HOMOGENEOUS POLYSACCHARIDE ESP-B4, DERIVED FROM HERBA EPHEDRA, PREPARATION METHOD AND MEDICAL USE THEREOF

Abstract

Provided is a homogeneous polysaccharide ESP-B4, method for producing the same and use thereof. The ESP-B4 is a polysaccharide monomer, which is isolated from the traditional Chinese medicine Ephedra sinica Stapf, Ephedra intermedia Schrenk et C. A. Mey, or Ephedra equisetina Bge. HPSEC-ELSD shows a single symmetrical peak. The chemical construction of ESP-B4 is an acidic heterosaccharide. It is composed of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid with a molar ratio of 1.0:4.5:1.0:2.0:5.5:1.5:50. The molecular weight of ESP-B4 is 2.3710.sup.7 Da, and the content of galacturonic acid was 75.2%. The application of ESP-B4 is significant activity against infectious acute lung injury, respiratory distress syndrome, asthma, pneumonia, trachea and bronchitis. Therefore, it can be used for severe infectious acute lung injury caused by SARS virus, influenza a virus, avian influenza virus. Meanwhile, ESP-B4 has few side effects, simple preparation method, and it is suitable for industrial production.

Claims

1. A homogeneous polysaccharide ESP-B4 from Ephedra sinica for treating a respiratory disease, where in the respiratory disease includes acute lung injury and respiratory distress syndrome.

2. The polysaccharide ESP-B4 from Ephedra sinica according to claim 1, wherein the polysaccharide ESP-B4 is isolated and purified from the stems of Ephedra sinica Stapf, Ephedra intermedia Schrenk et C. A. Mey., or Ephedra equisetina Bge.

3. The polysaccharide ESP-B4 from Ephedra sinica according to claim 1, wherein the polysaccharide ESP-B4 is acid heteropolysaccharide, the monosaccharide is composed of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid with a molar ratio of 1.0:4.5:1.0:2.0:5.5:1.5:50, and the molar percentage of galacturonic acid is 75.2%, the molecular weight of ESP-B4 is 2.3710.sup.7 Da.

4. A method for preparing the polysaccharide ESP-B4 from Ephedra sinica according to claim 1, comprising the following steps: Step (1): preparing total polysaccharides from Ephedra sinica by water extraction and alcohol precipitation; Step (2): separating the total polysaccharides by series chromatography of Anion and Cation resin column; and Step (3): separating polysaccharide ESP-B4 by separation of cellulose and sepharose chromatography column through ion exchange, adsorption and molecular sieve m.

5. The method according to claim 4, wherein: in step (1), the water extraction is performed by extracting 2-3 times with 5-10 times hot water, and the ethanol concentration of alcohol precipitation is 75%-87%; in step (2), anion and cation resin packing are preferably Amberlite FPC3500 and IRA-401; in step (3), the Cellulose column filler and DEAE-Sepharose F.F are preferably used; and the chromatographic packing materials used in step (2) and step (3) are not restricted to the above selection, regardless of the packing choice, the purity of the prepared ESP-B4 is more than 85%.

6. A pharmaceutical composition for treating acute lung injury and respiratory distress syndrome, comprising the polysaccharide ESP-B4 from Ephedra sinica and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is in the dosage form of oral preparation or parenteral preparation, the oral preparation is selected from tablet, medicinal granules, capsule, oral liquid or pill, the parenteral preparation is selected from injection or infusion solution, the capsule is selected from hard capsule and soft capsule, the pill is dropping pill, the injection is selected from injection solution, freeze-dried powder injection, water injection, the infusion solution is large volume injection.

8. A method for treating acute lung injury comprising administering to a subject in need thereof a therapeutically effective amount of the polysaccharide ESP-B4 according to claim 1.

9. The method according to claim 8, wherein the acute lung injury is selected from infectious acute lung injury and respiratory distress syndrome caused by infectious acute lung injury deterioration, the infectious acute lung injury is selected from major infectious acute lung injury caused by SARS virus, influenza A virus, avian influenza virus, as well as pulmonary infection caused by Escherichia coli, Pseudomonas aeruginosa, Pneumococcus, Staphylococcus aureus, Klebsiella pneumoniae, influenza virus, Chlamydia and Mycoplasma infection.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the examples of the present disclosure or the technical solutions in the prior art, the drawings used in the examples or the prior art will be briefly described below. Obviously, the drawings in the following description are only the examples of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without any creative work.

[0037] FIG. 1 is a plot elution curve with optical density value of ESP-B4 used in Example 1.

[0038] Plot elution curve with optical density value of ESP-B4 (A) HPCE-ELSD profiles of ESP-B4 (B).

DETAILED DESCRIPTION

[0039] The present invention is described in more detail below to facilitate understanding of the present invention.

Example 1: Extraction and Separation of Homogeneous Polysaccharide ESP-B4 from Ephedra sinica

[0040] The crude powder of dried herbaceous stem of Ephedra sinica is 200 kg. It is extracted by reflux with 95% ethanol for 3 times, each time for 3 hours, to remove alkaloids, pigments, polyphenols and oligosaccharides in Ephedra sinica. The residue is decocted with water for 3 times, each time for 3 hours. After cooling, they are filtered and combined with filtrate. The filtrate is recovered by decompression and freeze-dried to obtain 14 kg total polysaccharide of Ephedra sinica. Then, the total polysaccharides are separated by Amberlite FPC3500 and Amberlite IRA-401 anion-cation series resin column and monitored by phenol-sulfuric acid method. First, distilled water is used as eluent, and when no polysaccharide component flows out, 1M NaCl is used for elution. Collect 1 M NaCl eluent, concentrate, dialysis, freeze-drying to obtain elution site Fr.B.

[0041] Fr.B is eluted by Cellulose DE-52 column with gradient elution of water, 0.5 M NaCl and 1 M NaCl solution, respectively, at a flow rate of 2 mL/min, followed by phenol-sulfuric acid method. Collect 1 M NaCl main peak eluent, concentrate and freeze-drying. The obtained component is further separated and purified by cellulose DE-52 column using 1 M NaCl solution as eluent. The flow rate was 2 mL/min. Phenol-sulfuric acid method is also used to detect the main elution peaks. The symmetrical main elution peaks is obtained, and the eluents are merged and freeze-dried. The obtained polysaccharide component is named ESP-B4.

[0042] The chemical structure feature research of ESP-B4 is as follows:

[0043] (1) The polysaccharide of ESP-B4 contains mainly -glycosidic linkage. Its monosaccharides variety include xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid with the molar percentages 1.5%, 6.8%, 1.5%, 3.0%, 1.5%, 8.3%, 2.3% and 75.2%. ESP-B4 is a typical RG pectin polysaccharide. In addition, there are a few unidentified monosaccharides in constitution of ESP-B4.

[0044] (2) ESP-B4 contained the main part of homogalacturonan fragments as smooth regions, that is a repeating galacturonic acid interlinkage by .fwdarw.4)--D-GalpA-(1.fwdarw.). Rhamnose and galacturonic acid compose of the nonsmooth region of main chain with repeated structural segments, that is .fwdarw.4)--D-GalpA-(1.fwdarw.2)--Rha-(1.fwdarw.). In the nonsmooth region, about 70% of the rhamnose residues branch at C-4, and 25.2% galacturonan residues branch at C-3, at which position be substituted by side chains.

[0045] (3) The branched structure of ESP-B4 mainly contains arabinan and galactan. Among them, arabinoglycan were (1.fwdarw.5) linkage as the skeleton part, 43.8% of (1.fwdarw.5) Araf branch at C-3. Galactan were (1.fwdarw.3) linkage and (1.fwdarw.4) linkage as the skeleton part, among them, 40.2% of galactan is (1.fwdarw.3) connected, 35.9% of galactan is (1.fwdarw.4) connected, and 70.3% of (1.fwdarw.3) galactan has branch points in its C-6 position, 54.5% of (1.fwdarw.4) galactan has branch points at C-6.

Example 2: A Tablet Effective Against Respiratory Diseases, Infectious Acute Lung Injury, Respiratory Distress Syndrome, Asthma, Pneumonia, Trachea and Bronchitis

[0046] Take an appropriate amount of ESP-B4 prepared in example 1, mixed with diluent, disintegrating agent and other auxiliary materials to form granules, tabletting, coating or thin film coating.

Example 3: An Oral Liquid Effective Against Respiratory Diseases, Infective Acute Lung Injury, Respiratory Distress Syndrome, Asthma, Pneumonia, Trachea and Bronchitis

[0047] Appropriate amount of ESP-B4 prepared in example 1 is added with appropriate water solution, mixing, adding flavoring agents and preservatives, filling, capping and sterilization.

Example 4: An Infusion Agent Effective Against Respiratory Diseases, Infective Acute Lung Injury, Respiratory Distress Syndrome, Asthma, Pneumonia, Trachea and Bronchitis

[0048] Take an appropriate amount of the ESP-B4 prepared in example 1, add a little water for injection to dissolve it, then add an appropriate amount of sodium chloride, dissolve it, then add water for injection to the specified amount, filter, seal and sterilize it.

Experiment 1: Acute Toxicity Test of ESP-B4

[0049] Experimental animals: BALB/c mice (half female and half male), weighing 18-22 g, supplied by drugs safety evaluation center in Heilongjiang University of Chinese Medicine. Feeding temperature: 22.01.1 C., humidity: 58.811.0%, ventilation times 15 times/h, free feeding and drinking, feed and drinking water compliance with standards.

[0050] Methods: Twenty mice, half male and half female, were fed at a volume of 0.4 mL/10 g, ESP-B4 dosage 20 g/kg/d, (up to the maximum dosage and concentration). The mice were fed twice in a day with an interval of 6 hours, and the activity and the number of deaths were observed continuously for 14 days.

[0051] Results: LD.sub.50 of ESP-B4 was not be detected in mice after oral administration. The maximum dosage of ESP-B4 was 20 g/kg to mice, which was 200 times the dosage of clinical adults. After 14 days of continuous observation, it was found that no mice died, no abnormal changes happened on appearance, physical signs, behavioral activities, mental state, diet, defecation, etc, no abnormal secretions in mouth, eyes, nose, etc.

[0052] Conclusion: The acute toxicity test showed that ESP-B4 was safe and non-toxic.

Experiment 2: Anti-LPS-Induced Acute Lung Injury by ESP-B4

[0053] Experimental animals: BALB/c mice, weighing 18-22 g, supplied by drugs safety evaluation center in Heilongjiang University of Chinese Medicine. Feeding temperature: 22.01.1 C., humidity: 58.811.0%, ventilation times 15 times/h, free feeding and drinking, feed and drinking water compliance with standards.

[0054] Methods: Fifty BALB/c mice, half male and half female, after one week of adaptive feeding, were randomly divided into five groups: control group, model group, ESP-B4 low-dosage and high-dosage group and positive control group (dexamethasone group). The mice were anesthetized with 5% chloral hydrate (0.1 mL/20 g) and fixed on the mouse plate apparatus. Blunt separation exposed trachea with tweezers through a small opening at neck, inject 30 L LPS (5 mg/kg) saline solution into the trachea, rotate the plate for 1 min, and then suture the wound to establish acute lung injury model. The dosage of dexamethasone was 50 mg/kg, ESP-B4 low-dosage group was 50 mg/kg and ESP-B4 high-dosage group was 100 mg/kg in the. Each group intraperitoneal injection. The control group was given the same volume of normal saline. The mice were given continuous administration for 7 days. After the last administration for 2 hours, the mice were executed by cervical dislocation. The left lung tissues were quickly removed and grounded on ice to obtain homogenate to detect levels of the inflammatory factors TNF-, IL-6, IL-8 and Tp (Total protein), as follows:

[0055] Statistical Methods: All data were expressed as meanSD, and t-test was used for comparison between groups.

TABLE-US-00001 TABLE 1 Effects of ESP-B4 on LPS-induced acute lung injury in mice Group TNF- (pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) Tp (g/mL) Control group 793.46 50.93.sup. 331.94 30.20 26.21 5.87 164.44 27.44 Model group 1233.49 27.91.sup..box-tangle-solidup..box-tangle-solidup. 905.52 43.31.sup..box-tangle-solidup..box-tangle-solidup. 153.23 11.24.sup..box-tangle-solidup..box-tangle-solidup. .sup.304.78 18.96.sup..box-tangle-solidup..box-tangle-solidup. ESP-B4 low-dose group 1189.58 19.38.sup. 423.46 20.18** 133.84 12.69 282.24 19.89 ESP-B4 high-dose group 977.89 85.92* 366.18 35.41** 84.84 10.31** 211.04 15.61* Dexamethasone group 861.41 71.04** 255.18 35.63** 52.7 9.99** 202.40 16.54** Compared to the control group, .sup..box-tangle-solidup.P < 0.05; .sup..box-tangle-solidup..box-tangle-solidup.P < 0.01, Compared to model group *P < 0.05, **P < 0.01.

[0056] Results: The levels of inflammatory factors TNF-, IL-6, IL-8 and Tp in lung tissue of acute lung injury model group were significantly higher than control group (P<0.01), indicating that the model was successful. Compared with the model group, the levels of TNF-, IL-6, IL-8 and Tp in the ESP-B4 high-dose group and dexamethasone group of the present invention are significantly reduced. Among them, the levels of Tp and dexamethasone groups are roughly the same. The levels of TNF-, IL-8 and Tp in the ESP-B4 low-dose group are also reduced, and the differences of IL-6 are significant.

[0057] Conclusion: ESP-B4 showed an obvious dose-dependent therapeutic effect on LPS-induced acute lung injury in mice.

Experiment 3: Anti-E. coli-Induced Pneumonia Effect of ESP-B4

[0058] Experimental Animals: BALB/c mice, weighing 18-22 g, supplied by drugs safety evaluation center in Heilongjiang University of Chinese Medicine. Feeding temperature: 22.01.1 C., humidity: 58.811.0%, ventilation times 15 times/h, free feeding and drinking, feed and drinking water compliance with standards.

[0059] Methods: Fifty BALB/c mice were randomly divided into five groups: control group, model group, ESP-B4 low-dose group, ESP-B4 high-dose group and gentamicin sulfate positive control group. The pneumonia model was established by injection of E. coli. The mice were anesthetized with 5% chloral hydrate (0.1 ml/20 g) and fixed on the mouse plate apparatus. Blunt separation exposed trachea with tweezers through a small opening at neck, inject 30 L E. coli. (210.sup.8 CFU/mL) into the trachea, rotate the plate for 1 min, and then suture the wound to establish pneumonia model. Each group of mice was administered intragastrically. The dosage of gentamicin sulfate group was 100 mg/kg, ESP-B4 low-dose group was 75 mg/kg, and ESP-B4 high-dose group was 150 mg/kg. After 7 days of continuous intragastric administration, the mice were executed by cervical dislocation after 2 hours of the last administration. The left lung tissue was quickly removed and grounded on ice to obtain homogenate to detect levels of the inflammatory factors TNF-, IL-6, IL-8 and Tp, as follows:

[0060] Statistical Methods: All data were expressed as meanSD, and t-test was used for comparison between groups.

TABLE-US-00002 TABLE 2 of ESP-B4 on Escherichia coli-induced pneumonia in mice Group TNF- (pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) Tp (g/mL) Control group 803.23 75.82 397.55 32.22 30.06 4.85 151.15 28.06 Model group 1405.65 93.03.sup..box-tangle-solidup..box-tangle-solidup. 953.36 55.25.sup..box-tangle-solidup..box-tangle-solidup. 175.65 12.02.sup..box-tangle-solidup..box-tangle-solidup. .sup.327.25 20.12.sup..box-tangle-solidup..box-tangle-solidup. ESP-B4 low dose group 1112.58 88.55* 488.56 40.02** 138.38 13.99* 292.12 22.26 ESP-B4 high dose group 902.18 74.22** 387.36 39.39** 88.69 11.07** 223.26 15.52* Gentamycin Sulfate group 834.10 70.10** 285.16 32.71** 49.36 10.01** 197.05 14.20** Compared to control group, .sup..box-tangle-solidup.P < 0.05; .sup..box-tangle-solidup..box-tangle-solidup.P < 0.01, Compared to model group *P < 0.05, **P < 0.01.

[0061] Results: The levels of inflammatory factors TNF-, IL-6, IL-8 and Tp in lung tissue of pneumonia model group were significantly higher than control group (P<0.01), indicating that the model was successful. Compared with the model group, TNF-, IL-6 and IL-8 in the ESP-B4 low-dose and high-dose group and gentamicin sulfate group were significantly decreased (P<0.05) and showed a dose-effect relationship, while the Tp content in the high-dose group and gentamicin sulfate group was significantly decreased (P<0.05).

[0062] Conclusion: ESP-B4 has obvious therapeutic effect on E. coli-induced pneumonia in mice.

Experiment 4: Anti-H5NI-Induced Acute Lung Injury by ESP-B4

[0063] Laboratory Animals: BALB/c mice, weighing 18-22 g, supplied by drugs safety evaluation center in Heilongjiang University of Chinese Medicine. Feeding temperature: 22.01.1 C., humidity: 58.811.0%, ventilation times 15 times/h, free feeding and drinking, feed and drinking water compliance with standards.

[0064] Methods: After a week of adaptive feeding, fifty BALB/c mice, half male and half female were randomly divided into five groups: control group, model group, ESP-B4 low-dose group, ESP-B4 high-dose group and oseltamivir phosphate positive drug group. Acute lung injury was induced by intratracheal injection of H5N1. The mice were anesthetized with 5% chloral hydrate (0.1 ml/20 g) and fixed on the mouse plate apparatus. Blunt separation exposed trachea with tweezers through a small opening at neck, inject 30 L H5N1 (510.sup.4LD.sub.50/mL) into the trachea, rotate the plate for 1 min, and then suture the wound to establish pneumonia model. Each group of mice was administered intragastrically. The dosage of oseltamivir phosphate group was 50 mg/kg, ESP-B4 low-dose group was 50 mg/kg, and ESP-B4 high-dose group was 100 mg/kg. After 7 days of continuous administration, the mice were executed by cervical dislocation after 2 hours of the last administration. The left lung tissues were quickly removed and grounded on ice to obtain homogenate to detect levels of the inflammatory factors TNF-, IL-6, IL-8 and Tp, as follows:

[0065] Statistical Methods: All data were expressed as meanSD, and t-test was used for comparison between groups.

TABLE-US-00003 TABLE 3 Effects of ESP-B4 on H5NI-induced acute lung injury in mice Total protein Group TNF- (pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) (g/mL) Control group 745.41 48.81.sup. 368.56 35.53 31.65 7.85 134.64 17.55 Model group 1389.52 38.99.sup..box-tangle-solidup..box-tangle-solidup. 1003.35 60.15.sup..box-tangle-solidup..box-tangle-solidup. 201.85 20.77.sup..box-tangle-solidup..box-tangle-solidup. .sup.289.89 16.46.sup..box-tangle-solidup..box-tangle-solidup. ESP-B4 low dose group 1107.12 89.29.sup. 565.63 44.42** 127.51 15.84* 244.34 20.41 ESP-B4 high dose group 988.81 70.23* 402.21 40.15** 91.52 11.77** 207.51 17.88* Oseltamivir phosphate 901.55 68.58** 328.21 34.25** 45.64 7.75** 176.62 17.69** group Compared to the control group, .sup..box-tangle-solidup.P < 0.05; .sup..box-tangle-solidup..box-tangle-solidup.P < 0.01, Compared to model group *P < 0.05, **P < 0.01.

[0066] Results: The levels of inflammatory factors TNF-, IL-6, IL-8 and Tp in lung tissue of acute lung injury model group were significantly higher than those of control group (P<0.01), indicating that the model was successful. Compared with the model group, TNF-, IL-6, IL-8 and Tp in the ESP-B4 high dose group and oseltamivir phosphate group are significantly reduced, and there are significant differences (P<0.05). Compared with the model group, TNF- and Tp in the ESP-B4 low dose group are also decreased, and IL-6 and IL-8 are significantly different.

[0067] Conclusion: ESP-B4 has obvious therapeutic effect on H5NI-induced acute lung injury in mice.

[0068] The preferred embodiments of the present invention are described above, but they are not intended to define the present invention. Technicians in the research field may make improvements and changes to the implementation scheme disclosed herein without departing from the scope and spirit of the present invention.