Zanthoxylum bungeanum aromatic water as well as preparation method and application thereof

Abstract

The invention relates to the technical field of extraction of traditional Chinese medicine components, and particularly discloses a preparation method of zanthoxylum bungeanum aromatic water, which comprises the following steps: S1, weighing zanthoxylum bungeanum; s2, soaking the weighed red pepper in water of which the mass is 8-12 times that of the red pepper for 30-180 minutes; s3, heating and extracting the soaking solution in which the red pepper is soaked for 60-180 minutes; and S4, collecting the heated reflux liquid to obtain the red pepper aromatic water. The zanthoxylum bungeanum aromatic water can be applied as a medicine for promoting learning and memory ability. The invention aims to optimize the preparation method of the zanthoxylum bungeanum maxim aromatic water through an orthogonal experiment, so that effective components in zanthoxylum bungeanum maxim can be more accurately extracted, the biological activity of the prepared zanthoxylum bungeanum maxim aromatic water can be comprehensively displayed, and animal experiments prove that the zanthoxylum bungeanum maxim aromatic water has the effects of improving the learning and memory ability and improving the health care effect. The compound has remarkable drug-likeness, has the drug potential for treating brain and mental diseases such as senile dementia and the like, and has wide market prospects and research values.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine ingredient extraction, and specifically refers to a fragrant water of Zanthoxylum bungeanum Maxim. and its preparation method and application. Background Art

[0002] Fragrant water, also known as hydrosol, is a by-product of plant essential oils and is a saturated essential oil aqueous solution obtained by distilling aromatic plants. Fragrant water is rich in plant essential oils, can be drunk, and can also be used for skin care, aromatherapy, etc., and has a variety of active and health care applications.

[0003] Zanthoxylum bungeanum Maxim., anciently known as Sichuan pepper, is the pericarp of Zanthoxylum bungeanum Maxim. of the Rutaceae family and the Zanthoxylum genus. Its essential oil has a variety of activities, such as antidepressant and anti-anxiety pharmacological and health care effects. In past studies, more attention has been paid to the pharmacological effects and activities of the water extract or essential oil of Zanthoxylum bungeanum Maxim., while there have been no relevant reports on the biological activities of the fragrant water of Zanthoxylum bungeanum Maxim. and its possible active ingredients. It can be seen that the fragrant water of Zanthoxylum bungeanum Maxim. still has great research potential and awaits further exploration. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method that can more accurately extract the effective ingredients in Zanthoxylum bungeanum Maxim.

[0005] Another purpose of the present invention is the fragrant water of Zanthoxylum bungeanum Maxim. prepared by the above preparation method.

[0006] The present invention also aims to provide the specific application of the prepared fragrant water of Zanthoxylum bungeanum Maxim.

[0007] The present invention is achieved by the following technical solutions: A preparation method of a fragrant water of Zanthoxylum bungeanum Maxim., comprising the following steps:

[0008] Step S1: Weigh Zanthoxylum bungeanum Maxim.;

[0009] Step S2: Soak the weighed Zanthoxylum bungeanum Maxim. in 8 - 12 times the mass of water for 30 min - 180 min;

[0010] Step S3: Heat and extract the soaking solution soaked with Zanthoxylum bungeanum Maxim. for 60 min - 180 min;

[0011] Step S4: Collect the reflux liquid after heating to obtain the fragrant water of Zanthoxylum bungeanum Maxim.

[0012] To better implement the method of the present invention, further, the orthogonal design method is adopted, with the water addition amount, soaking time, and extraction time as influencing factors, and the volatile oil content in the aromatic water of Zanthoxylum bungeanum Maxim. as the evaluation index, to optimize the water addition amount, soaking time, and extraction time in the preparation process of the aromatic water of Zanthoxylum bungeanum Maxim. The optimized water addition amount in the preparation process of the aromatic water of Zanthoxylum bungeanum Maxim. is 8 times the mass of the weighed Zanthoxylum bungeanum Maxim., the soaking time of Zanthoxylum bungeanum Maxim. is 60 min, and the extraction time is 180 min.

[0013] To better implement the method of the present invention, further, the device for measuring the volatile oil content in the aromatic water of Zanthoxylum bungeanum Maxim. sequentially includes a hard round-bottom flask (1), a volatile oil detector (2), and a reflux condenser (3) from bottom to top. The upper part of the hard round-bottom flask (1) is connected to the lower part of the volatile oil detector (2) through a ground glass joint, and the upper part of the volatile oil detector (2) is connected to the lower part of the reflux condenser (3) through a ground glass joint.

[0014] To better implement the method of the present invention, further, the process for measuring the volatile oil content in the aromatic water of Zanthoxylum bungeanum Maxim. is as follows: After weighing the collected aromatic water of Zanthoxylum bungeanum Maxim., add it to the hard round-bottom flask (1), add 300 - 500 ml of water and several glass beads, shake and mix, then connect the volatile oil detector (2) and the reflux condenser (3). Add water from the upper end of the reflux condenser (3) to fill the graduated part of the volatile oil detector (2) until it overflows into the hard round-bottom flask (1); Heat the hard round-bottom flask (1) to boiling and maintain a gentle boil for about 5 hours until the amount of oil in the volatile oil detector (2) no longer increases, then stop heating, let it stand for a while, open the piston at the lower end of the volatile oil detector (2), and slowly release the water until the upper end of the oil layer reaches 5 above the 0 line of the scale; Let it stand for more than 1 hour, then open the piston of the volatile oil detector (2) again to lower the oil layer until its upper end is exactly flush with the 0 line of the scale, and read the amount of volatile oil.

[0015] To better implement the method of the present invention, further, the weighing of the collected aromatic water of Zanthoxylum bungeanum Maxim. needs to be accurate to 0.01 g.

[0016] To better implement the method of the present invention, further, an electric heating mantle is used to heat the hard round-bottom flask (1).

[0017] A red prickly ash aromatic water prepared by the method for preparing the above-mentioned red prickly ash aromatic water, the red prickly ash aromatic water contains aristolene, terpenic acid, 7-epi-terpenic acid, β-muurolene, modiphen, α-bulnesene, δ-elemene, trans-caryophyllene, γ-elemene, (Z,Z,Z)-1,5,9,9-tetramethyl-cycloundecatriene, 1,3,5-triisopropylbenzene, β-selinene, α-zingiberene, valenciaene, germacrene B, aromadendrin, β-amyrin, 7-propylidene-bicyclo[4.1.0]heptane, (E,E)-12-methyl-1,5,9,11-tridecatetraene, 2-hydroxy-2-cyclopenten-1-one, 1-hydroxy-1,7-dimethyl-4-isopropyl-cyclodecadiene, elemol, Γ-selinene, atractylon, epieudesmol, Γ-cadinene, agaro-wooden alcohol, elemol, 2-isopropenylnaphthalene, Beta-eudesmol, 2-(4a,8-dimethylhexahydronaphthalen-2-yl)-2-propanol, julolidone, 2,4-di-tert-butylphenol, 7,7-dimethylbicyclo[3.1.1]hept-3-ene-spiro-2,4'-(1',3'-dioxane), 5-fluoro-3-methylbenzothiophene, 4-methylbiphenyl, 8,9-dihydroneo-isolongifolene, trans-valerene acetate, 5-methyl-2-ethylamino-2-thiazoline, 2-(trimethylsilyl)naphthalene, 2-hydroxyfluorene, norhalichondrin, myrcene, dehydroaromadendrene, 1,1,1,3-tetrachloroacetone, diethyl 3-oxoheptanedioate, p-phenylbenzaldehyde, methyl 8-methoxy-α-1-naphthoacetate.

[0018] A drug for promoting learning and memory ability, which is prepared with the above-mentioned red prickly ash aromatic water as the main component and added with pharmaceutically acceptable medicinal carriers and / or excipient auxiliary components that are non-toxic and non-inert to humans and animals, and the drug is a spray.

[0019] An application of using the above-mentioned red prickly ash aromatic water as the main component to prepare a drug for treating Alzheimer's disease.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] The present invention aims to optimize the preparation method of red prickly ash aromatic water through orthogonal experiments, so that it can more accurately extract the effective components in red prickly ash, and then the prepared red prickly ash aromatic water can fully display its biological activity. And based on animal experiments, it is confirmed that the red prickly ash aromatic water has the ability to improve learning and memory ability, has significant drug-likeness, has the potential of a drug for treating brain diseases such as Alzheimer's disease, and has broad market prospects and research value. Description of the Drawings

[0022] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, purposes and advantages of the present invention will become more obvious:

[0023] Figure 1 This is the total ion current chromatogram of the aromatic water of Zanthoxylum bungeanum Maxim. in the present invention;

[0024] Figure 2 This is the residence time of the mice in the target area in the water maze in the present invention;

[0025] Figure 3 This is the number of times the mice cross the position of the target object in the water maze in the present invention;

[0026] Figure 4 This is the protein expression detection chart by Western blotting in the present invention;

[0027] Figure 5 This is the statistical chart of the expression level of cAMP response element-binding protein in the present invention;

[0028] Figure 6 This is the statistical chart of the expression level of brain-derived neurotrophic factor protein in the present invention;

[0029] Figure 7 This is the structural schematic diagram of the device for measuring the volatile oil content in the present invention. Detailed implementation manners

[0030] To make the purpose, process conditions and advantages of the present invention clearer, the present invention will be further described in detail below in combination with the following implementation examples. However, the implementation manners of the present invention are not limited thereto. Without departing from the above technical ideas of the present invention, various substitutions and changes made according to the common general knowledge and conventional means in the art shall all be included within the scope of the present invention. The specific implementation examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Example 1:

[0032] This example provides a preparation method of the aromatic water of Zanthoxylum bungeanum Maxim., and the specific process is as follows:

[0033] Step S1: Weigh Zanthoxylum bungeanum Maxim.;

[0034] Step S2: Soak the weighed Zanthoxylum bungeanum Maxim. with 8 - 12 times the mass of water for 30 min - 180 min;

[0035] Step S3: Heat and extract the soaking solution containing Zanthoxylum bungeanum Maxim. for 60 min - 180 min;

[0036] Step S4: Collect the reflux solution after heating to obtain the aromatic water of Zanthoxylum bungeanum Maxim.

[0037] Using the orthogonal design method, with the water addition amount, soaking time, and extraction time as influencing factors and the volatile oil content in the aromatic water as the evaluation index, the optimal preparation parameters for the preparation method of red prickly ash aromatic water were obtained, as shown in Table 1 and Table 2 below:

[0038] Table 1 Results of orthogonal experiments

[0039] Factor Water addition amount Soaking time (min) Extraction time (min) Experimental result (mL / L) 1 8 30 60 1.05 2 8 60 120 1.38 3 8 180 180 1.57 4 10 30 120 1.32 5 10 60 180 1.51 6 10 180 60 1.25 7 12 30 180 1.46 8 12 60 60 1.21 9 12 180 120 1.40 Mean 1 1.333 1.277 1.17 Mean 2 1.36 1.367 1.367 Mean 3 1.357 1.407 1.513 Range 0.027 0.13 0.343

[0040] Table 2 Results of variance analysis

[0041] Factor Sum of squared deviations Degree of freedom F ratio F critical value Significance Water addition amount 0.001 2 0.015 5.14 Soaking time 0.027 2 0.393 5.14 Extraction time 0.178 2 2.592 5.14 Error 0.21 6

[0042] From the range analysis of Table 1 based on the orthogonal design results, it can be seen that among the factors affecting extraction: extraction time > soaking time > water addition amount. According to the content of Table 2 in the variance analysis results, there is no significant difference in the factors affecting extraction. Through comprehensive analysis, the optimal extraction process for red prickly ash aromatic water is as follows: Weigh the red prickly ash medicinal materials, add 8 times the amount of water, soak for 60 minutes first, then heat and extract for 180 minutes, and collect the reflux extraction solution.

[0043] The components of red prickly ash aromatic water were analyzed by GC-MS, as Figure 1 , shown in Table 4.

[0044] Table 3 GC-MS analysis of red prickly ash aromatic water

[0045]

[0046]

[0047]

[0048]

[0049] Example 2:

[0050] In this example, in order to verify the specific effects of red prickly ash aromatic water, the following experiment was specifically designed:

[0051] The extracted aromatic water was diluted with normal saline to prepare samples of the original solution, 1 / 2 original solution, and 1 / 4 original solution, which were filled into spray bottles for later use.

[0052] ICR mice were divided into 4 groups, with 10 mice in each group, namely the normal group, low-dose, medium-dose, and high-dose groups of red prickly ash aromatic water. 10 minutes before the experiment, saline, 1 / 4 original solution of aromatic water, 1 / 2 original solution, and the original solution were sprayed into the noses of the mice 1 - 2 times, twice a day.

[0053] The Morris water maze is a circular pool with a black inner pool wall (120 cm in diameter and 50 cm in height), and 4 water entry points are marked on the inner pool wall. A movable cylindrical hidden platform (6.5 cm in diameter, 1 cm below the water surface) is placed at the center of the third quadrant.

[0054] The experimental procedures include: ① Place navigation experiment: lasting for 4 days, 2 times a day, with an interval of more than 20 minutes between each training. The mice are randomly placed into the water from 3 water entry points (except the quadrant where the platform is located) facing the barrel wall, and the time it takes for the mice to find the platform within 90 s is recorded, which is denoted as the escape latency; if the mice do not find the platform within 90 s, the timing is stopped and the mice are artificially induced to the platform and stay for 20 s, and the escape latency is recorded as 90 s. After each training, wipe the mice with toilet paper to avoid affecting the experimental results due to low temperature; ② Spatial exploration experiment: After training the mice as described above for 4 days, remove the platform, and place each group of mice into the water from the first quadrant facing the pool wall, and at the same time, the residence time of the mice in the target area within 60 s and the number of times passing through the platform are recorded.

[0055] The experimental result data are presented in the form of mean ± standard deviation (n = 10), *p < 0.05, **p < 0.01, vs. Normal. As Figure 2 , Figure 3 shown, under the influence of red prickly ash aromatic water spray with higher concentrations, normal mice have longer residence time in the target area, more times passing through the platform, and can find the platform faster. Thus, it can be seen that red prickly ash aromatic water spray can significantly promote the learning and memory ability of normal mice.

[0056] Example 3:

[0057] In this example, western blotting was used to detect brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) in the brains of the above experimental mice.

[0058] Among them, cAMP response element-binding protein is a protein that regulates gene transcription, and it mainly regulates the expression of brain-derived neurotrophic factor

[0059] Brain-derived neurotrophic factor is very important for maintaining the function of neurons. On the one hand, it can increase synaptic plasticity, thereby affecting long-term potentiation. On the other hand, it can promote neurogenesis, especially neurogenesis in the hippocampus. On the third hand, it can promote the survival of neurons, especially dopamine- and serotonin-related neurons

[0060] The western blotting method separates protein samples by polyacrylamide gel electrophoresis according to molecular weight, then transfers them to a hybridization membrane (blot), and then specifically detects the target protein through an antibody / secondary antibody complex. The specific process is as follows:

[0061] First, add lysis buffer (containing protease inhibitor and phosphatase inhibitor) to the brain tissues of each group of mice for lysis, measure the protein concentration, and prepare the protein concentrations of each group to the same concentration for convenient loading.

[0062] Then, separate the proteins by electrophoresis using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transfer them to a polyvinylidene fluoride membrane.

[0063] Finally, block the membrane with 5% bovine serum albumin (BSA), and then use the corresponding antibodies against brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) to detect the expression of the target protein, with β-actin as an internal control. The data are presented as mean ± standard deviation (n = 3), *p < 0.05, vs. the normal saline group.

[0064] The detection results are as Figure 4 shown, Figure 4 where the depth and width of the bands in the Figure 4 figure are the expression levels of the proteins. It can be clearly seen from the

[0065] Figure 5 figure that as the concentration of the aromatic water of Zanthoxylum bungeanum Maxim. increases, the expression levels of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) increase significantly, while there is no obvious change in β-actin.

[0066] Figure 6 This is a statistical chart of the expression level of cAMP response element-binding protein (CREB). It can be seen from the figure that as the concentration of the aromatic water of Zanthoxylum bungeanum Maxim. increases, the expression level of cAMP response element-binding protein (CREB) increases.

[0067] This is a statistical chart of the protein expression level of brain-derived neurotrophic factor (BDNF). It can be seen from the figure that as the concentration of the aromatic water of Zanthoxylum bungeanum Maxim. increases, the protein expression level of brain-derived neurotrophic factor (BDNF) increases.

[0068] Other parts of this embodiment are the same as those of the above embodiment and will not be described in detail.

[0069] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing red pepper aromatic water, characterized in that: The following steps are involved: Step S1: weigh red pepper; Step S2: Soak the weighed red peppercorns in water 8 to 12 times the mass of water for 30 to 180 minutes; Step S3: heating and extracting the soaking liquid soaked with red pepper for 60 min to 180 min; Step S4: collecting the heated reflux liquid to obtain red pepper aromatic water.

2. The method for preparing red pepper aromatic water according to claim 1, characterized in that: The orthogonal design method was adopted, with the amount of water added, soaking time and extraction time as influencing factors and the volatile oil content in red pepper aromatic water as the evaluation index to optimize the amount of water added, soaking time and extraction time in the preparation process of red pepper aromatic water. The amount of water added in the preparation process of the optimized red pepper aromatic water was 8 times the mass of the red pepper, the soaking time was 60 min, and the extraction time was 180 min.

3. The method for preparing red pepper aromatic water according to claim 2, characterized in that: The device for determining the volatile oil content in red pepper aromatic water comprises, from bottom to top, a hard round-bottom flask (1), a volatile oil determination device (2), and a reflux condenser tube (3); the upper portion of the hard round-bottom flask (1) is connected to the lower portion of the volatile oil determination device (2) via a glass ground joint, and the upper portion of the volatile oil determination device (2) is connected to the lower portion of the reflux condenser tube (3) via a glass ground joint.

4. The method for preparing red pepper aromatic water according to claim 3, characterized in that: The process for determining the volatile oil content in the red pepper aromatic water is as follows: weighing the collected red pepper aromatic water, adding it to a hard round-bottom flask (1), adding 300-500 ml of water and a few glass beads, shaking and mixing, connecting the volatile oil analyzer (2) and the reflux condenser (3), adding water from the upper end of the reflux condenser (3) to fill the scale part of the volatile oil analyzer (2) and overflow into the hard round-bottom flask (1); heating the hard round-bottom flask (1) to boiling, and keeping it slightly boiling for about 5 hours until the amount of oil in the volatile oil analyzer (2) no longer increases, stopping heating, leaving it for a while, opening the piston at the lower end of the volatile oil analyzer (2), and slowly releasing water until the upper end of the oil layer reaches 5 points above the scale 0 line; leaving it for more than 1 hour, and then opening the piston of the volatile oil analyzer (2) to allow the oil layer to drop until its upper end is just flush with the scale 0 line, and reading the volatile oil content.

5. The method for preparing red pepper aromatic water according to claim 4, characterized in that: The collected red pepper aromatic water is weighed accurately to 0.01 g.

6. The method for preparing red pepper aromatic water according to claim 4, characterized in that: The hard round-bottom flask (1) is heated using an electric heating mantle.

7. A red pepper aromatic water prepared by the method for preparing the red pepper aromatic water according to any one of claims 1 to 6, characterized in that: The red pepper aromatic water contains aristolochene, terpene acid, 7-epiterpene acid, β-malone, modifene, α-bulonene, δ-elemene, trans-caryophyllene, γ-elemene, (Z,Z,Z)-1,5,9,9-tetramethyl-cycloundecatriene, 1,3,5-triisopropylbenzene, β-serine, α-gingerene, valencia orangene, geraniol B, citronellol, β-amyrin, 7-propylene-bicyclo[4.1.0]heptane, (E,E)-12-methyl-1,5,9,11-tridetraene, 2-hydroxy-2-cyclopentene-1-one, 1-hydroxy-1,7-dimethyl-4-isopropyl-cyclodecadiene, galangal, Γ-caryophyllene, atractylodesone, epieucalyptol, Γ-cadinene, precipitate, Spiroalanol, 2-isopropenylnaphthalene, Beta-eudesmol, 2-(4a,8-dimethylhexahydronaphthalen-2-yl)-2-propanol, citriol, 2,4-di-tert-butylphenol, 7,7-dimethylbicyclo[3.1.1]hept-3-ene-spiro-2,4'-(1',3'-dioxane), 5-fluoro-3-methylbenzothiophene, 4-methylbiphenyl, 8,9-dihydroneoisoleifole, trans-valerenyl acetate, 5-methyl-2-ethylamino-2-thiazoline, 2-(trimethylsilyl)naphthalene, 2-hydroxyfluorene, nohalkentin, myrcene, dehydrocitriol, 1,1,1,3-tetrachloroacetone, diethyl 3-oxopimelate, p-phenylbenzaldehyde, 8-methoxy-α-1-naphthylacetic acid methyl ester.

8. A drug for improving learning and memory ability, characterized in that: The red pepper aromatic water according to claim 7 is used as the main component, and pharmaceutically acceptable, non-toxic and non-inert pharmaceutical carriers and / or excipient auxiliary components are added.

9. The drug for improving learning and memory ability according to claim 8, characterized in that: The medicine is a spray.

10. Use of the red pepper aromatic water as claimed in claim 7 as a main ingredient in preparing a drug for treating Alzheimer's disease.

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