Preparation method and application of diaphragma juglandis extract

Abstract

The invention relates to the field of natural product extraction, and particularly discloses a preparation method and application of a diaphragma juglandis extract. The preparation method comprises the following steps: crushing diaphragma juglandis, extracting with water, heating, evaporating and concentrating filtrate at the temperature of 60-70 DEG C, adding maltodextrin and sodium chloride into the concentrated solution, drying by adopting a cyclone separation spray dryer, and granulating to obtain diaphragma juglandis extract particles. The prepared diaphragma juglandis fructus extract particles can be mixed with a mulberry leaf extract, a bitter gourd extract, a radix puerariae extract, a Chinese yam extract and isomaltooligosacharide to prepare traditional Chinese medicine particles with the sleep assisting effect, and an application basis is provided for comprehensive utilization of diaphragma juglandis fructus.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product separation and extraction, and in particular to a preparation method and application of a walnut pith wood extract Background Art

[0002] Walnut pith wood refers to the woody septum of the ovary chamber of the walnut fruit. Walnut pith wood accounts for about 5% of the total weight of walnuts and is rich in bioactive substances such as flavonoids, polysaccharides, polyphenols, and minerals such as calcium, zinc, and iron. Pith wood tastes bitter, astringent, and has a neutral nature. It enters the spleen and kidney meridians and can be used as a traditional Chinese medicine.

[0003] At present, due to the low efficiency of separation and extraction of walnut pith wood, the comprehensive utilization rate of walnut pith wood is low, resulting in a waste of resources. By researching and developing functional products of walnut pith wood, turning waste into treasure, increasing the added value of raw materials, and promoting the utilization of walnut pith wood, it is of great significance to promote the healthy development of the walnut industry. Summary of the invention

[0004] The invention provides a preparation method of a walnut pith wood extract and application thereof, which solves the problems of low extraction rate and low comprehensive utilization rate of effective components in the walnut pith wood.

[0005] The present invention adopts the following technical scheme: a method for preparing a walnut pith wood extract, comprising the following steps:

[0006] (1) Extraction: grind the walnut pith wood and pass it through a 20-mesh sieve, add deionized water 8-12 times the weight of the walnut pith wood, reflux extract at 50-60° C. for 1-1.5 h, filter, extract twice, and combine the filtrate;

[0007] (2) Separation: The solution was decanted in a 0.45 μm plate and frame filter;

[0008] (3) Concentration: The filtrate is heated to 60-70°C for evaporation and concentration until the relative density of the concentrated solution reaches 1.05-1.07;

[0009] (4) spray drying: adding maltodextrin and sodium chloride to the concentrated solution in step (3), drying in a cyclone separation spray dryer, setting the air inlet temperature to 160-170° C., the air outlet temperature to 85° C., and the negative pressure in the dryer cavity to -30 to -40 Pa, to obtain walnut wood extract powder;

[0010] (5) Granulation: The walnut wood extract powder obtained by spray drying in step (4) is dry granulated, lubricant magnesium stearate and flow aid micro-powder silica gel are added, and the roller pressure of the granulator is set to 1.5 MPa and the roller speed is set to 15 Hz to prepare walnut wood extract particles.

[0011] Furthermore, in step (4), the amount of maltodextrin added is 8-10% of the mass of the concentrated solution, and the amount of sodium chloride added is 1-3% of the mass of the concentrated solution.

[0012] Furthermore, the product yield after spray drying in step (4) is 95%.

[0013] Furthermore, the added amount of magnesium stearate is 0.8-1.0% of the mass of the walnut wood extract powder, and the added amount of micro-powder silica gel is 0.5-1.0% of the mass of the walnut wood extract powder.

[0014] The application of walnut wood extract is used to prepare Chinese medicine granules with the effect of assisting sleep.

[0015] The components of the Chinese herbal granules with the function of assisting sleep include, by weight, 40-45 parts of walnut wood extract, 8-10 parts of mulberry leaf extract, 8-10 parts of bitter melon extract, 12-15 parts of kudzu root extract, 8-10 parts of yam extract and 8-10 parts of isomaltooligosaccharide.

[0016] In the present invention, the extract of the radix strychnifoliae is rich in polyphenols and flavonoids, has the effects of clearing away heat and detoxifying and nourishing, can resist inflammation and improve immunity, and is the main active ingredient in the formula; the mulberry leaf extract has good anti-inflammatory activity; the pueraria root is rich in puerarin, soy isoflavones, peanut and other ingredients, has the effects of promoting body fluid and stopping diarrhea, and is often used to treat spleen deficiency diarrhea, damp-heat diarrhea and other diseases; oligosaccharide is a prebiotic and can effectively regulate intestinal flora.

[0017] The extraction method of the invention is simple and the steps are easy to operate. After concentration and spray drying, the product yield is greater than 95%, the extract has high purity and good activity. Experiments have confirmed that the walnut wood extract can significantly improve the movement speed of mice and prolong the sleep time of mice. Zebrafish experiments have proved that the walnut wood extract can significantly improve the bottom-dwelling behavior of zebrafish, enhance its desire to explore, and improve its lurking and dark-moving behaviors. Comprehensive analysis shows that the walnut wood extract has a good effect of improving sleep, and provides an application basis for the comprehensive utilization of the walnut wood extract. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution will be clearly and completely described below in conjunction with specific embodiments.

[0019] Embodiment 1: A method for preparing a walnut core wood extract, comprising the following steps:

[0020] (1) Extraction: The walnut core wood was crushed and passed through a 20-mesh sieve, and deionized water 8 times the weight of the walnut core wood was added and refluxed at 55°C for 0.5 h, filtered, extracted twice, and the filtrates were combined;

[0021] (2) Separation: remove the solution through a 0.45 μm plate and frame filter and take the clear solution;

[0022] (3) Concentration: Take the clear liquid after plate and frame filtration, concentrate at 60°C, and stop when the relative density reaches 1.05;

[0023] (4) spray drying: adding maltodextrin and sodium chloride to the concentrated solution in step (3), wherein the amount of maltodextrin added is 10% of the mass of the concentrated solution, and the amount of sodium chloride added is 1% of the mass of the concentrated solution; using a cyclone separation spray dryer, setting the inlet air temperature to 160° C., the outlet air temperature to 85° C., and the negative pressure in the dryer cavity to -30 Pa; the product yield after spray drying is 95%, and walnut wood extract powder is obtained;

[0024] (5) Granulation: The walnut wood extract powder obtained after spray drying in step (4) is dry granulated, and a lubricant, magnesium stearate, and a flow aid, micro-powder silica gel, are added. The amount of magnesium stearate added is 0.8% of the mass of the walnut wood extract powder, and the amount of micro-powder silica gel added is 0.5% of the mass of the walnut wood extract powder. The roller pressure of the granulator is set to 1.5 MPa, and the roller speed is set to 15 Hz.

[0025] The forming rate of the walnut wood extract particles is 72.5%, and the particle friability is 63.4%.

[0026] Embodiment 2: A method for preparing a walnut core wood extract, comprising the following steps:

[0027] (1) Extraction: The walnut core wood was crushed and passed through a 20-mesh sieve, and deionized water (12 times the weight of the walnut core wood) was added and refluxed at 60°C for 0.5 h, filtered, extracted twice, and the filtrates were combined;

[0028] (2) Separation: remove the solution through a 0.45 μm plate and frame filter and take the clear solution;

[0029] (3) Concentration: Take the clear liquid after plate and frame filtration, concentrate at 70°C, and stop when the relative density reaches 1.05-1.07;

[0030] (4) spray drying: adding maltodextrin and sodium chloride to the concentrated solution in step (3), wherein the amount of maltodextrin added is 8% of the mass of the concentrated solution, and the amount of sodium chloride added is 3% of the mass of the concentrated solution; using a cyclone separation spray dryer, setting the inlet air temperature to 170° C., the outlet air temperature to 85° C., and the negative pressure in the dryer cavity to -40 Pa; the product yield after spray drying is 95%, and walnut wood extract powder is obtained;

[0031] (5) Granulation: The walnut wood extract powder obtained after spray drying in step (4) is dry granulated, and a lubricant magnesium stearate and a flow aid micro powder silica gel are added, wherein the amount of magnesium stearate added is 1.0% of the mass of the walnut wood extract powder, and the amount of micro powder silica gel added is 1.0% of the mass of the walnut wood extract powder. The roller pressure of the granulator is set to 2.5 MPa, and the roller speed is set to 9 Hz.

[0032] Example 3: A Chinese herbal granule with the effect of assisting sleep, wherein the components, by weight, include 45 parts of walnut wood extract, 10 parts of mulberry leaf extract, 10 parts of bitter melon extract, 15 parts of kudzu root extract, 10 parts of yam extract and 10 parts of isomaltooligosaccharide.

[0033] The preparation process includes the following steps:

[0034] (1) Raw material pretreatment: accurately weigh each component according to its weight, crush some agglomerated or crystallized raw materials, and ensure that all raw materials can pass through an 80-mesh sieve;

[0035] (2) Mixing: Put the processed raw materials into a three-dimensional mixer, adjust the mixer speed to 20 Hz / min, and mix for 20 min. Check whether the materials are fully mixed and whether there are color spots to obtain Chinese herbal granules with sleep-aiding effect.

[0036] Example 4: Component Analysis of Walnut Pyrite Extract Using UPLC-MS

[0037] (1) Sample processing

[0038] Accurately weigh 50 g of the crushed spatholobi, add 500 mL of pure water (g: mL), extract under hot reflux for 1 h, separate the filtrate, extract the residue again according to the above method and separate the filtrate, combine the two filtrates, rotary evaporate, and freeze-dry to obtain spatholobi water extract powder;

[0039] (2) Sample preparation

[0040] Accurately weigh 0.1 g of the extract powder and add 1 mL of water to dissolve it. Centrifuge at 4°C and 12,000 r / min for 10 min. Take the supernatant and place it in an injection vial for later use.

[0041] (3) Chromatographic and mass spectrometry conditions

[0042] Liquid phase conditions: chromatographic column: Hyperil Gold AQ (100×2.1mm, 1.9μm); column temperature: 35℃, injection volume: 2μL. Mobile phase: 0.1% formic acid solution (A)-0.1% formic acid acetonitrile (B), gradient elution: 0~3min (95%~75% B); 3~13min 75%~20% B); 13~23min (25%~5% B); 23~24min (5~95% B); 24~25min (95% B); flow rate 0.3mL min-1.

[0043] Mass spectrometry conditions: acquisition mode is positive and negative ion mode; scanning range m / z 100-1500, resolution: 120000; ESI source conditions: ion source temperature 350℃; positive and negative ion mode voltages are 4 and 3 kV respectively; sheath gas flow rate: 45 Arb; auxiliary gas flow rate: 10 Arb; purge gas flow rate: 0 Arb.

[0044] The results of component analysis in the walnut wood extract are shown in Table 1.

[0045] Table 1 Chemical composition analysis results of walnut wood extract

[0046]

[0047] As shown in Table 1, the specific components of the walnut wood were analyzed and identified by UPLC-MS technology, and a total of 20 phenolic acid compounds were identified, including ellagic acid, caffeic acid, isosakura glycosides, quercetin, astilbin, dihydroquercetin, vanillic acid, naringenin, kaempferol, quercetin, protocatechuic acid, p-hydroxybenzoic acid, methyl gallate, isoquercetin, hyperoside, gallic acid, chlorogenic acid, 2'-O-galloyl epicatechin gallate, 1,2,3,4,6-O-pentagalloyl glucose, and (-)-epicatechin gallate. Example 5: Detection of Chinese medicine granules of walnut walnut wood extract

[0048] (1) Sensory indicators

[0049] Appearance: Uniform particles, consistent color, no moisture absorption and agglomeration.

[0050] Smell: Pure smell, no peculiar smell.

[0051] (2) Physical and chemical indicators

[0052] Total phenolic acid content (based on ginsenolic acid Re) ≥ 20%; arsenic ≤ 0.5 mg / kg; lead ≤ 1 mg / kg; copper ≤ 5 mg / kg; moisture ≤ 2.0%; the difference in filling amount shall not exceed 5%.

[0053] (3) Microbiological indicators

[0054] Total colony count (CFU / g) ≤ 100; coliform group (CFU / g) ≤ 10; pathogenic bacteria shall not be detected.

[0055] Example 6: Experimental study on the efficacy of Chinese medicinal granules of walnut wood extract

[0056] 1. Mouse open field test

[0057] After the mice were purchased, they were fed adaptively for at least 7 days to adapt them to the laboratory environment and light-dark cycle. At the same time, they were stroked for a certain period of time every day to reduce the impact of nonspecific stress stimulation on the experiment. On the morning of the experiment, the weight of the mice was weighed and recorded. The mice were brought into the laboratory at least 3 hours in advance to reduce their anxiety about the new environment. At the same time, the experimental box was cleaned to ensure that there was no odor interference. Hold the mouse by 1 / 3 of the base of its tail and take it out of the cage, making sure it faces away from the experimenter, and avoid violent operations and other factors that cause animal stress. The mouse was quickly and gently placed in the central area of ​​the experimental box, and the recording system was started at the same time.

[0058] Grouping and dosing: The mice were gavaged with Chinese medicinal granules of walnut wood extract at low, medium and high doses (40 mg / L, 80 mg / L and 160 mg / L), and the blank group was gavaged with an equal amount of normal saline.

[0059] Observe behavior: Observe the movement trajectory, residence time, distance and other behavioral indicators of mice in the open field through video or manual recording. During the experiment, it is necessary to maintain low light and a quiet environment, and the observer should be separated from the test mice as much as possible. Set the experimental time to 15 minutes (adjustable as needed). After the experiment, stop and save the video, take the mouse out of the experimental box, and then put it back in the breeding cage. After each test, it is necessary to clean up the excrement of the previous mouse and remove the odor with 75% alcohol. Wait until the box is dry and odorless before conducting the experiment on the next mouse. The results are shown in Table 2.

[0060] Data analysis: Use professional software to analyze the recorded data, such as calculating the mice’s residence time in different areas, movement speed, etc., to evaluate their exploratory behavior and anxiety state.

[0061] Table 2 Effects on open field test in mice

[0062]

[0063] Note: Compared with the blank control group, * indicates P < 0.05

[0064] The results are shown in Table 2. The distance and speed of the mice in each dose group of walnut wood product in the open field were shorter than those in the blank group. The difference between the high dose group and the blank group was statistically significant (P < 0.05).

[0065] 2. Threshold dose sodium pentobarbital synergistic sleep experiment

[0066] The mice were randomly divided into several groups with the same number of mice in each group. According to the experimental design, different doses of sodium pentobarbital were given to the mice in different groups.

[0067] Grouping and drug administration: The subjects were given oral administration of Chinese medicine granules of walnut wood extract at low, medium and high doses (40 mg / L, 80 mg / L and 160 mg / L), and the blank group was given oral administration of the same amount of normal saline.

[0068] Injection and observation: Sodium pentobarbital was intraperitoneally injected into mice, and the timing was started after the injection was successful. The righting reflex of the mice was observed, and the time required for the righting reflex to disappear (sleep latency) and the time from the loss of the righting reflex to the recovery of the righting reflex (sleep time) were recorded. At the same time, the proportion of mice in each group whose righting reflex disappeared for more than 1 minute within a specific time was counted, that is, the sleep rate.

[0069] Data recording and analysis: The observation results were recorded in a table and statistically analyzed. The sleep latency, sleep time, and sleep rate of mice in different groups were compared to evaluate the synergistic effect of the test drug and sodium pentobarbital.

[0070] The synergistic results of subthreshold doses of sodium pentobarbital are shown in Table 3.

[0071] Table 3 Effects of subthreshold dose of sodium pentobarbital on sleep in mice

[0072]

[0073] From the results in Table 3, it can be seen that in the subthreshold dose sodium pentobarbital synergistic sleep time experiment, the sleeping rate of mice in the blank control group was 0, while the number of mice in other groups and the sleeping rate were significantly increased.

[0074] The synergistic results of suprathreshold doses of sodium pentobarbital are shown in Table 3.

[0075] Table 4 Effect of threshold dose of sodium pentobarbital on sleep time in mice

[0076]

[0077] Compared with the blank control group, *P<0.05

[0078] As shown in Table 4, in the threshold dose sodium pentobarbital synergistic sleep time experiment, the positive drug diazepam, high-dose and low-dose walnut wood products can significantly shorten the sleep latency of mice (P < 0.05), and the sleep duration of the medium-dose group also showed a shortening trend, but there was no statistical difference (P > 0.05). There was no significant difference in the sleep duration of mice in each group (P > 0.05), but the positive group and the high-dose walnut wood product group also showed a prolongation trend.

[0079] 3. Zebrafish Experiment

[0080] Wild-type AB adult zebrafish aged 4 months were randomly divided into 4 groups: blank group, model group, fluoxetine group and fenxin wood group, with 10 in each group. Except for the blank group, each group was treated with 40 mg / L reserpine for 20 minutes to establish the model; after stress treatment, zebrafish in the fluoxetine group were exposed to 0.01 mg / L fluoxetine and soaked for medication; after stress treatment, zebrafish in the fenxin wood group were exposed to 40 mg / L fenxin wood solution and soaked for medication. All the above 4 groups were fed normally, and the dressing was changed once a day for 14 consecutive days.

[0081] The novel tank (NTT) experiment and light-dark box (LDB) experiment were used to observe the differences in behavioral indicators of zebrafish in each group.

[0082] NTT experiment: A new water tank (15 cm high × 28 cm top × 23 cm bottom × 7 cm wide) was divided into two parts according to height. Zebrafish were placed into the new water tank with the water flow 20 minutes after the end of drug administration. One zebrafish was placed at a time and allowed to stand for 10 minutes until the zebrafish adapted to the new environment. The bottom lurking time and the number of times the zebrafish crossed the midline were recorded with a high-definition camera for 5 minutes. The surroundings were kept quiet throughout the process.

[0083] LDB experiment: The water tank (20cm high × 30cm long × 20cm wide) was divided at half of the height, with half of it shielded from light and half of it transparent. 20 minutes after the end of the administration of zebrafish, one zebrafish was put into the water tank with the water flow, and left to stand for 10 minutes. After the zebrafish adapted to the new environment, a high-definition camera was used to record the dark area lurking time of the zebrafish in the light-dark box and the number of times it entered the light box. The recording time was 5 minutes, and the surroundings were kept quiet throughout the process.

[0084] The results of the new environment water tank experiment are shown in Table 5.

[0085] Table 5 Results of zebrafish novel environment tank experiment ( n=10)

[0086]

[0087] Note: Compared with the blank group, * P<0.05, **P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0088] Compared with the blank group, the bottom lurking time of zebrafish in the model group was significantly increased (P<0.01), and the number of crossing the midline was significantly reduced (P<0.01), indicating that the zebrafish depression model was successfully established. Treatment with fluoxetine and walnut wood products can significantly shorten the bottom lurking time of zebrafish (P<0.01, P<0.05), increase the number of crossing the midline (P<0.01, P<0.05), and have a significant effect on improving the bottom-dwelling behavior of zebrafish and enhancing their desire to explore.

[0089] The results of the light-dark box experiment are shown in Table 6.

[0090] Table 6 Results of zebrafish light-dark box experiment ( n=10)

[0091]

[0092] Note: Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0093] Compared with the blank group, the dark box latency time of zebrafish in the model group was significantly increased (P<0.01), and the number of entries into the light box was significantly reduced (P<0.01); compared with the model group, the fluoxetine group and the walnut wood product group could significantly shorten the dark box latency time of zebrafish (P<0.01, P<0.05), increase the number of entries into the light box (P<0.05, P<0.05), and improve the lurking and dark-moving behaviors of zebrafish.

[0094] The above experiments prove that walnut wood products can significantly improve the movement speed of mice, prolong the sleep time of mice, and the high dose has the best effect. Zebrafish experiments prove that walnut wood products can significantly improve the bottom-dwelling behavior of zebrafish, enhance their desire to explore, and improve their lurking and dark-moving behaviors. Comprehensive analysis shows that walnut wood products have a good effect on improving sleep.

Claims

1. A method for preparing a walnut core wood extract, characterized in that: The steps include: (1) Extraction: grind the walnut pith wood and pass it through a 20-mesh sieve, add deionized water 8-12 times the weight of the walnut pith wood, reflux extract at 50-60° C. for 1-1.5 h, filter, extract twice, and combine the filtrate; (2) Separation: The solution was decanted in a 0.45 μm plate and frame filter; (3) Concentration: The filtrate is heated to 60-70°C for evaporation and concentration until the relative density of the concentrated solution reaches 1.05-1.07; (4) spray drying: adding maltodextrin and sodium chloride to the concentrated solution in step (3), drying in a cyclone separation spray dryer, setting the air inlet temperature to 160-170° C., the air outlet temperature to 85° C., and the negative pressure in the dryer cavity to -30 to -40 Pa, to obtain walnut wood extract powder; (5) Granulation: dry granulate the walnut wood extract powder obtained after spray drying in step (4), add lubricant magnesium stearate and flow aid micro-powder silica gel, set the roller pressure of the granulator to 1.5-2.5 MPa and the roller speed to 9-18 Hz, and prepare walnut wood extract particles.

2. The method for preparing the walnut core wood extract according to claim 1, characterized in that: In the step (4), the amount of maltodextrin added is 8-10% of the mass of the concentrated solution, and the amount of sodium chloride added is 1-3% of the mass of the concentrated solution.

3. The method for preparing the walnut core wood extract according to claim 1, characterized in that: The product yield after spray drying in step (4) is 95%.

4. The method for preparing the walnut core wood extract according to claim 1, characterized in that: The added amount of magnesium stearate is 0.8-1.0% of the mass of the walnut wood extract powder, and the added amount of micro powder silica gel is 0.5-1.0% of the mass of the walnut wood extract powder.

5. Use of the walnut pith wood extract obtained according to the preparation method of the walnut pith wood extract according to any one of claims 1 to 4 for preparing traditional Chinese medicine granules with sleep-aiding effect.

6. The use of the walnut core wood extract obtained by the preparation method of the walnut core wood extract according to claim 5, characterized in that: The components of the Chinese medicine granules with the effect of assisting sleep include 40-45 parts of walnut wood extract, 8-10 parts of mulberry leaf extract, 8-10 parts of bitter melon extract, 12-15 parts of kudzu root extract, 8-10 parts of yam extract and 8-10 parts of isomaltooligosaccharide by weight.

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