阅读说明：本技术 1-羧基-2-羟基-3-亚氨基丙烷及其提取方法 (1-carboxy-2-hydroxy-3-iminopropane and process for its extraction ) 是由 孙秀兰 洪茜茜 徐德平 张银志 耿树香 于 2020-12-21 设计创作，主要内容包括：本发明公开了1-羧基-2-羟基-3-亚氨基丙烷及其提取方法,属于食品、保健食品、医药技术领域。本发明所述提取1-羧基-2-羟基-3-亚氨基丙烷的方法,包括如下步骤：(1)将核桃分心木粉末加入乙醇溶液中进行提取,过滤得到上清液；将上清液减压浓缩得到核桃醇提物；(2)将步骤(1)的滤渣加入水中进行提取,过滤得到上清液；将上清液减压浓缩得到核桃水提物；(3)将核桃醇提物和醇提物混合得到核桃分心木混合提取物；之后依次经过AB-8型大孔树脂柱、MCI柱、ODS柱,洗脱,分离得到沉淀即1-羧基-2-羟基-3-亚氨基丙烷。本发明首次在核桃分心木中发现了1-羧基-2-羟基-3-亚氨基丙烷,且其具有良好的镇静催眠活性。(The invention discloses 1-carboxyl-2-hydroxyl-3-imino propane and an extraction method thereof, belonging to the technical field of food, health food and medicine. The method for extracting 1-carboxyl-2-hydroxyl-3-imino propane comprises the following steps: (1) adding walnut diaphragma juglandis powder into an ethanol solution for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain Juglandis alcoholic extract; (2) adding the filter residue obtained in the step (1) into water for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain walnut aqueous extract; (3) mixing the walnut alcohol extract and the alcohol extract to obtain a walnut diaphragma juglandis mixed extract; then sequentially passing through an AB-8 type macroporous resin column, an MCI column and an ODS column, eluting, and separating to obtain a precipitate, namely the 1-carboxyl-2-hydroxyl-3-imino propane. The invention discovers 1-carboxyl-2-hydroxyl-3-imino propane in the diaphragma juglandis for the first time, and the diaphragma juglandis has good sedative and hypnotic activity.)

1. 1-carboxy-2-hydroxy-3-iminopropane having the formula I:

2. a method for extracting 1-carboxy-2-hydroxy-3-iminopropane according to claim 1 from diaphragma juglandis, characterized by comprising the following steps:

(1) adding walnut diaphragma juglandis powder into an ethanol solution for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain Juglandis alcoholic extract;

(2) adding the filter residue obtained by filtering in the step (1) into water for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain walnut aqueous extract;

(3) mixing the walnut alcohol extract obtained in the step (1) and the alcohol extract obtained in the step (2) to obtain a walnut diaphragma juglandis mixed extract; then, the mixed extract of the walnut diaphragma juglandis sequentially passes through an AB-8 type macroporous resin column, an MCI column and an ODS column, and is sequentially eluted by ethanol with different concentrations, and the precipitate, namely the 1-carboxyl-2-hydroxyl-3-imino propane, is obtained by separation.

3. The method according to claim 2, wherein the ratio of the walnut diaphragma powder and the ethanol solution of step (1) is 1: 8-12.

4. The method according to claim 2 or 3, wherein the extraction conditions of step (1) and step (2) are extraction at 55-65 ℃ for 3-4 h.

5. The method according to any one of claims 2 to 4, wherein the concentration under reduced pressure in step (1) and step (2) is carried out to a solid content of 20 to 30%.

6. The method according to any one of claims 2 to 5, wherein the step (3) of passing through a macroporous resin column of AB-8 type, and the ethanol solution used comprises an aqueous ethanol solution with a volume fraction of 0, 30%, 50% or 70%.

7. The method of any one of claims 2-6, wherein the step (3) of passing through the MCI column uses an ethanol solution comprising 0, 10%, 30%, 50% ethanol in water by volume fraction.

8. The method according to any one of claims 2 to 7, wherein said step (3) of passing through the ODS column employs an ethanol solution comprising 0, 5%, 10%, 20% by volume of an aqueous ethanol solution.

9. Use of 1-carboxy-2-hydroxy-3-iminopropane according to claim 1 in the medical field.

10. A pharmaceutical preparation comprising the 1-carboxy-2-hydroxy-3-iminopropane according to claim 1.

Technical Field

The invention relates to 1-carboxyl-2-hydroxyl-3-imino propane and an extraction method thereof, belonging to the technical field of food, health food and medicine.

Background

Diaphragma juglandis Fructus (Diaphragma juglandis Fructus) is also called as walnut membrane and walnut valve. The traditional medicinal application of diaphragma juglandis fructus comprises treating and preventing diabetes, kidney deficiency, diarrhea and urogenital system diseases. In vivo and in vitro researches find that the diaphragma juglandis contains functional components such as flavone (3.89-5.44%), phenols (2.39-3.54%), saponins (1.86-5.98%), lignans and the like, and the diaphragma juglandis endows the diaphragma juglandis with various functional activities such as bacteriostasis, antioxidation, anti-tumor, blood sugar and blood fat reduction and the like; can be used for the product development in the related fields of health food, medicine and the like.

However, the components of the walnut tree are complex, and it cannot be expected at all which components are present therein and which components can play what role.

Disclosure of Invention

In order to solve at least one problem, the invention separates 1-carboxyl-2-hydroxy-3-imino propane with potential activity from diaphragma juglandis, which has sedative and hypnotic effects; and the extraction method is simple and easy to implement.

It is a first object of the present invention to provide a 1-carboxy-2-hydroxy-3-iminopropane having the formula:

the second purpose of the invention is a method for extracting 1-carboxyl-2-hydroxy-3-imino propane from diaphragma juglandis, which comprises the following steps:

(1) adding walnut diaphragma juglandis powder into an ethanol solution for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain Juglandis alcoholic extract;

(2) adding the filter residue obtained by filtering in the step (1) into water for extraction, and filtering to obtain a supernatant; concentrating the supernatant under reduced pressure to obtain walnut aqueous extract;

(3) mixing the walnut alcohol extract obtained in the step (1) and the alcohol extract obtained in the step (2) to obtain a walnut diaphragma juglandis mixed extract; then, the mixed extract of the walnut diaphragma juglandis sequentially passes through an AB-8 type macroporous resin column, an MCI column and an ODS column, and is sequentially eluted by ethanol with different concentrations, and the precipitate, namely the 1-carboxyl-2-hydroxyl-3-imino propane, is obtained by separation.

In one embodiment of the invention, the ratio of the walnut diaphragma powder and the ethanol solution in the step (1) is 1: 8 to 12, more preferably 1: 10.

in one embodiment of the present invention, the ethanol solution in step (1) is an ethanol aqueous solution, and the concentration is 70 to 100%, and more preferably 70%.

In one embodiment of the present invention, the extraction conditions in step (1) are 55-65 ℃ for 3-4h, and more preferably 60 ℃ for 4 h.

In one embodiment of the invention, the walnut diaphragma juglandis powder in the step (1) is obtained by crushing and sieving with a 40-mesh sieve.

In one embodiment of the present invention, the extraction process in step (1) may be repeated for 2-4 times.

In one embodiment of the present invention, said concentration under reduced pressure in step (1) is carried out to a solid content of 20 to 30%.

In one embodiment of the invention, the mass ratio of the filter residue to the water in the step (2) is 1: 8 to 12, more preferably 1: 10.

in one embodiment of the present invention, the extraction conditions in step (2) are 60 ℃ for 3-4 h.

In one embodiment of the present invention, the extraction process in step (2) may be repeated for 2-4 times.

In one embodiment of the present invention, said concentration under reduced pressure in step (2) is carried out to a solid content of 20 to 30%.

In one embodiment of the invention, the ethanol solution used in step (3) is an ethanol aqueous solution with the volume fraction of 0, 30%, 50% and 70% after passing through an AB-8 type macroporous resin column (10cm × 150 cm).

In one embodiment of the present invention, the ethanol solution used in step (3) passing through the MCI column comprises 0, 10, 30, 50% ethanol aqueous solution by volume fraction.

In one embodiment of the present invention, the step (3) of passing through the ODS column comprises 0, 5%, 10%, 20% by volume of an aqueous ethanol solution.

In one embodiment of the present invention, the eluted fraction is collected after each elution in step (3), and the sample is again loaded after concentration under reduced pressure to a solid content of 15 to 20%.

In one embodiment of the present invention, the column chromatography and the elution in step (3) are repeated 3 to 10 times.

The third object of the present invention is the use of the 1-carboxy-2-hydroxy-3-iminopropane according to the invention in the medical field.

In one embodiment of the invention, the application utilizes 1-carboxyl-2-hydroxy-3-imino propane with sedative and hypnotic effects.

The fourth object of the present invention is to provide a pharmaceutical preparation, the composition of which comprises the 1-carboxy-2-hydroxy-3-iminopropane according to the present invention.

The invention has the beneficial effects that:

(1) the invention discovers a new compound 1-carboxyl-2-hydroxyl-3-imino propane in the diaphragma juglandis for the first time, and the pharmacological experiment proves that the compound has good sedative-hypnotic activity.

(2) The 1-carboxy-2-hydroxy-3-iminopropane according to the present invention shortens the sleep latency by 24.62% (P <0.05) and prolongs the sleep duration by 61.49% (P < 0.05).

Drawings

FIG. 1 shows a preparation of the compound obtained in example 1¹H-NMR spectrum.

FIG. 2 shows the preparation of the compound obtained in example 1¹³C-NMR spectrum.

FIG. 3 shows the preparation of the compound obtained in example 1¹³⁵DEPT-NMR spectrum.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

Example 1

A method for extracting 1-carboxyl-2-hydroxyl-3-imino propane from diaphragma juglandis, which comprises the following steps:

(1) weighing 10kg of diaphragma juglandis, crushing, sieving with a 40-mesh sieve, and mixing according to the weight ratio of 1: adding ethanol solution with volume fraction of 70% into 10(kg/L) of the feed liquid, stirring and extracting at 60 deg.C for 4 hr, filtering, collecting filtrate, extracting the residue with ethanol again according to the above method, and collecting filtrate; mixing the filtrates, concentrating under reduced pressure to solid content of 30% to obtain Juglandis Mandshuricae ethanol extract, and freezing at-20 deg.C for storage;

(2) the filter residue after the alcohol extraction of the diaphragma juglandis is extracted is as follows: adding water into 10(kg/L) feed liquid according to the mass volume ratio, stirring and extracting for 4 hours at the temperature of 60 ℃, and filtering to obtain filtrate; extracting the filter residue with water again according to the above method, and collecting the filtrate; mixing the two filtrates, and concentrating under reduced pressure to solid content of 30%; namely the water extract of the diaphragma juglandis fructus;

(3) mixing the walnut alcohol extract obtained in the step (1) and the alcohol extract obtained in the step (2) to obtain a walnut diaphragma juglandis mixed extract; taking 1000g of a mixed extract of diaphragma juglandis fructus, loading the mixed extract on an AB-8 type macroporous resin column (10cm multiplied by 150cm), performing gradient elution by using water and 30%, 50% and 70% of ethanol in sequence, collecting a water elution part, concentrating the water elution part under reduced pressure until the solid content is 20%, loading the concentrated water to an MCI column, performing gradient elution by using water and ethanol with the volume fractions of 10%, 30% and 50% in sequence, collecting a water elution part, concentrating the solid content under reduced pressure until the solid content is 20%, loading the concentrated water to an ODS column, eluting and collecting the eluted water and ethanol with the volume fractions of 5%, 10% and 20% in sequence, then repeatedly purifying and eluting, and separating to obtain a precipitate, namely the 1-carboxyl-2-hydroxy-3-.

The resulting precipitate (white amorphous powder) was examined and the results were as follows:

FIG. 1 shows a preparation of the compound obtained in example 1¹H-NMR spectrum. As can be seen from fig. 1: the spectrum had a total of 5 hydrogen signals, of which delta_H6.63(1H, s), 4.07(1H, s), 3.86(1H, s, J ═ 4HZ), 2.79(1H, d, J ═ 15HZ), 2.63(1H, dd, J ═ 7.5,8HZ), where δ is_H4.07 is the hydrogen signal of the carbon with oxygen, 2.79 and 2.63 are-CH₂-hydrogen signal of;

FIG. 2 shows the preparation of the compound obtained in example 1¹³C-NMR spectrum. As can be seen from fig. 2: the spectrogram totally shows 4 carbon signals, namely delta 181.2(-COOH), 135.5, 70.2 and 42.5;

FIG. 3 shows the preparation of the compound obtained in example 1¹³⁵DEPT-NMR spectrum. As can be seen from fig. 3: δ 135.5 is the carbon signal of-CH-, it is inferred from chemical shifts that-CH-is attached to the imino group with a double bond, δ 70.2 is the oxygen linked carbon signal, δ 42.5 is the carbon signal of-CH 2-;

from FIGS. 1 to 3, it was confirmed that the compound 1-carboxy-2-hydroxy-3-iminopropane was indeed obtained by extraction in example 1.

Example 21 application of carboxy-2-hydroxy-3-iminopropane

The 1-carboxy-2-hydroxy-3-iminopropane obtained in example 1 was subjected to a sedative-hypnotic activity test, as follows:

after adaptively feeding 30 SPF-grade 4-week-old male ICR mice for 1 week, they were randomly divided into 3 groups of 10 mice each. The test group is respectively irrigated with a stomach test group (aqueous solution of 1-carboxyl-2-hydroxy-3-iminopropane) and a blank group (water) at a dosage of 1.0 g/kg-bw, and the positive control group is irrigated with a stomach of 2 mg/kg-bw of an estazolam aqueous solution. After 14 days of continuous gavage, the test was developed. The test was carried out in a quiet environment at around 25 ℃.

The mice were subjected to behavioral tests as follows:

1. open field experiment

Experiments were performed using an open field maze of 4 cells (50 cm x 38cm per cell) consisting of 4 living spaces, 4 mice being tested at a time. The open field space was wiped clean with alcohol before the experiment, and the experiment was started 30min after the administration of the sample group (15 min for the positive control group). In the experiment, a rat tail is lightly grasped, a mouse is placed in the middle of an open-field labyrinth of each unit, the mouse is adapted to the labyrinth for 5min, then a software start button is clicked to activate software, the moving path of the mouse is tracked, the average speed of movement, the movement time and the residence time in the central area within 10min are used as indexes, and data analysis is carried out by using EthoVision XT 11 software. After each test is finished, urine and excrement are cleaned, 5% -10% of ethanol is sprayed on the bottom surface and the inner wall of the maze, the maze is wiped completely by paper towels, the smell left by the mice is eliminated, and then the next group of mice is tested, so that the influence of the smell and residues on the next mouse is avoided.

2. Direct observation of sleep experiment

Direct sleep observation was performed the evening after the end of the open field experiment. And after the intragastric administration is carried out for 30min, monitoring, shooting and observing and recording the sleeping time of each group of mice within 12h after the intragastric administration.

3. Pentobarbital sodium induced sleep test

Before the formal experiment, the pre-experiment is carried out to determine the intraperitoneal injection dose of the sodium pentobarbital, the dose with moderate sleep duration of all mice after the intraperitoneal injection is taken as a standard, and the formal experiment is carried out according to the dose (50 mg/kg-bw). The mice are induced to sleep by injecting pentobarbital sodium into the abdominal cavity after 30min of last gastric lavage, the sleep latency period of the mice is recorded as the time from the injection of the pentobarbital sodium to the disappearance of the righting reflex, the time from the disappearance of the righting reflex to the reappearance of the righting reflex is recorded as the sleep time of the mice, and whether the latency period of each group is shortened or not and whether the sleep time is prolonged or not are observed.

The test results were as follows:

table 1 shows the results of the test of the rate of movement, the time of movement, and the residence time in the central area of the mice in the blank group, experimental group, and positive control group. As can be seen from table 1: 1-carboxy-2-hydroxy-3-iminopropane significantly reduced the locomotor speed and locomotor time of the mice (P <0.05), significantly decreasing the residence time of the mice in the central zone (P < 0.01).

TABLE 1 test results (x. + -. s) of the speed of movement, the time of movement and the residence time in the central zone of mice in the blank group, experimental group and positive control group

Note: p <0.05, P <0.01, compared to the blank group.

Table 2 shows the test results (x + -s) of the sleeping time of 12h, the latency period of pentobarbital sodium induced sleep and the sleeping time of mice in the blank group, the experimental group and the positive control group. As can be seen from table 2: the total duration of sleep in 12h of 1-carboxy-2-hydroxy-3-iminopropane was extended compared to the blank group and was statistically different (P < 0.05). The results of sleep experiments induced by sodium pentobarbital show that 1-carboxyl-2-hydroxy-3-iminopropane shortens the sleep latency by 24.62% (P <0.05) and prolongs the sleep duration by 61.49% (P < 0.05). The 1-carboxyl-2-hydroxyl-3-imino propane is proved to have remarkable sedative-hypnotic activity.

TABLE 2 test results (x + -s) of sleep duration of 12h in natural condition, sleep latency induced by pentobarbital sodium and sleep time in mice in blank group, experimental group and positive control group

Note: p <0.05, P <0.01, compared to the blank control group.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.