阅读说明：本技术 一种白芥子提取物及其制备方法和应用 (White mustard seed extract and preparation method and application thereof ) 是由 方向 张赟彬 冯奇 许静 于 2021-07-21 设计创作，主要内容包括：本发明涉及一种白芥子提取物及其制备方法和应用,其制备方法为：将白芥子用乙醇提取后,去除乙醇,得乙醇提取物,之后将乙醇提取物加水溶解,用乙酸乙酯萃取,再去除乙酸乙酯溶剂,即得所述白芥子提取物,该提取物应用于制备乙酰胆碱酯酶抑制剂中,抑制剂为胶囊、片剂或颗粒。与现有技术相比,本发明的白芥子提取物为从常见香辛料中提取,可长期服用,相较于常规治疗药物副作用小。白芥子提取物对乙酰胆碱酯酶的强抑制力为开发新的治疗老年痴呆病提供了新的思路。(The invention relates to a white mustard seed extract and a preparation method and application thereof, wherein the preparation method comprises the following steps: extracting semen Sinapis Albae with ethanol, removing ethanol to obtain ethanol extract, dissolving ethanol extract in water, extracting with ethyl acetate, and removing ethyl acetate solvent to obtain semen Sinapis Albae extract, which can be used in preparation of acetylcholinesterase inhibitor in the form of capsule, tablet or granule. Compared with the prior art, the white mustard seed extract is extracted from common spices, can be taken for a long time, and has small side effect compared with the conventional treatment medicines. The strong acetylcholinesterase inhibition ability of the brassica alba boiss extract provides a new idea for developing a new method for treating senile dementia.)

1. A method for preparing a white mustard seed extract is characterized by comprising the following steps: extracting semen Sinapis Albae with ethanol, removing ethanol to obtain ethanol extract, dissolving the ethanol extract in water, extracting with ethyl acetate, and removing ethyl acetate solvent to obtain the semen Sinapis Albae extract.

2. The method for preparing a semen Sinapis Albae extract as claimed in claim 1, wherein the volume ratio of semen Sinapis Albae to ethanol is 1 (10-15), the extraction temperature is 65-72 deg.C, and the extraction time is 100-140 min.

3. The method of claim 1, wherein the number of extractions with ethyl acetate is 2-4.

4. A mustard extract prepared by the method of any one of claims 1-3.

5. Use of the extract of Brassica alba boiss as claimed in claim 4 for the preparation of an acetylcholinesterase inhibitor.

6. The use of a mustard extract as claimed in claim 5, wherein the inhibitor comprises the mustard extract and pharmaceutically acceptable excipients.

7. The use of a mustard extract as claimed in claim 5, wherein the inhibitor is in the form of a capsule, tablet or granule.

8. The use of a semen Sinapis Albae extract as claimed in claim 7, wherein the tablet is prepared by: mixing semen Sinapis Albae extract and starch slurry, sieving, mixing, adding starch slurry, sieving, granulating, drying until water content is lower than 1%, sieving, grading, adding magnesium stearate, mixing, and tabletting to obtain semen Sinapis Albae extract tablet.

9. The use of a semen Sinapis Albae extract as claimed in claim 7, wherein the capsule is prepared by the steps of: vacuum drying semen Sinapis Albae extract, making into powder, mixing semen Sinapis Albae extract and starch, adding magnesium stearate, and filling into hard capsule with controlled filling amount to obtain semen Sinapis Albae extract capsule.

10. The use of a semen Sinapis Albae extract as claimed in claim 7, wherein the granule is prepared by: adding sucrose and dextrin into semen Sinapis Albae extract, mixing, sieving, drying, grading, and sieving to obtain semen Sinapis Albae granule.

Technical Field

The invention relates to the field of biological medicines, and in particular relates to a white mustard seed extract and a preparation method and application thereof.

Background

Alzheimer's disease, commonly known as senile dementia, is a neurological disorder of the elderly. The clinical manifestations are that the cognitive and memory functions are continuously deteriorated, and the normal life of the elderly is serious. In recent years, the number of patients with AD in the world is exponentially increased, great economic pressure and social burden are brought to families, public medical treatment and the like of the patients, and the method is a research hotspot of the current world medical treatment. The main theory on the pathogenesis of alzheimer's disease is the "cholinergic theory", acetylcholine is a neurotransmitter, which transmits signals between neurons. Acetylcholine in the brain of a patient is immediately hydrolyzed by acetylcholinesterase once it is released into the interstitium, resulting in the loss of the neurotransmitter acetylcholine, which results in the failure of central nervous signal transmission and ultimately the development of alzheimer's disease.

Therefore, the acetylcholinesterase inhibitor can inhibit the acetylcholinesterase of the brain of a patient, so that the concentration of acetylcholine is increased, and the cognitive ability of the patient can be improved. Currently, the approved alzheimer disease treatment drugs are acetylcholinesterase inhibitors. However, clinical tests show that the existing medicine can cause adverse reactions such as nausea, anorexia, vomiting and the like after being taken for a long time, has general curative effect and is not suitable for being taken for a long time. Therefore, more and more research at home and abroad focuses on extracting the acetylcholinesterase inhibitor from natural products, so that the acetylcholinesterase inhibitor is safer and is suitable for long-term administration.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a white mustard seed extract which is used as an acetylcholinesterase inhibitor instead of the prior therapeutic drugs, has small toxic and side effects and particularly has obvious effect on senile dementia, a preparation method and application thereof.

The purpose of the invention can be realized by the following technical scheme:

the inventor knows that white mustard seed, also called pepperweed, is the seed of the plant white mustard, is dark yellow granules, and is a relatively common traditional Chinese spice. In addition, it can also be used as a medicine for warming lung and resolving phlegm and relieving cough, and recorded in Ben Cao fen, pungent and warm herbs enter lung. Passing through meridians, inducing sweat and dispelling cold, warming middle energizer and activating qi, and eliminating phlegm. Mainly produced in Anhui, Henan, Sichuan and Shandong, and cultivated in various parts of the country.

Based on the above facts, the following specific schemes are proposed:

a method for preparing a white mustard seed extract comprises the following steps: extracting semen Sinapis Albae with ethanol, removing ethanol to obtain ethanol extract, dissolving the ethanol extract in water, extracting with ethyl acetate, and removing ethyl acetate solvent to obtain the semen Sinapis Albae extract. The mass yield of the mustard seed extract is generally 1.3-1.7%. The medicinal preparation prepared from the white mustard seed extract has obvious effect and relatively small toxic and side effect, and can effectively improve the senile dementia.

Further, when ethanol is adopted for extraction, the volume ratio of the white mustard seeds to the ethanol is 1 (10-15), the extraction temperature is 65-72 ℃, and the extraction time is 100-140 min.

Further, the number of extractions with ethyl acetate was 2 to 4.

A semen Sinapis Albae extract prepared by the above method is provided.

The application of the white mustard seed extract in preparing acetylcholinesterase inhibitors is disclosed. The acetylcholinesterase inhibitor can be used for preventing and treating senile dementia.

Further, the inhibitor comprises a white mustard seed extract and pharmaceutically acceptable auxiliary materials.

Further, the inhibitor is a capsule, a tablet or a granule.

Further, the preparation method of the tablet comprises the following steps: mixing semen Sinapis Albae extract and starch slurry, sieving, mixing, adding starch slurry, sieving, granulating, drying until water content is lower than 1%, sieving, grading, adding magnesium stearate, mixing, and tabletting to obtain tablet containing semen Sinapis Albae extract 0.2 g.

Specifically, the white mustard seed extract and starch slurry are firstly mixed according to the mass ratio of 1 (0.8-1.2), the mixture passes through a sieve of about 80 meshes, then starch slurry with the volume of 9-12% of the mixture is added at one time, the mixture is granulated by a sieve of about 14 meshes, the mixture is dried at 50-55 ℃ until the moisture content is lower than 1%, the dry granules are granulated by a sieve of about 16 meshes, the prepared dry granules are added with 2-5% of magnesium stearate to be uniformly mixed, and the tablet of the white mustard seed extract is prepared by tabletting, wherein the weight of each tablet is controlled to be 0.2 g.

Further, the preparation method of the capsule comprises the following steps: vacuum drying semen Sinapis Albae extract, making into powder, mixing semen Sinapis Albae extract and starch, adding magnesium stearate, and filling into hard capsule with controlled filling amount to obtain semen Sinapis Albae extract capsule.

Specifically, after vacuum drying the white mustard seed extract, preparing the white mustard seed extract into powder, uniformly mixing the white mustard seed extract and starch according to the mass ratio (1.8-2.2) to 1, adding magnesium stearate accounting for 0.45-0.55% of the total weight, filling the mixture into hard capsules, and controlling the filling amount to be 0.2-0.3g to obtain the capsules of the white mustard seed extract.

Further, the preparation method of the granules comprises the following steps: adding sucrose and dextrin into semen Sinapis Albae extract, mixing, sieving, drying, grading, and sieving to obtain semen Sinapis Albae granule.

Specifically, sucrose and dextrin are added into the white mustard seed extract according to the mass ratio of 1 (1.8-2.2) to 0.8-1.2, the white mustard seed extract and the dextrin are uniformly mixed, sieved by a sieve with about 16 meshes, dried, granulated and sieved to obtain white mustard seed particles.

Compared with the prior art, the white mustard seed extract has strong acetylcholinesterase inhibition activity, can be taken for a long time and has small side effect compared with the conventional treatment medicines because the white mustard seed extract is extracted from common spices. The strong acetylcholinesterase inhibition ability of the brassica alba boiss extract provides a new idea for developing a new method for treating senile dementia.

Drawings

FIG. 1 is a diagram of the preliminary identification of the extract of Sinapis alba by thin layer chromatography.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

Preparation of white mustard seed extract:

white mustard seed: the white mustard seeds used in the invention are purchased from Anhui Anqing and identified as dry mature seeds of the cruciferous plant white mustard Sinapis alba L.

Extraction and separation of the white mustard seed extract: soaking and extracting semen Sinapis Albae with 80% ethanol at a volume ratio of 1:12 for 60min, extracting at 70 deg.C under reflux for 120min, concentrating the extractive solution under reduced pressure, recovering ethanol to obtain ethanol extract, dissolving in water, extracting with ethyl acetate for 3 times, mixing extractive solutions, and recovering solvent under pressure to obtain ethyl acetate extract. Generally, the yield of mustard extract from mustard is generally 1.3-1.7%.

The thin-layer chromatography of the white mustard seed extract is preliminarily identified:

(1) preparation of a standard substance:

and (3) catechin standard substance: accurately weighing 6.70mg of catechin standard control sample, adding 60% ethanol to constant volume to 10mL to obtain 0.670mg/mL catechin standard.

Rutin standard substance: accurately weighing 7.92mg of rutin standard control sample, adding 60% ethanol to constant volume to 10mL to obtain 0.792mg/mL rutin standard.

Oleanolic acid standards: accurately weighing 1.02mg of an oleanolic acid standard control sample, adding 60% ethanol, and diluting to 10mL to obtain 0.690mg/mL of oleanolic acid standard.

(2) Preparing a color developing agent: 45mL of ethanol and 5mL of concentrated sulfuric acid are prepared into a 10% concentrated sulfuric acid ethanol solution.

(3) And (3) thin-layer chromatography spotting: the main substance classes of the active substances in the extract are qualitatively identified by respectively using standard catechin, rutin and oleanolic acid as the contrast of polyphenols, flavonoids and triterpenoids. Taking catechin, oleanolic acid and rutin as reference standard substances, adding into the same silica gel GF254 thin layer plate, adding into developing agent chloroform: ethyl acetate: methanol: formic acid 6: 8: 4: and 2, unfolding the unfolding system, airing and spraying a 10% concentrated sulfuric acid ethanol solution after unfolding, heating in a constant-temperature drying box at 105 ℃, taking out after color development, observing, and calculating the Rf value.

(4) The identification result of semen Sinapis Albae extract by thin layer chromatography is shown in figure 1, and the semen Sinapis Albae extract is primarily identified by thin layer chromatography, and the specific shift value and color reaction are shown in the above figure. It can be seen from the observation that the Rf value of the spots after the color development of the sample is close to the ratio shift value of catechin and rutin, and the typical color is presented, polyphenols are presented as brick red spots, and flavonoids are presented as yellow spots. Therefore, the extract of the white mustard seed mainly contains polyphenol and flavonoid compounds which have no toxicity and little side effect.

Experimental study

1. Research on in-vitro acetylcholinesterase inhibition of brassica alba boiss extract

1.1 Experimental materials

Extracts were tested for their IC50 values for acetylcholinesterase inhibition using the method of Ellman (Ellman, g.l.courtney, k.d.andres, v.et al.biochem.pharmacol.1961,7, 88.). All tests were carried out using a Microplate Reader model SpectraMax M2(Multi-Mode Microplate Reader), Molecular Devices USA, at 37 ℃. Data analysis software data processing was performed using Origin software, using galantamine hydrobromide as a control.

1.2 Experimental methods

1) Preparation of an inhibitor stock solution: the inhibitors tested were formulated as a 10mmol/L solution of mustard extract in dimethyl sulfoxide (DMSO).

2) Preparing an enzyme stock solution: acetylcholinesterase was purchased from Sigma; the resulting mixture was adjusted to 0.1mg/mL and 2mg/mL, respectively, using a phosphate buffer solution having a pH of 8.0.

3) Preparation of a substrate stock solution: acetylmercaptocholine (acetylcholinesterase substrate) was purchased from Sigma; the resulting mixture was adjusted to 2mg/mL and 4mg/mL, respectively, using a phosphate buffer solution having a pH of 8.0.

4) Preparing a color developing agent stock solution: the color reagent 5,5' -dithiobis (2-nitrobenzoic acid), abbreviated as DTNB, was purchased from Sigma; the resulting mixture was adjusted to 4mg/mL using a phosphate buffer solution having a pH of 8.0.

5) And (3) testing: the volume of each test was 150 μ L of phosphate buffer pH 8.0. Adding 6 mu L of developer stock solution into a 96-well enzyme label plate, then respectively adding 15 mu L of inhibitor solutions with different concentrations (diluting the inhibitor stock solution by using a phosphate buffer solution with the pH value of 8.0), supplementing to 139 mu L by using a phosphate buffer solution with the pH value of 8.0, then adding 5 mu L of enzyme stock solution, preserving the temperature in a 37 ℃ enzyme label instrument for 12min, immediately adding 6 mu L of substrate stock solution, and immediately measuring the change of absorbance at the position of the lambda value of 405nm (slope Kt) after mixing uniformly. The reference solution was a phosphate buffered solution at pH 8.0 and the change in absorbance at λ 405nm was measured for one minute (slope K0). Calculating a formula according to the inhibition ratio:

and obtaining the inhibition rates of the inhibitors with different concentrations, and obtaining the IC50 of the inhibitory activity of the tested substance on the acetylcholinesterase by nonlinear fitting.

In detail, IC50 is the 50% inhibition rate, which has been reiterated previously, and different concentrations of inhibitor solutions were added to the experiment, thus corresponding inhibition rates were obtained. Whereas the IC50, i.e., 50% inhibition, was obtained by non-linear fitting of the data.

The IC50 of the acetylcholinesterase inhibitory activity of the substance to be tested was determined.

1.3 Experimental results and analysis

TABLE 1 Effect of Brassica alba extracts on Acetylcholinesterase Activity

Group of	Name of article	Acetylcholinesterase IC50(μmol/L)
			Group of the invention	White mustard seed extract	0.92
Control group	Galanthamine hydrobromide	0.85

As can be seen from Table 1, the extract of Sinapis alba of the present invention has a strong inhibitory activity against acetylcholinesterase, and its measured IC50 value is close to that of galantamine hydrobromide approved by FDA, and the extract of Sinapis alba of the present invention is extracted from common spicery, and can be taken for a long time with less side effects compared with conventional therapeutic drugs. The strong acetylcholinesterase inhibition ability of the brassica alba boiss extract provides a new idea for developing a new method for treating senile dementia.

Example 1

Mixing the white mustard seed extract and starch slurry according to the mass ratio of 1:1, mixing, sieving with a 80-mesh sieve, mixing, adding 10% starch slurry, sieving with a 14-mesh sieve, granulating, drying at 52 deg.C until the water content is less than 1%, and sieving with a 16-mesh sieve to obtain the final product. Adding 3% magnesium stearate into the dry granules, mixing, and tabletting to obtain tablet containing semen Sinapis Albae extract, wherein the weight of each tablet is controlled to be 0.2 g.

Example 2

Vacuum drying the white mustard seed extract, preparing the white mustard seed extract into powder, and mixing the white mustard seed extract with starch according to a mass ratio of 2:1, mixing, adding magnesium stearate with a total weight of 0.5%, and filling with hard capsule, wherein the filling amount is controlled to be 0.2-0.3g, thus obtaining the capsule of the white mustard seed extract.

Example 3

Adding sucrose and dextrin (mass ratio of 1:2:1) into semen Sinapis Albae extract, mixing, sieving with 16 mesh sieve, drying, and sieving to obtain semen Sinapis Albae granule.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.