CN106589149B

CN106589149B - Extraction method of momordica polysaccharide, product and application thereof

Info

Publication number: CN106589149B
Application number: CN201611038930.1A
Authority: CN
Inventors: 齐素华; 马菊云; 郭鹏
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2016-11-12
Filing date: 2016-11-12
Publication date: 2020-04-14
Anticipated expiration: 2036-11-12
Also published as: CN106589149A

Abstract

The invention provides an extraction method of momordica charantia polysaccharide, a product and an application thereof, wherein the method comprises the steps of adding momordica charantia powder into water with the temperature of 90-100 ℃ according to the material-liquid ratio of 1: 35-45, regulating the pH value to keep 7.5-8.5, then carrying out ultrasonic extraction, adding α -amylase accounting for 3-10% of the mass of a substrate into a mixed solution, reacting for 15-25 min at the temperature of 35-40 ℃, transferring the whole system into water with the temperature of 90-100 ℃ for 4-6 min, adding trichloroacetic acid, filtering and concentrating to obtain a concentrated solution, and then cleaning and drying.

Description

Extraction method of momordica polysaccharide, product and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an extraction method of momordica polysaccharide, a product and an application thereof.

Background

Learning and memory ability is one of the basic abilities of human beings to recognize the objective world, and is also an important function of human brain. At present, the pressure of life and work of young people is increased due to social competition, and the phenomenon of decline of learning and memory is caused; besides various senile diseases, a considerable number of the elderly often suffer from learning and memory impairment, even learning and memory disorder. Vascular dementia can also be caused by cerebral infarction, cerebral ischemia, cerebral hemorrhage, etc. caused by cardiovascular and cerebrovascular diseases. In addition to this, learning and memory disorders caused by accidents, wounds, and the like, and even dementia are common. Regardless of the cause of the decline of brain function, the decline and disorder of learning and memory ability can be caused to different degrees.

The balsam pear has good edible value and obvious medicinal function, and is called as medicinal vegetable. Momordica polysaccharide (MCP) is a polysaccharide component extracted from Momordica charantia and has high bioactivity. At present, the application of the momordica polysaccharide is mainly focused on the aspects of enhancing immunity, resisting tumors, resisting oxidation, reducing blood sugar and the like.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above and/or other technical problems associated with the prior art methods for extracting momordica polysaccharide.

Therefore, one of the objectives of the present invention is to solve the deficiencies of the prior art and provide a method for extracting momordica polysaccharide.

The extraction method comprises the following steps of dissolving bitter gourd powder in water, adjusting the pH value to be 7.5-8.5, placing the mixture into a water bath at 90-100 ℃, simultaneously carrying out ultrasonic extraction, adding α -amylase accounting for 3-10% of the mass of a substrate into the mixed solution, reacting at 35-40 ℃ for 15-25 min, transferring the whole system into the water bath at 90-100 ℃ for 4-6 min, adding trichloroacetic acid, filtering and concentrating to obtain a concentrated solution, cleaning and drying, adding a 95% ethanol solution into the concentrated solution, stirring, carrying out low-temperature sedimentation, taking out, carrying out refrigerated centrifugation, cleaning a precipitate with acetone, carrying out refrigerated centrifugation, cleaning the precipitate with diethyl ether, carrying out refrigerated centrifugation, dissolving the solid obtained by centrifugation in water, carrying out dialysis, and carrying out refrigerated drying to obtain the bitter gourd polysaccharide.

The preferable scheme of the extraction method of the momordica polysaccharide is that: the bitter gourd powder is prepared by crushing dried bitter gourd slices, sieving with a 35-45-mesh sieve, leaching with 80% ethanol, carrying out suction filtration, keeping the vacuum degree of 0.07-0.09 MPa and the temperature of 50-60 ℃ for 30-60 min, cooling and collecting.

The preferable scheme of the extraction method of the momordica polysaccharide is that: the ultrasonic extraction is carried out for 2-4 hours under the conditions of 30-50 KHz and 40-60W of power.

The preferable scheme of the extraction method of the momordica polysaccharide is that: and adding a 95% ethanol solution into the concentrated solution and stirring, wherein the volume of the 95% ethanol solution is 2-4 times that of the concentrated solution, and stirring for 8-12 min at 100-200 rpm.

The preferable scheme of the extraction method of the momordica polysaccharide is that: adding trichloroacetic acid, namely adding a trichloroacetic acid solution with the mass fraction of 2.0-2.5% and the same volume as the whole system, and stirring at the temperature of 20-30 ℃ and the speed of 250-350 rpm for 15-25 min.

The preferable scheme of the extraction method of the momordica polysaccharide is that: and the freeze drying is to freeze the polysaccharide solution after dialysis at-85 to-75 ℃ for 1 to 3 hours, then freeze the polysaccharide solution at-45 to-35 ℃ for 38 to 42 hours, finally freeze the polysaccharide solution at-15 to-5 ℃ for 7 to 9 hours, and reduce the pressure of the system to 0.360 to 0.370mbar for dehydration.

Therefore, another object of the present invention is to solve the deficiencies of the prior art and to provide a momordica polysaccharide product.

In order to solve the technical problems, the invention provides the following technical scheme: a product of momordica charantia polysaccharide comprises, by mass, 0.04-0.08% of rhamnose, 0.16-0.20% of xylose, 0.02-0.04% of mannose, 0.6-1.0% of glucose, 0.20-0.30% of galactose and 93.0-94.0% of galacturonic acid.

Still another object of the present invention is to provide an application of momordica polysaccharide in improving learning and memory ability.

It is a further object of the present invention to provide a pharmaceutical formulation prepared from momordica polysaccharide comprising (1) an effective amount of momordica polysaccharide as an active ingredient; (2) optionally a pharmaceutically acceptable carrier.

As a preferable embodiment of the pharmaceutical preparation of the present invention, wherein: the dosage of the momordica polysaccharide is 150-300 mg of momordica polysaccharide per kg of body weight.

The invention has the beneficial effects that:

1. the momordica charantia polysaccharide extracted by the method has high biological activity, and the Morris water maze experiment shows that the momordica charantia polysaccharide has an improvement effect on learning and memory abilities of a mouse aging model caused by D-galactose; the momordica polysaccharide has an effect of improving the learning and memory ability of a rat stroke model; the Momordica charantia polysaccharide has effect in enhancing learning and memory ability of normal rat. The invention widens the application field of the momordica polysaccharide and provides experimental basis for the clinical application of the momordica polysaccharide in the aspect of preparing the medicine for improving the learning and memory ability.

2. The extraction method for extracting the momordica charantia polysaccharide has the advantages that the extraction rate of the momordica charantia polysaccharide is high, the extraction purity is high, the highest extraction rate can reach 30.02%, and the purity of the extracted momordica charantia polysaccharide can reach 80.20%.

3. The prepared momordica polysaccharide has no dark color and can be utilized without further decolorization.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a graph showing a comparison of behavioral indicators of mice in the normal group and the aging model group in example 6 (wherein A: escape latency; B: pathway efficiency; C: number of platform crossings; D: efficiency of platform-finding trajectories);

FIG. 2 is a graph showing comparison of behavioral indexes of mice in the aging model group and the positive control group (4.2%) given by the model in example 6 (wherein A: escape latency; B: path efficiency; C: number of times of crossing a platform; D: efficiency of finding a trajectory of the platform);

FIG. 3 is a graph showing comparison of behavioral indexes of mice in the group of the aging model and the group of the model administered with a low dose of Momordica charantia polysaccharide (1%) in example 6 (wherein A: escape latency; B: path efficiency; C: number of times of crossing a platform; D: efficiency of finding a trajectory of the platform);

FIG. 4 is a graph showing comparison of behavioral indexes of mice in the group of aging models and the group of high doses of Momordica charantia polysaccharide (3%) in example 6 (wherein A: escape latency; B: path efficiency; C: number of times of crossing platforms; D: efficiency of finding a trajectory of a platform);

FIG. 5 is a graph comparing behavioral indicators of rats in the normal group and the stroke model group in example 7 (wherein A: escape latency; B: path efficiency; C: number of times of crossing the platform; D: efficiency of finding the trajectory of the platform);

FIG. 6 is a graph comparing behavioral indicators of rats in the stroke model group and the model administration group in example 7 (wherein A: escape latency; B: path efficiency; C: number of times of crossing the platform; D: efficiency of finding the trajectory of the platform);

FIG. 7 is a graph comparing behavioral indicators of rats in the normal group and the administered group in example 8 (wherein A: escape latency; B: path efficiency; C: number of times of crossing the platform; D: efficiency of finding the trajectory of the platform);

FIG. 8 is a standard high performance liquid chromatogram in which 1 is mannose, 2 is ribose, 3 is rhamnose, 4 is glucuronic acid, 5 is galacturonic acid, 6 is glucose, 7 is galactose, 8 is xylose, 9 is arabinose, and 10 is fucose;

FIG. 9 is a high performance liquid chromatogram obtained after sample injection of a Momordica charantia polysaccharide sample prepared in example 1, wherein 5 is galacturonic acid, 6 is glucose, and 7 is galactose;

FIG. 10 is a LC-MS plot of rhamnose (a), xylose (b) in the polysaccharide sample of Momordica charantia prepared in example 1;

FIG. 11 is a LC-MS plot of mannose (c), glucose (d) in a sample of Momordica charantia polysaccharides prepared in example 1;

FIG. 12 is a LC-MS graph of galactose (e) and galacturonic acid (f) in the polysaccharide samples of Momordica charantia prepared in example 1.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

Crushing the dried bitter gourd slices, sieving with a 40-mesh sieve, leaching with 80% ethanol, filtering, keeping the vacuum degree of 0.08MPa and the temperature of 55 deg.C for 35min, and cooling to obtain bitter gourd powder.

Adding 10g of the prepared bitter gourd powder into 95 ℃ water (40 ml of water is prepared for each gram of bitter gourd powder), adjusting the pH value to 8.0, performing ultrasonic extraction for 3h under the condition of 40KHz and 50W, adding α -amylase accounting for 5% of the mass of a substrate into the mixed solution, reacting for 20min at 37 ℃, transferring the whole system into 95 ℃ water bath for 5min, adding trichloroacetic acid solution with the mass fraction of 2.5% and the volume equal to that of the whole system into the water bath, stirring for 20min at 300rpm at 25 ℃, filtering and concentrating to obtain concentrated solution, concentrating the filtrate to 1/10 mbar of the raw material solution, wherein 300ml of 95% ethanol solution is added into the concentrated solution, stirring for 10min at 150rpm, placing the concentrated solution in a refrigerator at 4 ℃ for overnight precipitation, taking out, performing refrigerated centrifugation for 20min at 8000rpm, taking out the precipitate, cleaning the precipitate with acetone, centrifuging for 20min at 8000rpm, taking out the precipitate, performing refrigerated dialysis for 20min at 8000rpm, centrifuging the polysaccharide for 20min, performing refrigerated dialysis for 20 h at 40-80 hours, and finally performing refrigerated dialysis for the polysaccharide solution at 40-80 hours, and performing refrigerated dialysis for 20 h at 40-40 ℃ for a refrigerated dialyzing to obtain polysaccharide.

The components of the prepared momordica charantia polysaccharide sample are analyzed, as shown in fig. 8-12, and the momordica charantia polysaccharide sample prepared in the example 1 is obtained by calculation and arrangement, and consists of 6 monosaccharides, namely rhamnose, xylose, mannose, glucose, galactose and galacturonic acid, wherein the content percentages of the monosaccharides are 0.06% of rhamnose, 0.18% of xylose, 0.03% of mannose, 0.8% of glucose, 0.28% of galactose and 93.7% of galacturonic acid.

Example 2

Crushing the dried bitter gourd slices, sieving with a 35-mesh sieve, leaching with 80% ethanol, filtering, keeping the vacuum degree of 0.07MPa at 60 deg.C for 60min, cooling, and collecting to obtain bitter gourd powder.

Adding 10g fructus Momordicae Charantiae powder into 95 deg.C water (35 ml per gram fructus Momordicae Charantiae powder), adjusting pH to 7.5, performing ultrasonic extraction for 2 hr under 30KHz and power of 60W, adding α -amylase 3% of the substrate mass into the mixed solution, reacting at 35 deg.C for 20min, transferring the whole system into 95 deg.C water bath for 5min, adding trichloroacetic acid solution 2.0% of the same volume as the whole system, stirring at 25 deg.C and 300rpm for 15min, filtering, concentrating to obtain concentrated solution, concentrating the filtrate to 1/10 mbar of the raw material solution, adding 200ml 95% ethanol solution into the concentrated solution, stirring at 100rpm for 12min, standing in 4 deg.C refrigerator for overnight precipitation, taking out, performing refrigerated centrifugation at 7500rpm for 25min, taking out the precipitate, cleaning with acetone, centrifuging at 8500rpm for 15min, taking out the precipitate, cleaning with diethyl ether, centrifuging at 8500rpm for 15min, centrifuging at 8500 deg.C, dialyzing the polysaccharide, cooling, dialyzing at 360-0 mbar, dialyzing at 1-0 mbar, and finally obtaining polysaccharide, and dialyzing at 1-0 mbar.

The components of the prepared momordica charantia polysaccharide sample are analyzed, and the momordica charantia polysaccharide sample prepared in the example 2 is obtained by calculation and arrangement, and consists of 6 monosaccharides including rhamnose, xylose, mannose, glucose, galactose and galacturonic acid, wherein the content percentages of the monosaccharides are 0.08% of rhamnose, 0.17% of xylose, 0.04% of mannose, 1.0% of glucose, 0.20% of galactose and 94.0% of galacturonic acid.

Example 3

Crushing the dried bitter gourd slices, sieving with a 45-mesh sieve, leaching with 80% ethanol, filtering, keeping the vacuum degree of 0.07MPa at 60 deg.C for 30min, cooling, and collecting to obtain bitter gourd powder.

Adding 10g fructus Momordicae Charantiae powder into 95 deg.C water (45 ml per gram fructus Momordicae Charantiae powder), adjusting pH to 8.0, performing ultrasonic extraction for 2 hr under 50KHz and power of 60W, adding α -amylase 10% of the substrate mass into the mixed solution, reacting at 37 deg.C for 25min, transferring the whole system into 95 deg.C water bath for 6min, adding trichloroacetic acid solution 2.0% of the same volume as the whole system, stirring at 25 deg.C and 250rpm for 25min, filtering, concentrating to obtain concentrated solution, concentrating the filtrate to 1/10 mbar of the raw material solution, adding 400ml 95% ethanol solution into the concentrated solution, stirring at 150rpm for 10min, standing in 4 deg.C refrigerator for overnight precipitation, taking out, performing refrigerated centrifugation at 7500rpm for 25min, taking out the precipitate, cleaning with acetone, centrifuging at 8500rpm for 15min, taking out the precipitate, cleaning with diethyl ether, centrifuging at 8000 min, performing refrigerated dialysis at 8000rpm, and dialyzing at 75-75 deg.C for 5 hr to obtain polysaccharide, and finally performing refrigerated dialysis at 75-5 hr.

The components of the prepared momordica charantia polysaccharide sample are analyzed, and the momordica charantia polysaccharide sample prepared in the example 2 is obtained by calculation and arrangement, and consists of 6 monosaccharides including rhamnose, xylose, mannose, glucose, galactose and galacturonic acid, wherein the content percentages of the monosaccharides are 0.04% of rhamnose, 0.20% of xylose, 0.03% of mannose, 0.95% of glucose, 0.30% of galactose and 93.0% of galacturonic acid.

A great deal of research has been done on these monomers, but the results are less than ideal. The research shows that the action effect of each monomer is far less remarkable than that of the momordica polysaccharide compound.

Example 4 (comparative example)

Carrying out hot water bath reflux extraction on the commercially available bitter gourd powder by using 80% ethanol, wherein the material-liquid ratio is 1:3, extracting for 2h at 80 ℃, carrying out suction filtration to obtain bitter gourd powder cakes, heating and drying in vacuum, and then placing in a dryer for storage. Taking 10g of balsam pear powder cakes, adjusting the pH value to 8.0 according to the material-liquid ratio of 1:50, extracting with hot water at 95 ℃ for 5h, filtering, concentrating the filtrate to 1/10 (about 100 ml) of the raw material liquid, and adding 95% ethanol for alcohol precipitation (the volume of the balsam pear liquid is 1:3 of the ethanol). After stirring uniformly, the mixture is placed in a refrigerator at 4 ℃ and stands overnight. The next day, the alcohol precipitated crude sugar was centrifuged by a refrigerated centrifuge (8000rpm, 20min), and the precipitate was washed twice with ethanol, refrigerated, centrifuged, and stored in a desiccator under vacuum.

Example 5

The content of the momordica charantia polysaccharide products prepared in the embodiments 1 to 4 is measured according to a phenol sulfate method, the momordica charantia polysaccharide products prepared in the embodiments 1 to 4 have a red color reaction when measured by the phenol sulfate method, the extraction rate and the purity of the momordica charantia polysaccharide products prepared in the embodiments 1 to 4 are calculated according to OD values measured by the polysaccharide color reaction, and the calculation results are shown in the following table.

Group of	Example 1	Example 2	Example 3	Example 4
					Polysaccharide extraction Rate/% of Momordica charantia	30.02	26.62	28.44	8.70
Purity/% of Momordica charantia polysaccharide	80.20	75.21	76.79	50.21

The color comparison of the momordica charantia polysaccharide products prepared in examples 1-4 shows that the momordica charantia polysaccharide product prepared in example 4 is dark in color and can be used only after further dehydration, while the momordica charantia polysaccharide products prepared in examples 1-3 can be directly used.

Example 6

Effect of Momordica charantia polysaccharides (prepared in example 1) on learning and memory abilities of mouse aging model due to D-galactose

1. Laboratory animal

Male Kunming Mice (KM), 25-40 g in weight, clean grade, provided by the laboratory animals center of Xuzhou medical university, laboratory animals production license: SCXK (SU) 2010-0003.

2. Primary reagent

D-galactose: purchased from Biotechnology engineering (Shanghai) Ltd;

piracetam (Naofukang): purchased from the national pharmaceutical factory of Jinling, pharmaceutical industries, Ltd.

3. Instrument for measuring the position of a moving object

Morris water maze (model: XR-XM101-R), ANY-maze animal behavior analysis system.

4. Data processing

The experimental data are expressed as means ± standard deviation (Mean ± SD) using SPSS16.0 analytical statistical software. Statistical analysis adopts One-Way analysis of variance (One Way ANOVA), Dunnet test is adopted for comparing a plurality of experimental groups with a control group, q test is adopted for pairwise comparison between the experimental groups, and P <0.05 represents that the difference has statistical significance.

5. Establishment, grouping and administration of aging model of experimental animal

Kunming mice were randomly divided into: a blank control group, a D-galactose model group (an aging model group), a momordica polysaccharide high-dose group (300mg/kg), a momordica polysaccharide low-dose group (100mg/kg) and a piracetam (Naofukang, 420mg/kg) positive control group, wherein each group contains 25 momordica polysaccharide. One week after acclimation feeding, placebo group: injecting normal saline 5ml/kg subcutaneously at the abdomen every day, and infusing normal saline 10ml/kg after 1 h; aging model group: 5ml/kg of 2.5 percent D-galactose is injected subcutaneously into the abdomen every day, and 10ml/kg of normal saline is perfused after 1 hour; positive control group: 5ml/kg of 2.5 percent D-galactose is injected subcutaneously at the abdomen every day, and 10ml/kg of 4.2 percent piracetam is perfused after 1 hour; administration group: 5ml/kg of 2.5 percent D-galactose is injected subcutaneously into the abdomen every day, and 10ml/kg of momordica polysaccharide with different doses is injected into the stomach after 1 hour. Each group was administered 1 time daily for 40 days. Animals were weighed once every 3 days and the dosage was adjusted according to changes in body weight.

6. Water maze learning and memory behavior test

After 40 days of Kunming mouse perfusion treatment, a Morris water maze test method is adopted to carry out a positioning navigation and space exploration test. The experiment was carried out for a total of 7 days during which the water temperature was maintained at (22 + -0.5) ° c while maintaining consistency of the ambient parameters of the experiment per day.

The first 5 days of continuous positioning navigation training, 4 equidistant points on the wall of the water pool are taken as test starting points, the water pool is equally divided into 4 quadrants, the mouse is respectively put into the water from 4 water inlet points facing the wall of the water pool for four times every day, and the required time for successfully entering the platform (the mouse finds the platform and stays the platform for 5 s) within 60s is measured, namely the escape latency is obtained. The shorter the escape latency, the stronger the learning ability of the mouse. If the mice were unable to successfully mount the platform within 60s, the experimenter brings them up and holds them for 10s, recording the escape latency as 60s and the interval between each training as 120 s.

And (3) space exploration test: and after 5 days of the water maze experiment, removing the platform, respectively putting the mouse into water from the middle point of the opposite side pool wall of the quadrant of the original platform on 6 th and 7 th days, recording the swimming time and the swimming track of the mouse in the quadrant of the original platform within 1min, and calculating the number of times that the mouse jumps over the platform and the swimming time of each quadrant. The more number of platform crossings indicates the stronger memory of the mouse.

7. Results of the experiment

The water maze test results are shown in the first graph (namely A, E, I and M) of each graph 1-4 respectively, the latency of each group of mice in the experiment shows a descending trend in the 5-day positioning navigation training, although the latency is increased in the 5 th day, the curves of each group are consistent, and the mean value of the latency of the last day of each group is smaller than that of the mice in the beginning of the training, so that the mice in each group are prompted to have normal learning ability. In addition, the baseline of each treatment group (the momordica polysaccharide low-dose group, the momordica polysaccharide high-dose group, and the piracetam positive control group) was always below the aging model group during the training process, the momordica polysaccharide low-dose group and the piracetam positive control group were above the blank group after day 3, and the momordica polysaccharide high-dose group was always below the blank group.

And the third graph (namely C, G, K and Q) in each of the graphs 1-4 is that after 5 days of training, the platform is removed, the times of crossing the quadrant of the platform in 60s of the mice on the 6 th and 7 th days are recorded, the memory capacity of the mice to the original platform is inspected, and the times of crossing the platform are obviously increased compared with the low-dose momordica charantia polysaccharide group, the high-dose momordica charantia polysaccharide group and the piracetam positive control group.

The second graph (F, J, N) of each of fig. 2-4 is the pathway efficiency of the first entry into the target quadrant, and the learning ability of the mice was examined, and comparing the different graphs, it can be seen that the pathway efficiency was improved in the high momordica polysaccharide dose group compared to the aging model group, the low momordica polysaccharide dose group, and the piracetam positive control group.

Therefore, the results obtained with reference to fig. 1, 2, 3 and 4 are:

(1) for the momordica polysaccharide prepared in example 1, mice in both the low dose group (100mg/kg) and the high dose group (300mg/kg) had enhanced memory learning ability compared to the aging model group;

(2) for the momordica polysaccharide prepared in example 1, the learning and memory ability of the high dose group (300mg/kg) mice is enhanced compared with the positive control group piracetam (Naofukang, 420 mg/kg);

(3) with respect to the polysaccharide isolated from Momordica charantia prepared in example 1, the learning and memory abilities of the high dose group (300mg/kg) mice were enhanced as compared with the low dose group (100 mg/kg).

Example 7

Effect of Momordica charantia polysaccharide (prepared in example 1) on learning and memory ability of rat Stroke model

1. Establishment, grouping and administration of experimental animal models

SD rats were randomly divided into: blank group, stroke model group, model administration group, 30 per group. Since the number of rats was too large, the experiment was performed in 2 batches: the first batch was a blank and stroke model group, and the second batch was a stroke model group and a model administration group.

Establishment of artery occlusion line embolism Model (MCAO) in SD rat cerebral apoplexy: after intraperitoneal injection of anesthesia with 20% chloral hydrate, a median incision was made in the neck, surrounding tissues were isolated, the Common Carotid Artery (CCA) was exposed and ligated proximal, the External Carotid Artery (ECA), the Internal Carotid Artery (ICA), and the pterygopalatine artery were isolated distal, and the ECA and pterygopalatine arteries were ligated, keeping the only branch of the common carotid artery open. And closing the CCA distal end by using a arteriole clamp, cutting a small opening at the proximal end of the CCA distal end by holding anatomical scissors at an angle of 45 degrees, inserting a standard thread plug corresponding to the body weight from the CCA cut opening carefully, loosening the arteriole clamp, adjusting the thread inlet direction to avoid pterygopalatine artery, entering the ICA neck inner segment until the beginning of the middle cerebral artery, and timing. The insertion depth of the suture pin was about 17mm according to the animal body weight, until slight resistance was met, and then the ICA was fastened and fixed, and the skin was sutured in full layers. After 90 minutes of cerebral ischemia, the rat was taken out of the suppository and re-irrigated for 2 hours.

Dissolving Momordica charantia polysaccharide in ddH₂In O, the bitter gourd polysaccharide with the gavage of 200mg/kg is administrated 30min before the model, and the water maze experiment is carried out after the model is administrated once a day and continuously gavage for 2 weeks.

2. Water maze learning and memory behavior test

In order to evaluate the effect of momordica charantia polysaccharide in improving neurological functions after cerebral ischemia, the neurological functions of cerebral apoplexy rats were evaluated in a behavioral manner, which was divided into sensorimotor behavior evaluation and cognitive behavior evaluation. Aiming at sensorimotor behaviors, a classical Longa scoring method is adopted to carry out preliminary model identification and screening on MCAO rats, 50 rats are selected in total, then the water maze (Morris water maze) cognitive behavior detection method is applied, 25 rats are randomly grouped and dosed to the screened rats, and the learning and memory abilities are detected.

The behavioral experiment is divided into 2 batches, the number of rats in each group is counted finally (n is more than or equal to 20), and finally, the rats are compared two by two: blank and stroke model MCAO (see fig. 3A-D), MCAO and MCAO + charantin (see fig. 4A-D). Each group detects four indexes: escape latency, efficiency of a path entering a target quadrant for the first time, number of times of crossing a platform quadrant, and track of a sought platform. The first 2 indexes are contained in the first part of the water maze experiment to detect and position navigation and mainly detect learning ability, and the last 2 indexes belong to the second part of the detection space exploration and mainly detect memory ability. The influence of the momordica charantia polysaccharide on learning and memory of rats after stroke is judged by combining the behavioral indexes.

3. Results of the experiment

To prove the reliability of the stroke model, the behavioural tests of the rats in the first blank group and the model group were performed, as shown in fig. 5A-D, the MCAO model group rats had a significantly improved average latency, significantly reduced pathway efficiency, dramatically reduced number of times of crossing the platform quadrant, and almost no swimming trajectory involved the region of the target quadrant, compared to the blank group rats. The four detection indexes are integrated to obtain an experimental result: the MCAO model group rats have impaired learning and memory functions.

Similarly, the results of the comparison between the second MCAO model group and the model administration group are shown in fig. 6A-D, and it can be seen from the four graphs that the mean latency of the MCAO model administration group rats is relatively shorter, the path efficiency is improved, the number of times of platform crossing is obviously increased, and the swimming tracks are denser than those of the MCAO model group rats. The four detection indexes are integrated to obtain an experimental result: the learning and memory abilities of the rats in the model administration group (the momordica polysaccharide prepared in example 1) are improved compared with those of the rats in the simple model group.

Example 8

Momordica charantia polysaccharide (prepared in example 1) for improving learning and memory ability of normal rat

1. Grouping and administration of laboratory animals

SD rats were randomly divided into: normal group (i.e. control group), administration group, 25 per group. Dissolution of Momordica charantia polysaccharide in ddH₂In O, the bitter gourd polysaccharide of 200mg/kg is administrated once a day, and the water maze experiment is carried out for 2 weeks after continuous gavage.

2. Water maze test results

As shown in fig. 7A-D, it can be seen from the graph a that the latency of the rats in the normal group and the rats in the administration group both showed a downward trend in the 5-day positioning navigation training, although the latency was increased by the 5 th day, the curves of the two groups were more consistent, and the mean value of the latency of the last day of each group was smaller than that of the initial training, indicating that the rats in the two groups both had normal learning ability, and in addition, the baseline of the administration group was always below the normal group during the training; graph B compares the path efficiencies of the two groups, and the path efficiency of the administration group is improved. The two figures combined with A, B show that rats in the group to which the Momordica charantia polysaccharide prepared in example 1 was administered have improved learning ability compared with those in the normal group.

C, after 5 days of training, removing the platform, recording the times of the rat passing through the quadrant where the platform is located within 60s, and inspecting the memory capacity of the rat on the original platform, wherein the times of the administration group are obviously increased compared with the times of the normal group as can be seen from a bar chart; graph D shows representative swimming traces of two groups of rats, and it is apparent from the graph that the swimming traces of the rats in the administered group are more concentrated in the target quadrant than those in the normal group, and therefore, the memory of the rats in the administered group (momordica charantia polysaccharide prepared in example 1) is enhanced as compared with that in the normal group as shown in the graph C, D.

Example 9

The experiments (the dose of the momordica charantia polysaccharide is 3%) carried out in examples 6 to 8 on the momordica charantia polysaccharide prepared in examples 2 to 4 comprise ① influence on learning and memory ability of a mouse aging model caused by D-galactose ② influence on learning and memory ability of a rat stroke model ③ influence of the momordica charantia polysaccharide on the learning and memory ability of a normal rat.

The method comprises the steps of detecting three indexes including escape latency, path efficiency entering a target quadrant for the first time and the number of times of crossing a platform quadrant, comparing index data of ①②③ experiments of examples 2-4 with corresponding index data of example 1 one by one, taking the index data of example 1 as a reference, calculating a multiple relation between the effect achieved by the examples 2-4 and the corresponding index data of example 1 in percentage, wherein the time dimension data is data, and the result is the average of average effect index percentage of each day.

The results are shown in the following tables, respectively.

① influence on learning and memory ability of mouse aging model caused by D-galactose

Detecting the index	Example 1	Example 2	Example 3	Example 4
					Escape latency	100％	90.5％	95.5％	50.5％
Path efficiency for first entry into target quadrant	100％	85.6％	92.6％	60.8％
					Number of passes through platform quadrant	100％	100％	95.0％	85.6％

② influence of Momordica charantia polysaccharide on learning and memory ability of rat apoplexy model

③ improvement of learning and memory ability of rat with normal momordica charantia polysaccharide

Detecting the index	Example 1	Example 2	Example 3	Example 4
					Escape latency	100％	96.0％	96.5％	70.2％
Path efficiency for first entry into target quadrant	100％	92.4％	94.0％	60.0％
					Number of passes through platform quadrant	100％	100％	95.0％	75.5％

Therefore, the momordica polysaccharide product prepared in the example 1 has the best effect on learning and memory capacity, and compared with the momordica polysaccharide product prepared by the traditional method, the momordica polysaccharide product prepared in the examples 2 and 3 times has the outstanding effect on enhancing the learning and memory capacity.

After the starch and the protein are separated, the protein disulfide bonds in the separated bitter gourd powder are scattered and recombined, and the trans-action is weakened, so that the starch and the protein are separated by adopting α -amylase, the enzyme ratio is controlled to be 3-10%, the reaction of the organic solvent TCA sedimentation protein is mild, the conformational change speed is slow, and the influence on the bitter gourd polysaccharide is small.

In the freeze drying process, water in the balsam pear polysaccharide extracting solution is directly sublimated in an ice crystal state, a framework of the balsam pear polysaccharide extracting solution maintains the original shape, and micropores are formed in the ice crystal part. The freezing time is too short, the freezing temperature is too high, and ice crystals cannot be generated; the freezing time is too long, the freezing temperature is too low, the color of the polysaccharide is deepened, the biological activity of the polysaccharide is also reduced, and the required energy consumption is also increased.

In conclusion, the momordica charantia polysaccharide extracted by the method has high biological activity, and the Morris water maze experiment shows that the momordica charantia polysaccharide has an improvement effect on learning and memory abilities of mouse aging models caused by D-galactose; the momordica polysaccharide has an effect of improving the learning and memory ability of a rat stroke model; the Momordica charantia polysaccharide has effect in improving learning and memory ability of normal mice. The invention widens the application field of the momordica polysaccharide and provides experimental basis for the clinical application of the momordica polysaccharide in the aspect of preparing the medicine for improving the learning and memory ability.

According to the method for extracting the momordica polysaccharide, provided by the invention, the extraction rate and the purity of the momordica polysaccharide are high, the extraction rate can reach 30.02% at most, and the purity of the extracted momordica polysaccharide can reach 80.20%; the prepared momordica polysaccharide has no dark color and can be utilized without further decolorization.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for extracting polysaccharide from fructus Momordicae Charantiae comprises pulverizing dried fructus Momordicae Charantiae, sieving with 40 mesh sieve, extracting with 80% ethanol, vacuum filtering, maintaining at 55 deg.C under 0.08MPa for 35min, cooling to obtain fructus Momordicae Charantiae powder, adding 10g of fructus Momordicae Charantiae powder into 95 deg.C water, regulating pH to 8.0, performing ultrasonic extraction at 40KHz and 50W for 3 hr, adding α -amylase 5% of substrate mass, reacting at 37 deg.C for 20min, transferring the whole system into 95 deg.C water bath for 5min, adding trichloroacetic acid solution 2.5% of the whole system mass fraction, stirring at 25 deg.C for 20min at 300rpm, filtering, concentrating the filtrate to 1/10% of the raw material solution, adding 95% ethanol solution 300ml into the concentrated solution, stirring at 150rpm for 10min for 20min, centrifuging at 20min for 20min, centrifuging at 8000-8000 hr, freeze-dialyzing at 8000 hr, centrifuging at 20-8000 hr, freeze-dialyzing at 8000 hr for 20 hr at 8000 hr, freeze-20 hr, centrifuging at 8000 hr, dialyzing at 8000 hr to obtain polysaccharide, freeze-dialyzing at 30 hr, freeze-10 rpm, centrifuging at 8000 hr to obtain polysaccharide precipitate at 8000 hr, freeze-20 hr, freeze dialyzing at 8000 hr at 150rpm, freeze-60 hr;

the prepared momordica polysaccharide comprises rhamnose, xylose, mannose, glucose, galactose and galacturonic acid, and the content percentages are as follows: rhamnose 0.06%, xylose 0.18%, mannose 0.03%, glucose 0.8%, galactose 0.28% and galacturonic acid 93.7%.

2. A pharmaceutical formulation, comprising,

(1) an effective amount of the polysaccharide of Momordica charantia of claim 1 as an active ingredient;

(2) optionally a pharmaceutically acceptable carrier.

3. A pharmaceutical formulation according to claim 2, wherein the dose of Momordica charantia polysaccharide is 150 to 300mg Momordica charantia polysaccharide/kg body weight.