CN112961262A

CN112961262A - Passiflora edulis pericarp acidic polysaccharide, preparation method and application thereof

Info

Publication number: CN112961262A
Application number: CN202110404688.XA
Authority: CN
Inventors: 孙永进; 李霞; 彭琴; 关媛; 李丽芬
Original assignee: Guilin University of Technology
Current assignee: Guilin University of Technology
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-06-15

Abstract

The invention discloses passion fruit peel acidic polysaccharide, a preparation method and application thereof, and belongs to the technical field of plant extraction and medicines. The preparation method of the passion fruit peel acidic polysaccharide comprises the following steps: pulverizing passion fruit peel, adding ethanol, condensing, reflux-extracting, collecting residue, drying, soaking in water, extracting, mixing extractive solutions, concentrating, cooling, adding 95% ethanol, precipitating, and centrifuging to obtain passion fruit peel crude polysaccharide; performing chromatography with DEAE cellulose ion exchange column, sequentially eluting with distilled water and NaCl solution, separating and purifying to obtain passion fruit peel acidic polysaccharide. The passion fruit peel acidic polysaccharide prepared by the method has the activity of reducing blood sugar, and can be used as a medicine for treating diabetes.

Description

Passiflora edulis pericarp acidic polysaccharide, preparation method and application thereof

Technical Field

The invention relates to passion fruit peel acidic polysaccharide, a preparation method and application thereof, and belongs to the field of plant extraction and the technical field of medicines.

Background

Polysaccharides are sugars which are present in nature in abundant resources, and are widely present in organisms such as plants, animals, and microorganisms. The polysaccharide as a natural polymer extract almost without toxic and side effects has unique effects in preventing and treating many diseases, especially some stubborn diseases and stubborn diseases, such as tumors, hypoimmunity, hyperglycemia, aging, viral diseases and the like.

Passiflora edulis, also known as passion fruit, is a subtropical special fruit, has high fruit pulp acidity, rich flavor and rich nutrition, and is an excellent subtropical fruit juice processing raw material. However, after the pulp is processed by the passion flower processing industry, the vast majority of the peel is discarded. The fruit peel as the processing waste accounts for about 50 percent of the whole passion fruit, the waste of the fruit peel not only increases the enterprise cost and reduces the benefit, but also wastes a large amount of agricultural resources and pollutes the environment, and the utilization of the fruit peel is very necessary and significant. The existing research shows that the polysaccharide in the passion fruit peel is one of the main functional components, and is highly concerned by scholars at home and abroad because the passion fruit peel has multiple active functions of antianxiety, sedation, anti-inflammation, anti-addiction, antioxidation, cell immunity enhancement, tumor inhibition, blood sugar reduction and the like. It has been demonstrated that polysaccharides have a wide range of biological effects, and are directly related to acidic polysaccharides contained therein, which are the main components for their functions, and the amount of sulfate groups contained in the polysaccharides determines the strength and weakness of the functions of the polysaccharides.

The passion fruit peel is a abundant biological material which is abandoned by the food processing industry, and although the activity of polysaccharide ingredients in the passion fruit peel is researched at present, the extraction preparation of the main active ingredient acidic polysaccharide and the biological function of the acidic polysaccharide are researched less. Therefore, the development of the passion fruit peel not only can change waste into valuable to obtain economic benefits, but also can improve the environment to obtain good social benefits, and more importantly, the development of the potential medicinal value of the passion fruit peel has profound significance and broad prospect for fully utilizing resources and expanding medicine sources.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of passion fruit peel acidic polysaccharide.

The second purpose of the invention is to provide the passion fruit peel acidic polysaccharide prepared by any one of the preparation methods.

The third purpose of the invention is to provide the application of the passion fruit peel acidic polysaccharide in preparing a pharmaceutical composition.

A fourth object of the present invention is to provide a pharmaceutical composition.

In order to achieve the purpose, the invention provides the following scheme:

a preparation method of passion fruit peel acidic polysaccharide comprises the following steps:

(1) pulverizing dried passion fruit peel, sieving, extracting with 80% ethanol under reflux, collecting residue, and drying;

(2) mixing the residue obtained in the step (1) with water according to a feed-liquid ratio of 1 g: mixing 16mL of the mixed solution, soaking overnight, extracting, combining concentrated extracting solutions, cooling, and adding 95% ethanol with three times of volume;

(3) filtering the ethanol precipitated extracting solution prepared in the step (2), centrifuging, and freeze-drying the precipitate to obtain crude passion fruit peel polysaccharide;

(4) and (3) dissolving the passion fruit peel crude polysaccharide obtained in the step (3) in Water, carrying out DEAE-cellulose ion exchange chromatography column chromatography separation, eluting with distilled Water, discarding the eluent, eluting with a chloride solution, collecting the eluent, concentrating, dialyzing, and freeze-drying to obtain the passion fruit peel acidic polysaccharide (WPEP).

Further, in the step (1), the extraction is performed for 1-3 times by condensation reflux, and each time lasts for 1-3 hours.

Further, the extraction temperature in the step (2) is 90-120 ℃, and the extraction is carried out for 1-3 times, 2-4 hours each time.

Further, in the step (3), the filtered extracting solution is centrifuged for 10-20 min under the condition of 3000-5000 rpm.

Further, in the step (4), the elution flow rate is 15mL/min, the chloride solution is 0.7mol/L NaCl solution, and the running water dialysis is carried out for 48 hours.

Further, the chromatographic separation in the step (4) adopts a CL-type DEAE-cellulose ion exchange chromatographic column.

Meanwhile, the invention claims the passion fruit peel acidic polysaccharide prepared by the preparation method.

Furthermore, the invention claims the application of the passion fruit peel acidic polysaccharide in the preparation of hypoglycemic drugs or health care products.

Furthermore, the dosage form of the medicine or the health care product is tablets, granules, hard capsules, soft capsules, oral liquid, powder, mixture or pills.

The invention also claims a pharmaceutical composition which contains the passion fruit peel acidic polysaccharide and a pharmaceutically acceptable carrier or auxiliary material.

The invention discloses the following technical effects:

1) the invention innovatively utilizes the passion fruit peel biomass resources at a high value to prepare the passion fruit peel acidic polysaccharide with the blood sugar reducing effect, and the prepared passion fruit peel acidic polysaccharide has a good application prospect.

2) The method comprises the steps of alcohol extraction, water extraction, alcohol precipitation, precipitation centrifugation and the like, impurities in passion fruit peel and proteins, carbohydrates, flavones, cellulose, polyphenols and the like contained in the passion fruit peel are separated, DEAE cellulose ion exchange column chromatography is used, elution is carried out by using distilled water, neutral polysaccharide is eluted to achieve the purpose of coarse extraction and fine extraction, then sodium chloride solution with the concentration of 0.7mol/L is used for eluting acid polysaccharide, and the concentrated eluent is subjected to running water dialysis, so that sodium chloride and micromolecular impurities in the acid polysaccharide can be removed.

3) The method for preparing the passion fruit peel acidic polysaccharide is to stir at a certain temperature and extract by DEAE cellulose ion exchange column chromatography, saves intermediate steps for removing protein and other small molecular impurities, reduces extraction time and improves utilization rate compared with the traditional extraction process.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described 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 without inventive exercise.

FIG. 1 is a graph showing the change in daily food intake of mice in each group;

FIG. 2 is a graph showing the change in daily weight gain of mice in each group;

FIG. 3 is the blood glucose levels in the mice of each group during the first week;

FIG. 4 is the blood glucose levels in the four week group of mice;

FIG. 5 shows the serum insulin levels of the mice of the fourth week group;

FIG. 6 shows the serum Triglyceride (TG) content of mice in the peripheral groups;

FIG. 7 is a graph showing the change in blood glucose values after each group of mice received insulin.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The method for measuring the uronic acid content, daily feed intake, daily weight gain, blood sugar content, insulin content in serum, triglyceride content and blood sugar level in the passion fruit peel acidic polysaccharide in the animal experiment is a conventional technical means in the field, is not an inventive main point, and is not described herein any more.

The percentage numbers of the 95% ethanol, the 80% ethanol and the like are volume fractions unless otherwise specified.

The technical solution of the present invention is further illustrated by the following examples.

Extraction and determination of passion fruit peel acidic polysaccharide

Example 1

(1) Pulverizing 100g dried passion fruit peel, sieving, extracting with 80% ethanol at 85 deg.C under reflux for 3 times, each for 2 hr, collecting residue, and drying; taking the residue and water according to the material-liquid ratio of 1 g: mixing 16mL of the raw materials, soaking overnight, extracting for 2 times at 90 ℃ for 4 hours each time, mixing the extracting solutions, concentrating the extracting solution to be viscous, cooling, and adding 95% ethanol with three times of volume; filtering the ethanol precipitated extractive solution, centrifuging at 4000rpm for 15min, and freeze drying the precipitate to obtain crude Passiflora edulis pericarp polysaccharide;

(2) dissolving 2g of crude passion fruit peel polysaccharide in 200mdL water, loading the crude passion fruit peel polysaccharide on a CL-type DEAE-cellulose ion exchange chromatographic column, eluting with distilled water at the flow rate of 15mL/min, discarding the eluent, eluting with a NaCl aqueous solution with the concentration of 0.7mol/L, collecting the eluent, concentrating, dialyzing with running water for 48h, and freeze-drying to obtain 0.228g of passion fruit peel acidic polysaccharide (WPEP-2), wherein the yield is 11.4%;

(3) the content of uronic acid in the passion fruit peel acidic polysaccharide is determined by adopting an m-hydroxybiphenyl method, and the purity of the obtained acidic polysaccharide is 77.14%.

Example 2

(1) Pulverizing 100g dried passion fruit peel, sieving, extracting with 80% ethanol at 85 deg.C under reflux for 3 times, each for 2 hr, collecting residue, and drying; taking the residue and water according to the material-liquid ratio of 1 g: mixing 16mL of the raw materials, soaking overnight, extracting for 2 times at 100 ℃ for 3 hours each time, mixing the extracting solutions, concentrating the extracting solution to be viscous, cooling, and adding 95% ethanol with three times of volume; filtering the ethanol precipitated extractive solution, centrifuging at 4000rpm for 15min, and freeze drying the precipitate to obtain crude Passiflora edulis pericarp polysaccharide;

(2) dissolving 2g of crude passion fruit peel polysaccharide in water, loading the crude passion fruit peel polysaccharide to a CL-type DEAE-cellulose ion exchange chromatographic column, eluting with distilled water at the flow rate of 15mL/min, discarding the eluent, sequentially eluting with a NaCl aqueous solution with the concentration of 0.7mol/L, collecting the eluent for concentration, dialyzing with running water for 48 hours, and freeze-drying to obtain 0.206g of passion fruit peel acidic polysaccharide (WPEP-2), wherein the yield is 10.3%;

(3) the content of uronic acid in the passion fruit peel acidic polysaccharide is determined by adopting an m-hydroxybiphenyl method, and the purity of the obtained acidic polysaccharide is 75.03%.

Example 3

(1) Pulverizing 100g dried passion fruit peel, sieving, extracting with 80% ethanol at 85 deg.C under reflux for 3 times, each for 2 hr, collecting residue, and drying; taking the residue and water according to the material-liquid ratio of 1 g: mixing 16mL of the raw materials, soaking overnight, extracting for 2 times at 100 ℃ for 3 hours each time, mixing the extracting solutions, concentrating the extracting solution to be viscous, cooling, and adding 95% ethanol with three times of volume; filtering the ethanol precipitated extractive solution, centrifuging at 5000rpm for 10min, and freeze drying the precipitate to obtain crude Passiflora edulis pericarp polysaccharide;

(2) dissolving 2g of crude passion fruit peel polysaccharide in water, loading the crude passion fruit peel polysaccharide to a CL-type DEAE-cellulose ion exchange chromatographic column, eluting with distilled water at the flow rate of 15mL/min, discarding the eluent, sequentially eluting with a NaCl aqueous solution with the concentration of 0.7mol/L, collecting the eluent for concentration, dialyzing with running water for 48 hours, and freeze-drying to obtain 0.215g of passion fruit peel acidic polysaccharide (WPEP-2), wherein the yield is 10.8%;

(3) the content of uronic acid in the passion fruit peel acidic polysaccharide is determined by adopting an m-hydroxybiphenyl method, and the purity of the obtained acidic polysaccharide is 72.33%.

Example 4

(1) Pulverizing 100g dried passion fruit peel, sieving, extracting with 80% ethanol at 85 deg.C under reflux for 3 times, each for 2 hr, collecting residue, and drying; taking the residue and water according to the material-liquid ratio of 1 g: mixing 16mL of the raw materials, soaking overnight, extracting for 1 time at 100 ℃ for 2 hours each time, mixing the extracting solutions, concentrating the extracting solution to be viscous, cooling, and adding 95% ethanol with three times of volume; filtering the ethanol precipitated extractive solution, centrifuging at 3000rpm for 10min, and freeze drying the precipitate to obtain crude Passiflora edulis pericarp polysaccharide;

(2) dissolving 2g of crude passion fruit peel polysaccharide in water, loading the crude passion fruit peel polysaccharide to a CL-type DEAE-cellulose ion exchange chromatographic column, eluting with distilled water at the flow rate of 15mL/min, discarding the eluent, sequentially eluting with a NaCl aqueous solution with the concentration of 0.7mol/L, collecting the eluent for concentration, dialyzing with running water for 48 hours, and freeze-drying to obtain 0.192g of passion fruit peel acidic polysaccharide (WPEP-2), wherein the yield is 9.6%;

(3) the content of uronic acid in the passion fruit peel acidic polysaccharide is determined by adopting an m-hydroxybiphenyl method, and the purity of the obtained acidic polysaccharide is 71.4%.

Second, evaluation of hypoglycemic action of passion fruit peel acidic polysaccharide

Animal experiments:

the experiment shared 30 mice, among which the normal blood sugar mice: 5, diabetic mice: 25 are provided. 5 mice of normoglycemic mice were used as the normal group; diabetic mice were randomly divided into five groups of 5 mice each, each group being: model control group, positive control group, WPEP-2 low dose group, WPEP-2 medium dose group and WPEP-2 high dose group, mice freely drink water and food during feeding period.

And groups of mice were treated as follows:

NC: in the normal group, C57BLKS/JNju mice are subjected to distilled water intragastric administration;

MC: performing stomach irrigation treatment on a model control group and db/db mice by using distilled water;

PC: in the positive control group, the metformin lavage treatment is carried out on db/db mice, and the dosage is 50 mg/kg/d;

l: in the low-dose group, the WPEP-2 of db/db mice is subjected to intragastric administration, and the dose is 50 mg/kg/d;

m: in the middle dose group, the WPEP-2 of db/db mice is treated by gastric lavage, and the dose is 100 mg/kg/d;

h: in the high-dose group, db/db mice are subjected to WPEP-2 intragastric administration, and the dose is 200 mg/kg/d.

(1) Measurement of daily food intake

The effect of WPEP-2 on the daily food intake of diabetic mice was studied, and the change curve of the daily food intake of each group of mice is shown in FIG. 1. The food intake of the mice in each group during week 4 is shown in Table 1, wherein the food intake of the positive control group during week 4 is 40.08g/d, which is lower than the food intake of the model control group; the intake of the mice in the WPEP-2 treatment group is lower than that of the model control group, and the daily intake of the mice in the low, medium and high dose groups is 55.15 g/d, 50.24g/d and 45.50g/d respectively. It is demonstrated that WPEP-2 can reduce the daily food intake of mice.

TABLE 1

(2) Determination of daily weight gain

The daily gain of body weight of each group of mice is shown in FIG. 2. The initial and post-experiment body weights of each group of mice are shown in table 2, wherein the initial weights of each group of diabetic mice are higher than those of the normal group; after the experiment (week 4), the weight average of each group showed different increases, and the weight of the normal group mice and the model control group mice increased 11.89% and 32.84% respectively compared with week 1, and the weight of the low, medium and high dose groups and the positive control group increased 29.67%, 32.62%, 28.57% and 33.47% respectively. It is shown that WPEP-2 can reduce the body weight of diabetic mice.

TABLE 2

(3) Measurement of blood glucose in mice

In vivo, the effect of WPEP-2 on changes in blood glucose was studied, and the blood glucose levels in mice in groups at week 1 are shown in FIG. 3, and in mice in groups at week 4 are shown in FIG. 4. The blood sugar content of the mice in each group at week 1 and week 4 is shown in table 3, the blood sugar level of the mice in the normal group is lower than that of the control group of the model, and the difference is very obvious (P < 0.001); the blood sugar level of the mice in the positive control group and the high-dose group is lower than that of the mice in the model control group, and the difference is very obvious (P < 0.01); the blood sugar level of mice in the low-dose group and the medium-dose group is lower than that of the model control group, and the difference is obvious (P < 0.05). Compared with the model control group, the WPEP-2 dry prognosis, the low dose, the medium dose and the high dose groups respectively reduce the blood sugar level of the mice by 32.49 percent, 32.70 percent and 36.60 percent. It is shown that WPEP-2 has the effect of lowering blood sugar level.

TABLE 3

(4) Determination of the Effect of WPEP-2 on insulin and Triglycerides (TG) in the serum of mice

The serum insulin levels of the mice in each group at week 4 are shown in FIG. 5. Compared with the model control group, the insulin levels of the mice in the medium and high dose groups are respectively reduced by 40.93 percent and 44.81 percent, and the effect is similar to that of the positive control group (37.77 percent). The WPEP-2 low dose group also has the effect of reducing blood sugar, but the reduction rate is 23.78 percent without obvious medium and high dose groups. It was shown that WPEP-2 reduces insulin resistance in diabetic mice.

The serum Triglyceride (TG) content of each group of mice at week 4 is shown in FIG. 6. The serum triglyceride of the normal group is obviously lower than that of the model control group, and the difference is extremely obvious (P < 0.01); after treatment with metformin and low, medium and high doses of WPEP-2, TG levels were significantly reduced in diabetic mice. It was shown that a medium or high dose of WPEP-2 has a potential effect on improving lipid levels. Table 4 shows the serum insulin and triglyceride levels of the mice at week 4.

TABLE 4

(5) Mouse insulin resistance (ITT) assay

The change curve of blood glucose value after each group of mice injected with 0.5U/kg insulin is shown in FIG. 7, and the blood glucose values before and after injection are shown in Table 5. Compared with the blood glucose value at 0, the blood glucose values of mice in the low, medium and high dose groups, the model control group and the positive control group are reduced by 22.51%, 14.57%, 2.46%, 24.23% and 29.07% at 30 min. It is shown that the low dose and the medium dose of WPEP-2 can reduce the insulin resistance of diabetic mice and delay the disease condition.

TABLE 5

Diabetes is a highly metabolic disease, and a patient may eat more food to increase blood sugar content, but the insufficient secretion of insulin makes the blood sugar not be used effectively, but only glycogen of the patient is decomposed, resulting in lipid metabolism disorder. According to the invention, the low, medium and high doses of passion fruit peel acidic polysaccharide are injected to sick mice, so that the feed intake of the mice is effectively controlled, and the problems of weight increase and high blood sugar value caused by the rise of the blood sugar content are solved; the level of insulin in serum of an insulin resistant mouse is relatively high, but the insulin can not play a role in reducing blood sugar due to the resistance of the mouse to the insulin, the content of the insulin in the serum of the mouse can be reduced by injecting different amounts of passion fruit peel acidic polysaccharide, the effects of reducing the insulin resistance of a diabetic mouse and relieving the disease condition are achieved, and the passion fruit peel acidic polysaccharide prepared by the method has a good blood sugar reducing effect through the effect verification.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A preparation method of passion fruit peel acidic polysaccharide is characterized by comprising the following steps:

(1) pulverizing dried passion fruit peel, sieving, extracting with ethanol under reflux, collecting residue, and drying;

(2) mixing the residue obtained in the step (1) with water according to a feed-liquid ratio of 1 g: mixing 10-20mL of the mixture, soaking overnight and extracting, combining concentrated extracting solutions, cooling, and adding ethanol with three times of volume to obtain an ethanol-precipitated extracting solution;

(4) and (3) dissolving the crude passion fruit peel polysaccharide obtained in the step (3) in water, carrying out chromatographic separation, eluting with distilled water, discarding the eluent, eluting with a chloride solution, collecting the eluent, concentrating, dialyzing, and freeze-drying to obtain the acidic passion fruit peel polysaccharide.

2. The preparation method of the passion flower pericarp acidic polysaccharide as claimed in claim 1, wherein the condensation reflux extraction in step (1) is performed for 1-3 times, each time for 1-3 hours.

3. The preparation method of the passion flower pericarp acidic polysaccharide as claimed in claim 1, wherein the extraction temperature in the step (2) is 90-120 ℃, and the extraction is carried out for 1-3 times, each time for 2-4 hours.

4. The method for preparing the passion fruit peel acidic polysaccharide in claim 1, wherein in the step (3), the filtered extracting solution is centrifuged at 3000-5000 rpm for 10-20 min.

5. The preparation method of the passion fruit peel acidic polysaccharide of claim 1, wherein the elution flow rate in the step (4) is 15mL/min, the chloride solution is 0.7mol/L NaCl solution, and the running water dialysis is performed for 48 h.

6. The method for preparing the passion flower pericarp acidic polysaccharide as claimed in claim 1, wherein the chromatography separation in step (4) adopts CL-type DEAE-cellulose ion exchange chromatography column.

7. The passion fruit peel acidic polysaccharide prepared by the preparation method of any one of claims 1-6.

8. The use of the passion fruit peel acidic polysaccharide of claim 7 in the preparation of a hypoglycemic medicament or health product.

9. The use of claim 8, wherein the pharmaceutical or nutraceutical is in the form of tablet, granule, hard capsule, soft capsule, oral liquid, powder, mixture or pill.

10. A pharmaceutical composition comprising the acidic passion fruit peel polysaccharide of claim 7 as an active ingredient, together with a pharmaceutically acceptable carrier or excipient.