CN113069466B - Application of purslane polysaccharide in preparation of acute lung injury resistant medicine - Google Patents

Abstract

The invention relates to the technical field of pharmacy, and discloses application of purslane polysaccharide in preparation of an anti-acute lung injury medicament. According to the invention, through the research on the effect of the purslane polysaccharide on lipopolysaccharide-induced acute lung injury of mice, the purslane polysaccharide can reduce the W/D ratio of the lung tissues of the mice and the MPO activity of the lung tissues, simultaneously reduce the contents of TNF-alpha, IL-1 beta and IL-6 inflammatory factors in the serum of the mice, and can relieve the degree of the lung tissue injury of the mice, which indicates that the purslane polysaccharide has a certain protection effect on the lipopolysaccharide-induced acute lung injury of the mice, and is expected to be used for preparing anti-acute lung injury medicines.

Description

Application of purslane polysaccharide in preparation of acute lung injury resistant medicine

Technical Field

The invention relates to the technical field of pharmacy, in particular to application of purslane polysaccharide in preparing an anti-acute lung injury medicament.

Background

Acute Lung Injury (ALI) is one of the most common critical diseases in emergency department, and is characterized by diffuse pulmonary interstitial and alveolar edema caused by injury of alveolar epithelial cells and capillary endothelial cells, and accompanied by protein exudation, proinflammatory mediators and factor release. During the ALI process, various cytokines and inflammation-related mediators are involved in the occurrence and development of the disease, such as tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), inducible Nitric Oxide Synthase (iNOS), nitric Oxide (NO), and the like. In recent years, despite great progress of medical treatment and scientific research, the ALI fatality rate still remains high, and the ALI fatality rate is up to 30-50% through research and statistics. At present, medicines such as glucocorticoid, litofylline and surfactant are commonly used for treating ALI clinically, but the side effects are large, and the treatment effect is not ideal, so that the research on effective measures for preventing and treating ALI is one of the hot spots of the current research.

Purslane (Portulaca Oleracea L) is an annual herb plant of Portulacaceae, also named as Changshou vegetable, wuxing grass, mafeng vegetable and the like, is cold in nature and sour in taste, has the effects of clearing heat and removing toxicity, cooling blood and stopping bleeding, and stopping dysentery, and has high medicinal and edible values. The herba Portulacae mainly contains polysaccharides, flavonoids, alkaloids, organic phenolic acids, terpenes, and coumarins. Modern pharmacological research finds that the purslane polysaccharide has the effects of resisting inflammation, protecting liver, easing pain, resisting oxidation and the like. However, no report is found on whether the polysaccharide has a protective effect on acute lung injury.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides the application of purslane polysaccharide in preparing an anti-acute lung injury medicament. The purslane polysaccharide has a certain protection effect on acute lung injury caused by LPS (lipopolysaccharide), and shows that the purslane polysaccharide can be expected to be used for preparing anti-acute lung injury medicines, and purslane belongs to herbaceous plants and has small toxic and side effects.

In order to achieve the purpose, the invention provides an application of purslane polysaccharide in preparing an anti-acute lung injury medicament.

Preferably, the preparation process of the purslane polysaccharide comprises the following steps: pulverizing herba Portulacae with a pulverizer, sieving with 10 mesh sieve to obtain coarse powder, adding 4 times of anhydrous ethanol, soaking for 24 hr, vacuum filtering, and oven drying the residue at 60 deg.C; adding 10 times of distilled water into the dried filter residue, soaking in 80 deg.C water bath for 2h, vacuum filtering, repeating the above steps twice, mixing filtrates, and concentrating under reduced pressure; adding absolute ethyl alcohol into the concentrated solution to 80%, standing overnight at 4 ℃, performing suction filtration, washing filter residue with 50mL of absolute ethyl alcohol, and collecting the filter residue to obtain the crude purslane polysaccharide; dissolving the obtained purslane crude polysaccharide with distilled water, adding activated carbon accounting for 1% of the weight of the purslane crude polysaccharide for decoloring, then fully oscillating the purslane crude polysaccharide with Sevage reagent in a separating funnel, removing protein contained in the crude polysaccharide, collecting supernatant, finally concentrating under reduced pressure, and carrying out vacuum freeze drying for 48 hours to obtain purslane polysaccharide, and storing at-20 ℃ for later use.

Preferably, the Sevage reagent is n-butanol: chloroform =4:1 (v/v).

Preferably, the vacuum freeze-drying temperature is-10 deg.C, and the vacuum freezing pressure is 100Pa.

LPS is a major component of the cell wall of gram-negative bacteria. LPS stimulation can activate macrophages, induce organisms to release a large amount of inflammatory cytokines such as TNF-alpha, IL-1 beta, IL-6 and the like, and further initiate acute inflammation, and related research reports suggest that imbalance of inflammatory response of lung tissues is the root cause of ALI. The invention adopts the method of instilling LPS solution in the trachea to establish the animal model of the mouse with acute lung injury, ALI can be induced within 2-4 hours after instillation, and the maximum lung injury can be caused within 24-48 hours. This method avoids systemic inflammatory responses and multiple organ failure induced by subcutaneous LPS injection.

One of the symptoms of ALI is pulmonary edema, and the wet/dry (W/D) ratio of lung tissue reflects the degree of pulmonary edema, which is greater the W/D ratio, which is calculated by W/D = (wet/dry) x 100%.

The purslane polysaccharide can reduce the edema degree of lung tissues and the activity of Myeloperoxidase (MPO) in the lung tissues, and simultaneously reduce the contents of TNF-alpha, IL-1 beta and IL-6 inflammatory factors in mouse serum, thereby indicating that the purslane polysaccharide has a certain protection effect on acute lung injury caused by LPS.

The invention has the advantage of providing a new medicine source for resisting acute lung injury.

Drawings

Figure 1 is the effect of purslane polysaccharide on the lung tissue wet/dry weight ratio (W/D) (n = 10).

Fig. 2 is the effect of purslane polysaccharide on Myeloperoxidase (MPO) activity in lung tissue (n = 10).

FIG. 3 is a graph of the effect of purslane polysaccharide on TNF- α, IL-1 β and IL-6 levels in serum (n = 10).

FIG. 4 is a graph of the effect of purslane polysaccharide on the pathomorphology of lung tissue (. Times.400).

Figure 5 is a graph of the effect of purslane polysaccharide on lung tissue injury score.

Detailed Description

The present invention will be described in detail below by way of examples, but the scope of the present invention is not limited thereto.

Example 1

1. Materials and apparatus

1.1 Reagent

Purslane (Zhang Zhongjing Dachun pharmacy); lipopolysaccharide (Sigma company, usa); dexamethasone (Shanghai-derived leaf Biotech, inc.); ELISA detection kit for tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6) (Shanghai Wenshan Biotech, inc.); myeloperoxidase (MPO) activity detection kit (Nanjing institute of bioengineering); sodium carboxymethylcellulose (miuiou chemical reagents ltd, department of tianjin); pentobarbital sodium (produced by Shanghai reagent II).

1.2 Animal(s) production

60 SPF-grade KM mice, each half male and female, with the weight of 18-20g, purchased from Beijing Wittingerli experiment animal technology Limited company, and the certification number SCXK (Jing) 2017-0005, are stored in an animal room of the yellow river academy of science and technology, are irradiated for 12h/d at the temperature of 20-23 ℃ and the relative humidity of 40-60%, and are used for experiments after being adaptively fed for 7 d.

Instrument for measuring the position of a moving object

Multifunctional microplate readers (Infinite, austria); high speed table refrigerated centrifuge (seimer feishell technologies ltd.); model FY135 Chinese herbal medicine grinder (Tester instruments, inc. of Tianjin); DHP-9052 electric heating constant temperature incubator (Shanghai-Hengshi Co., ltd.); BS-3000A series electronic balances (Shanghai friend Sound weighing machine, inc.); optical microscopy (OLYMPUS); model RM2125 paraffin wax microtome (Leica, germany); model L9 ultraviolet-visible spectrophotometer (shanghai instrument electric analyzer limited).

Method

2.1 Pharmaceutical preparation

2.1.1 Preparation of purslane polysaccharide and solution preparation

Pulverizing herba Portulacae with a pulverizer, sieving with a 10-mesh sieve to obtain coarse powder, weighing 500g herba Portulacae coarse powder, adding 4 times of anhydrous ethanol, soaking for 24h, vacuum filtering, and oven drying the residue at 60 deg.C; adding 10 times of distilled water into the dried filter residue, soaking in 80 deg.C water bath for 2h, vacuum filtering, repeating the above steps twice, mixing filtrates, and concentrating under reduced pressure; adding absolute ethyl alcohol into the concentrated solution to 80%, standing overnight at 4 ℃, performing suction filtration, washing filter residue with 50mL of absolute ethyl alcohol, and collecting the filter residue to obtain the crude purslane polysaccharide; dissolving the obtained purslane crude polysaccharide with distilled water, adding activated carbon accounting for 1% of the mass of the purslane crude polysaccharide for decoloring, and then mixing with n-butyl alcohol: chloroform =4: and (2) fully shaking the mixed solution of 1 (v/v) in a separating funnel, removing protein contained in the crude polysaccharide, collecting supernatant, finally concentrating under reduced pressure, and carrying out vacuum freeze drying for 48 hours under the conditions that the temperature is-10 ℃ and the vacuum pressure is 100pa to obtain the purslane polysaccharide, and storing at-20 ℃ for later use.

Accurately weighing 0.75 g, 1.5 g and 3g of purslane polysaccharide respectively, placing the purslane polysaccharide into a 50mL volumetric flask, adding 20mL of distilled water to completely dissolve the purslane polysaccharide, and then metering the volume to 50mL by using the distilled water, wherein the concentrations are respectively 0.015g/mL, 0.03g/mL and 0.06g/mL, namely low, medium and high concentrations for later use.

Preparation of dexamethasone solution

Precisely weighing 25mg of dexamethasone, placing the dexamethasone into a 50mL volumetric flask, adding 20mL of 0.5% sodium carboxymethylcellulose aqueous solution, uniformly mixing, and then using 0.5% sodium carboxymethylcellulose aqueous solution to fix the volume to 50mL of a solution with the concentration of 0.0005g/mL for later use, wherein the dexamethasone and the solution are uniformly mixed before each intragastric administration.

Grouping, modeling and administering

After 60 mice were acclimatized for 1 week, they were randomly divided into a normal group, a model group, a dexamethasone group (0.005 g/kg. D), and low-, medium-, and high-dose portulaca oleracea polysaccharide groups (0.15, 0.3, 0.6 g/kg/d), each of which contained 10 mice. And (3) performing intragastric administration according to the mass of 10mL/kg. D for 1 time/d for 14 days continuously, wherein the normal group and the model group are intragastric with 0.5% sodium carboxymethylcellulose aqueous solution. 2h after the last administration, the mice were anesthetized with a 1.5% pentobarbital sodium solution (in an amount of 0.1ml/10 g) by intraperitoneal injection, followed by removal of the median cervical hair, alcohol sterilization, median cervical skin incision, exposure and trachea isolation. A sample injector is adopted to slowly drip LPS normal saline solution 50uL (containing LPS 10 ug) in the trachea to establish an animal model of the mice with acute lung injury, normal groups are given normal saline with corresponding volume by the same method, and after dripping, the mice are disinfected and sutured on the skin. After fasting without water deprivation and 12h of molding, the mice were bled from the orbit, and then sacrificed and the lung tissue of the mice was rapidly removed for use.

Statistical method

Data processing was performed using SPSS 22.0 software. The measured data conforming to the normal distribution is represented by () and is tested by adopting one-factor variance analysis,Pdifferences < 0.05 are statistically significant.

Mouse Lung tissue Wet/Dry weight ratio (W/D) determination

Taking the upper right lung lobe of the mouse, removing connective tissues, sucking surface water and blood by using filter paper, and weighing to obtain the wet weight of the lung tissue; the lung tissue was then placed in an 80 ℃ oven for 48h continuous drying, the dry weight of the lung tissue was weighed and recorded, and the W/D was calculated, the results are shown in fig. 1.

As is clear from the results in FIG. 1, the W/D ratio was significantly increased in the model group mice as compared with that in the normal group mice ( ^## P< 0.01), the W/D ratio of the mice in each dose group of purslane polysaccharide is reduced compared with that in the model group, wherein the medium dose and the high dose groups have statistical difference ( ^* P<0.05 , ^** P<0.01 Showing that purslane polysaccharide can reduce ALI induced by LPS.

Determination of MPO Activity in mouse Lung tissues

Placing mouse lung tissue in a tissue homogenizer, adding normal saline, grinding in ice water bath, centrifuging to obtain supernatant, and detecting MPO activity in lung tissue according to MPO activity detection kit specification, with the result shown in FIG. 2.

As is clear from the results in FIG. 2, MPO activity was significantly increased in lung tissue of the model group mice as compared with that of the normal group mice ( ^## P<0.01 In mice with each dose of portulaca oleracea polysaccharide, the MPO activity was reduced in lung tissue compared to the model group, with statistical differences between the medium and high dose groups: ( ^* P<0.05 , ^** P<0.01）。

Determination of TNF-alpha, IL-1 beta and IL-6 content in mouse serum

Blood samples are taken and centrifuged for 10min under the condition of 3000r/min, then supernatant is taken and the contents of TNF-alpha, IL-1 beta and IL-6 in mouse serum are determined by an ELISA method according to the steps of each ELISA detection kit specification, and the result is shown in figure 3.

As is clear from the results in FIG. 3, the serum contents of TNF-. Alpha., IL-1. Beta. And IL-6 were significantly increased in the mice of the model group as compared with those of the normal group ( ^## P< 0.01), the serum contents of TNF-alpha, IL-1 beta and IL-6 in mice of each dose group of purslane polysaccharide are reduced compared with those of the model group, wherein the medium dose group and the high dose group have statistical differences ( ^* P<0.05 , ^** P<0.01). The pathogenic mechanism of ALI is not clear at present, but the relevant studies report that imbalance of the inflammatory response of lung tissues is the root cause of ALI. The research result shows that the purslane polysaccharide can reduce the contents of TNF-alpha, IL-1 beta and IL-6 inflammatory cytokines, and the protection of the purslane polysaccharide on acute lung injury induced by LPS is related to the inhibition of the secretion of inflammatory factors.

Pathological examination of lung tissue

Fixing the right lung lobes of the mice in a 10% neutral formaldehyde solution, taking out after 24 hours, and washing with distilled water until the mice are odorless; they were then subjected to conventional dehydration, waxing, embedding, sectioning, hematoxylin-eosin staining, mounting, and pathology observation under a microscope (fig. 4) and scored pathologically (fig. 5). The method comprises three indexes of alveolar wall thickness, lung tissue destruction and inflammatory cell infiltration, wherein each index is scored according to a 5-point system: the score 0 is the minimum injury, the score 1 is the mild injury, the score 2 is the moderate injury, the score 3 is the severe injury, the score 4 is the maximum injury, and the lung injury score is the sum of the scores of the 3 indexes.

From the results of fig. 4, it is clear in the lung tissue structure, no exudate, and no edema and inflammatory cell infiltration in the normal mice. The lung tissue of the model mouse has blood spots, pulmonary edema, widened alveolar wall and a large amount of inflammatory cell infiltration ( ^## P< 0.01). Compared with the model group of mice, the lung tissue damage of the mice of each dose group of the purslane polysaccharide is relieved to different degrees ( ^* P＜0.05 , ^** P＜0.01）。

The above experimental results show that: the purslane polysaccharide can reduce the W/D ratio of the lung tissue of a mouse and the MPO activity in the lung tissue, simultaneously reduce the contents of TNF-alpha, IL-1 beta and IL-6 inflammatory factors in the serum of the mouse, and can relieve the damage degree of the lung tissue of the mouse. The purslane polysaccharide has a certain protection effect on acute lung injury caused by LPS, and the action mechanism of the purslane polysaccharide is probably related to inhibition of secretion of cell inflammatory factors by the purslane polysaccharide. The purslane polysaccharide is expected to be used for preparing the anti-acute lung injury medicine.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (2)

1. The application of purslane polysaccharide as the only active component in preparing the medicine for resisting acute lung injury is characterized in that the preparation process of the purslane polysaccharide is as follows: pulverizing herba Portulacae with a pulverizer, sieving with 10 mesh sieve to obtain coarse powder, adding 4 times of anhydrous ethanol, soaking for 24 hr, vacuum filtering, and oven drying the residue at 60 deg.C; adding 10 times of distilled water into the dried filter residue, soaking in 80 deg.C water bath for 2h, vacuum filtering, repeating the above steps twice, mixing filtrates, and concentrating under reduced pressure; adding absolute ethyl alcohol into the concentrated solution to 80%, standing overnight at 4 ℃, performing suction filtration, washing filter residue with 50mL of absolute ethyl alcohol, and collecting the filter residue to obtain the crude purslane polysaccharide; dissolving the obtained purslane crude polysaccharide with distilled water, adding activated carbon accounting for 1% of the mass of the purslane crude polysaccharide for decoloring, and then fully oscillating the purslane crude polysaccharide and a Sevage reagent in a separating funnel, wherein the Sevage reagent is n-butyl alcohol: chloroform =4:1 (v/v), removing protein contained in the crude polysaccharide, collecting supernatant, finally concentrating under reduced pressure, and freeze-drying for 48h under vacuum to obtain purslane polysaccharide, and storing at-20 ℃ for later use.

2. The use of purslane polysaccharide as the sole active ingredient in the preparation of an anti-acute lung injury medicament according to claim 1, wherein the vacuum freeze-drying temperature is-10 ℃ and the vacuum freeze pressure is 100pa.

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