the excitability of the nervous system. The decrease of glutamate level in high-dose group mice corresponds to the abnormal performance in the behavioral experiment. The decrease of neural excitability may be related to the decrease of motor tendency in high-dose group mice. This shows that higher doses of tryptamine alkaloids in sea buckthorn seed meal may have an inhibitory effect on neural excitability. Moreover, the high-dose group of mice showed excessively high levels of monoamine neurotransmitters, which may have inhibited glutamate. From the experimental results, the glutamate level of the tryptamine alkaloid-rich fraction in sea buckthorn seed meal is promoted at low doses, but the inhibitory effect on glutamate level is stronger at high doses, which may be related to the excessively high level of monoamine neurotransmitters.

[0215] The results show that there is no significant difference in the activity of monoamine oxidase in the brain tissue of model mice and normal mice, which indicates that PCPA-induced sleep deprivation may have little effect on the activity of monoamine oxidase in mice. Among the four treatment groups, only the low-dose treatment group shows a significant inhibition of MAO, which indicates that tryptamine alkaloids in sea buckthorn seed meal can inhibit the activity of MAO at a certain concentration, which may promote the increase of the level of monoamine neurotransmitters.

[0216] The MAO activity level of the high-dose administration treatment group was significantly higher than that of the low-dose administration treatment group, which might be related to the excessive level of monoamine neurotransmitters, or might be explained by the mouse's own regulation, that is, the excessive level of monoamine neurotransmitters led to the feedback increase of MAO level to cope with the neurotransmitter imbalance caused by excessive monoamine neurotransmitters, which also showed that the drug intervention did not inhibit the ability to regulate MAO in the mouse brain tissue. At the same time, the slightly special situation of the high-dose group might also imply that the effect of seabuckthorn seed meal tryptamine alkaloid enrichment product on the mouse brain neurotransmitter level cannot be explained only by the MAO-related mechanism, but also involves other mechanisms that affect neurotransmitter levels.

[0217] Summary:

[0218] After sleep deprivation induced by PCPA, the spatial memory ability of mice was impaired. Compared with normal mice, hippocampal neuron cell damage increased, but no obvious inflammatory symptoms occurred. At the same time, sleep deprivation led to the disturbance of neurotransmitter levels in mice, including the decrease of 5-HT, DA and glutamate, and the increase of NA level. Moreover, the rhythm of mice was also disturbed, which was manifested as the decrease of melatonin level. After the intervention of tryptamine alkaloids in seabuckthorn seed meal, the melatonin level in the serum of mice was increased, and the neurotransmitter level in the hippocampus was improved, thereby improving the memory of sleep-deprived mice. The effect of seabuckthorn seed meal tryptamine alkaloids on neurotransmitters was strong, especially the improvement of monoamine neurotransmitter levels including 5-hydroxytryptamine, norepinephrine and dopamine. At a higher dose, the level of monoamine neurotransmitters was too high, which would inhibit the excitability of nerves. At a certain dose, it could inhibit the activity of monoamine oxidase in the hippocampus of mice, thereby affecting the metabolism of monoamine neurotransmitters in mice. In summary, seabuckthorn seed meal tryptamine alkaloids can promote the sleep rhythm and memory improvement of sleep-deprived mice, but the optimal drug dose and its specific mechanism of action need further study.

Claims

1. The use of sea buckthorn seed meal extract in the preparation of products for improving memory, wherein the sea buckthorn seed meal extract is prepared by the following method: (1) Seabuckthorn seed meal, acetic acid water, extraction, alcohol precipitation, and solvent removal from the filtrate to obtain crude extract; (2) The crude extract was eluted with macroporous adsorption resin, followed by water and 10% v / v ethanol. The ethanol-eluted portion was collected, and the solvent was removed to obtain the sea buckthorn seed meal extract.

2. The use according to claim 1, characterized in that, In step (1), the concentration of acetic acid water is 2.5%; ethanol is added until the alcohol content reaches 80% v / v for alcohol precipitation.

3. The use according to claim 1, characterized in that, In step (2), AB-8 macroporous adsorption resin is selected.

4. The use according to claim 1, characterized in that, The product that improves memory is one that increases the levels of monoamine neurotransmitters and glutamate in tissues.

5. The use according to claim 4, characterized in that, The monoamine neurotransmitters mentioned are serotonin and dopamine.

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