CN114573723A

CN114573723A - Tamarix chinensis polysaccharide and application thereof in preparation of body supplement inhibitor and medicine for preventing and treating viral pneumonia

Info

Publication number: CN114573723A
Application number: CN202011460635.1A
Authority: CN
Inventors: 卢燕; 陈道峰; 焦峪坤; 杨屹婷; 力弘; 朱海燕
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-12-02
Filing date: 2020-12-12
Publication date: 2022-06-03
Anticipated expiration: 2040-12-12
Also published as: CN114573723B

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and relates to tamarix chinensis 90 alcohol precipitation polysaccharide, six homogeneous polysaccharides and medicinal application thereof. According to the invention, 90 alcohol-precipitated polysaccharide and six homogeneous polysaccharides MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are separated from the tamarix chinensis, and experiments prove that the tamarix chinensis polysaccharide has a remarkable inhibitory effect on complement activation and can be further used as an active ingredient for preparing a novel complement inhibitor; the integral animal model test proves that the tamarisk polysaccharides MBAP90, MBAP-2, MBAP-3 and MBAP-5 can obviously improve the pathological state of viral pneumonia, have the effect of treating viral pneumonia, and can be further used as active ingredients to prepare the medicaments for preventing and treating viral pneumonia.

Description

Tamarix chinensis polysaccharide and application thereof in preparation of body supplement inhibitor and medicine for preventing and treating viral pneumonia

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and relates to polysaccharides, in particular to 90 alcohol precipitation crude polysaccharide and 6 homogeneous polysaccharides which are separated and prepared from tamarix chinensis, and application thereof in preparation of complement inhibitors and medicaments for preventing and treating viral pneumonia.

Background

The complement system is an important part of the human immune system, however, excessive activation of this system can cause excessive reaction of the human immune system, resulting in damage to the normal tissues of the human body. Excessive complement activation plays an important role in the pathogenesis of acute diseases such as rheumatoid arthritis, senile dementia, Systemic Lupus Erythematosus (SLE), ischemic reperfusion, acute myocardial infarction, Acute Respiratory Distress Syndrome (ARDS), multiple organ failure syndrome and the like. Currently, immunosuppressive agents such as glucocorticoids and the like used clinically are not specific complement inhibitors and also generate various complications and side effects, so that a novel complement inhibitor with high efficiency, low toxicity and specificity is urgently needed clinically.

Viral pneumonia (viral pneumoconia) is a serious infectious disease caused by lung infection by various viruses, and has the advantages of emergent onset, rapid disease development and high fatality rate. Viral pneumonia is pathologically changed into gas exchange disorder at the alveolar level, further causing Acute Lung Injury (ALI), resulting in respiratory distress and severe hypoxemia in patients. Complement overactivation is a common feature of respiratory distress syndrome, with C5a being significantly elevated in peripheral blood samples and has been identified as a marker for respiratory distress syndrome associated with severe sepsis, cytokine storm, and Multiple Organ Failure (MOF) (Polycarpou a, Howard M, Farrar C a. embo Molecular Medicine,2020,12(8): 1-15.). According to the results of the new coronary autopsy, the excessive consumption of the plasma complement of a patient causes the damage of the alveolar capillary wall, the permeability is increased, the release of inflammatory mediators is further enhanced, and the tissue damage is aggravated; therefore, the complement inhibitor can be used as a potential drug for treating viral pneumonia.

In the current common medicines for clinically treating viral pneumonia, antiviral medicines face the problems of rapid virus strain variation, long vaccine period and the like, and glucocorticoid anti-inflammatory medicines have obvious side effects. Because complement inhibition is one of potential therapeutic targets of viral pneumonia, the development of the viral pneumonia medicament using the high-efficiency low-toxicity complement inhibitor has important significance. Chinese medicine resources are rich, and many Chinese medicines have obvious regulating effect on immune system, so that the Chinese medicine is a valuable resource for searching complement inhibitors and viral pneumonia prodrugs.

Tamarix chinensis, also known as Tamarix occidentalis, Tamarix kwangsiensis and Tamarix aristolochia, is collected from the Ben Cao Tu Jing (materia Medica of materia Medica), is derived from branches and leaves of Tamarix chinensis Lour of Tamarix of Tamaricaceae, has sweet taste, has effects of dispelling pathogenic wind, relieving exterior syndrome, promoting eruption, relieving cough, clearing blood heat and promoting urination, and can be used for treating measles without adequate eruption, rheumatalgia, wind-heat type common cold, cough, joint rheumatism, chronic bronchitis, yellow water disease, nasopharyngeal carcinoma, etc. The tamarix chinensis prescription such as Maxingshigan decoction has obvious curative effect on measles complicated by pneumonia (Zhangzu union, Chinese and western medicine combined clinical journal, 1992,2(3): 41-41; Xiayaiyi, Shizhen Chinese medicine, 2000,11(6): 536-. And research has shown that the aqueous extract of Tamarix chinensis has anti-inflammatory and anti-herpesvirus activity (Zhao Runzhou, Sunshiyin, Chenfa Quizhi, Chinese herbal medicine, 1995,26(2): 85-85; Zhou Yuan, Yang Zhi, Zheng Xue, proceedings of Jiangxi medical college, 1988,24(28): 1-7.); however, the related researches on the structure, the anti-complement activity and the prevention and treatment effect on the viral pneumonia of the tamarix chinensis polysaccharide are not found.

Based on the current situation of the prior art, the inventor of the application intends to provide tamarix chinensis polysaccharide and application thereof in preparation of a complement inhibitor and a medicament for preventing and treating viral pneumonia.

Disclosure of Invention

The invention aims to provide an anticomplement active component for preventing and treating viral pneumonia, tamarix chinensis polysaccharide and application thereof in preparing a complement inhibitor and a medicament for preventing and treating viral pneumonia based on the current situation of the prior art. In particular to a tamarix chinensis 90 alcohol-precipitated polysaccharide and 6 tamarix chinensis homogeneous polysaccharides, a preparation method thereof and application thereof in preparing a body-supplementing inhibiting medicament and a viral pneumonia prevention and treatment medicament. The experiment of the invention proves the anti-complement activity of the tamarix chinensis polysaccharide and the protection effect on the influenza A virus induced acute lung injury mice.

Specifically, 90 alcohol-precipitated polysaccharide (MBAP90) and 6 homogeneous polysaccharides (MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6) are separated from branches and leaves of Tamarix chinensis Lour.

In the invention, an integral animal model test is carried out, and the result proves that 90 alcohol-precipitated polysaccharide (MBAP90) and homogeneous polysaccharide (MBAP-2, MBAP-3 and MBAP-5) can obviously improve the pathological state of viral pneumonia, have the effect of treating viral pneumonia, and can be used for preparing the medicine for preventing and treating viral pneumonia.

The structural characteristics of the tamarix chinensis 90 alcohol precipitation polysaccharide MBAP90 and homogeneous polysaccharides MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are described as follows:

(1) MBAP90 is composed of six monosaccharides, and has a molecular weight range of 30.2-274.1 kDa; the total sugar content was 64.36%; the protein content was 3.53%; the uronic acid content was 13.52%; the flavone content is 12.18%; contains no sulfuric acid group. Monosaccharide molar ratio D-glucose: d-galactose: l-arabinose: d-mannose: d-glucuronic acid: d-galacturonic acid (21.2: 8.12:9.31:4.14:2.07: 1.12).

(2) MBAP-1 is a polysaccharide composed of five monosaccharides and has a molecular weight of about 269.3 kDa; total sugar content 86.06%; the protein content is 1.90%; the uronic acid content is 9.64%; the flavone content is 12.03%, and the product does not contain sulfate group. Monosaccharide molar ratio D-glucose: d-galactose: l-arabinose: d-glucuronic acid: d-galacturonic acid ═ 54.54:4.21:18.18: 4.87:4.21. The methylation result shows that the structure comprises 1, 4-linked glucose, 1,3,4, 6-linked glucose, 1,2, 3-linked glucose, end-linked glucuronic acid, 1, 5-linked arabinose, end-linked arabinose, 1, 4-linked galactose and end-linked galacturonic acid, and the molar ratio is 7.89:2.01:1:0.95:1.1:1.95:2.24:0.99: 1.12. And quercetin is linked to the 6-position of 1,3,4, 6-linked glucose.

(3) MBAP-2 is composed of glucose and has a molecular weight of about 46.5 kDa; total sugar content 82.03%; the protein content is 2.06%; the flavone content is 15.96%, and the product does not contain sulfate group. Methylation results show that the sugar consists of 1, 4-linked glucose, 1,3, 6-linked glucose, end-linked glucose, 1, 6-linked glucose and 1,3, 4-linked glucose, and the molar ratio is 0.44:0.35:1:0.9: 0.63. And quercetin is linked to both the 6-position of 1,3, 6-linked glucose and the 1-position of 1, 6-linked glucose.

(4) MBAP-3 is a polysaccharide composed of five monosaccharides and has a molecular weight of about 80.1 kDa; the total sugar content was 89.01%; the protein content is 1.55%; the uronic acid content is 10.51%; the flavone content is 9.43%, and the product does not contain sulfate group. Monosaccharide molar ratio D-mannose: d-glucuronic acid: d-glucose: d-galactose: l-arabinose was 11.87:10.51:63.21:5.04: 10.21. Methylated sugar chain analysis shows that the sugar consists of end group connected arabinose, end group connected glucuronic acid, 1, 4-connected galactose, 1, 4-connected glucose, 1,3, 4-connected glucose, 1,4, 6-connected mannose and 1,3,4, 6-connected glucose, and the molar ratio is 2.01:1.89:1:10.42:2.11:1.16: 0.79. And myricetin is linked to the 4-position of the 1, 4-linked galactose.

(5) MBAP-4 is a polysaccharide composed of three monosaccharides and has a molecular weight of about 56.4 kDa. The total sugar content was 88.66%; the protein content is 1.49%; the uronic acid content is 11.02%; the flavone content is 9.84%, and the product does not contain sulfate group. Monosaccharide molar ratio D-glucuronic acid: d-glucose: d-galacturonic acid 6.35:87.86: 5.79. Methylated sugar chain analysis shows that the sugar consists of end-linked glucuronic acid, end-linked galacturonic acid, 1, 4-linked glucose, 1,3, 4-linked glucose and 1,4, 6-linked glucose, and the molar ratio is 1:1.01:14.93:0.79: 2.33. And myricetin is connected to the 6-position of the 1,4, 6-connecting glucose.

(6) MBAP-5 is a polysaccharide composed of four monosaccharides and has a molecular weight of about 153.6 kDa. The total sugar content was 83.98%; the protein content is 1.07%; uronic acid content of 24.86%; the flavone content is 14.94%, and the product does not contain sulfate group. Monosaccharide molar ratio D-glucose: d-glucuronic acid: d-galactose: l-arabinose 50.14:24.86:15.42: 9.58. Methylated sugar chain analysis showed that the sugar consisted of 1, 4-linked glucuronic acid, 1, 6-linked glucose, end-linked arabinose, 1,4, 6-linked glucose, end-linked galactose, 1,3, 4-linked glucose and 1,3,4, 6-linked glucose in a molar ratio of 4.86:5.06:1.79:2.24:3.11:1.96: 0.89. And quercetin is linked to the 4-position of 1,3,4, 6-linked glucose.

(7) MBAP-6 is a polysaccharide composed of two monosaccharides and has a molecular weight of about 34.4 kDa. Total sugar content 88.69%; the protein content is 0.91%; uronic acid content of 9.51%; the flavone content is 10.42%, and the product does not contain sulfate group. Monosaccharide molar ratio D-glucose: d-glucuronic acid 82.4: 17.6. Methylated sugar chain analysis showed that the sugar consisted of 1, 4-linked glucuronic acid, 1, 6-linked glucose, 1,3,4, 6-linked glucose, 1,3, 6-linked glucose and terminal linked glucose in a molar ratio of 4.15:3.67:0.92:3.13:2.15: 5.93. And quercetin is linked to the 3-position of the 1,3,4, 6-linked glucose.

The tamarix chinensis polysaccharides (MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6) are prepared by the following method:

extracting ramulus et folium Tamaricis with 95% ethanol, filtering, oven drying the residue, extracting with hot water, filtering the extractive solution, concentrating, adding 95% ethanol until ethanol concentration is 80%, standing, centrifuging to obtain supernatant, concentrating the supernatant, adding anhydrous ethanol until ethanol concentration is 90%, standing, centrifuging to remove supernatant, precipitating, adding water to redissolve, removing protein with trichloroacetic acid, centrifuging, adjusting the supernatant to neutrality, concentrating, dialyzing, and freeze drying to obtain 90-alcohol precipitated polysaccharide (MBAP 90).

90 dissolving the precipitated polysaccharide in distilled water, and performing primary separation by using DEAE-cellulose column chromatography. Eluting with distilled water, 0.2, 0.4, 0.8 and 2.0mol/L NaCl solution, collecting each fraction, concentrating, dialyzing and lyophilizing to obtain 5 secondary components: MBAP90-1, MBAP90-2, MBAP90-3, MBAP90-4 and MBAP 90-5.

Dissolving each secondary component in appropriate amount of water, centrifuging, and collecting supernatant with Sephacryl^TMS200 gel chromatography (molecular weight cut-off of 2kDa-400kDa) was used for separation and fractions were collected. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with separate tube, mixing the fractions, concentrating, and freeze drying to obtain homogeneous polysaccharide.

The invention carries out in vitro tests, and the results prove that the tamarix chinensis polysaccharides MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 all have obvious inhibition on cell hemolysis caused by the activation of the classical complement pathway, namely obvious anticomplementary action, CH₅₀The values (concentration of test sample required for 50% inhibition of hemolysis by the classical pathway) were 96. + -. 4. mu.g/mL, 33. + -. 4. mu.g/mL, 122. + -. 5. mu.g/mL, 72. + -. 3. mu.g/mL, 67. + -. 2. mu.g/mL, 56. + -. 2. mu.g/mL and 102. + -. 8. mu.g/mL, respectively.

In vivo animal experiments prove that the tamarix chinensis polysaccharides MBAP90, MBAP-2, MBAP-3 and MBAP-5 have obvious improvement effects on the weight, pathological damage of the lung, inflammatory reaction of the lung and the like of a mouse with H1N1 induced viral pneumonia, and have obvious treatment effects on viral pneumonia caused by influenza viruses.

The tamarix chinensis polysaccharide can be used for preparing a complement inhibitor and a medicament for preventing and treating viral pneumonia.

Drawings

FIG. 1 shows a process for separating homogeneous Tamarix chinensis polysaccharides.

FIG. 2 is a HPGPC chromatogram of MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5, and MBAP-6, wherein TSK-GEL GMPW_XLGel column (300X 7.6 mm); eluent: distilled water; flow rate: 0.8 ml/min).

Figure 3 effect of tamarix chinensis polysaccharide MBAP90 on body weight of H1N1 virus infected mice.

Fig. 4, effect of tamarix chinensis polysaccharide MBAP90 on H1N1 virus infected mouse lung index, wherein, # # # P <0.001, Normal Control vs Model Control, # P <0.05, # P <0.01, # P <0.001, vs Model Control.

FIG. 5, the effect of Tamarix chinensis polysaccharides MBAP90 on H1N1 virus infected mouse serum TNF- α, IL-18, C3C and IL-1 β, wherein, # # P <0.01, # # P <0.001, Normal Control vs Model Control, # P <0.05, # P <0.01, # P <0.001, vs Model Control.

FIG. 6, the effect of Tamarix chinensis polysaccharide MBAP90 on H1N1 virus infected mouse lung homogenate MCP-1, IL-8, IL-6, C3C, wherein, # # P <0.001, Normal Control vs Model Control, # P <0.05, # P <0.01, # P <0.001, vs Model Control.

FIG. 7, the effect of Tamarix chinensis polysaccharide MBAP90 on lung pathology in H1N1 virus-infected mice (200X).

FIG. 8, effect of tamarix chinensis homopolysaccharides (MBAP-2, MBAP-3, MBAP-5) on body weight of H1N1 virus-infected mice.

FIG. 9, Effect of Tamarix chinensis homopolysaccharides (MBAP-2, MBAP-3, MBAP-5) on H1N1 virus-infected mouse lung index, wherein, # # # P <0.001, Normal Control vs Model Control, # P <0.05, # P <0.01, # P <0.001, vs Model Control.

FIG. 10, effect of Tamarix chinensis homopolysaccharides (MBAP-2, MBAP-3, MBAP-5) on H1N1 virus infected mouse lungs to homogenize IL-8, IL-6, IL-18, IL-1 β, C3C, and C5b-9, wherein # P <0.001, Normal Control vs Model Control, # P <0.05, # P <0.01, # P <0.001, vs Model Control.

FIG. 11, the effect of tamarix chinensis homopolysaccharide (MBAP-2, MBAP-3, MBAP-5) on the pathological changes in the lungs of H1N1 virus-infected mice (200X).

Detailed Description

EXAMPLE 1 preparation of Tamarix chinensis polysaccharides MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6

Crushing 17kg of tamarix chinensis medicinal materials, extracting with 95% ethanol, filtering, extracting medicinal residues with an aqueous solution for 3 times, concentrating, centrifuging, adding 95% ethanol into supernate until the ethanol concentration reaches 80%, standing, centrifuging to obtain supernate, concentrating the supernate, adding absolute ethanol until the ethanol concentration reaches 90%, standing, centrifuging to remove the supernate, redissolving the precipitate with water, and recovering under reduced pressure to remove the ethanol; redissolving, removing free protein with trichloroacetic acid, centrifuging, adjusting supernatant to neutral, dialyzing, concentrating, and freeze drying to obtain crude polysaccharide MBAP 90. Dissolving 100g crude polysaccharide in distilled water, centrifuging, and subjecting the supernatant to preliminary separation by DEAE-cellulose column chromatography. Eluting with distilled water, 0.2, 0.4, 0.8 and 2.0mol/L NaCl solution, eluting with volume more than 2 times column volume (about 10L), flow rate 25mL/min, collecting each fraction, and detecting absorbance value at 490nm (after sulfuric acid-phenol method color development) with separate tubes. According to the chromogenic reaction of sugar and the result of ultraviolet detection, combining the fractions, concentrating, dialyzing and freeze-drying to obtain 5 secondary components: MBAP90-1, MBAP90-2, MBAP90-3, MBAP90-4 and MBAP 90-5.

Dissolving MBAP90-2(9.5g) in distilled water, centrifuging, and separating supernatant with Sephacryl^TMAnd (4) separating by S200 chromatography (molecular weight cut-off is 2kDa-400 kDa). Eluting with distilled water solution at flow rate of 0.5mL/min, and collecting fractions. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, mixing the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides MBAP-1(205mg) and MBAP-2(920 mg).

Dissolving MBAP90-4(5.5g) in distilled water, centrifuging, and separating supernatant with Sephacryl^TMAnd S200 chromatographic separation. Eluting with distilled water solution at flow rate of 0.5mL/min, and collecting each fraction. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with separate tube, mixing the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides MBAP-3(905mg) and MBAP-4(196 mg).

Dissolving MBAP90-5(4.2g) in distilled water, centrifuging, and separating supernatant with Sephacryl^TMAnd S200 chromatographic separation. Eluting with 0.1mol/L NaCl solution at a flow rate of 0.8mL/min, and collecting fractions. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, mixing the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides MBAP-5(930mg) and MBAP-6(228 mg).

MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are all homogeneous components detected by High Performance Gel Permeation Chromatography (HPGPC).

Example 2 structural characterization of Tamarix chinensis polysaccharides (MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6)

1) Determination of molecular weight

The method is characterized in that the relative molecular weight of polysaccharide samples is measured by High Performance Gel Permeation Chromatography (HPGPC), the basic principle is that homogeneous polysaccharide forms symmetrical chromatographic peaks through gel permeation chromatography, the peak-appearing time is related to the molecular weight, and calculation is carried out according to a calibration curve obtained by known molecular weight.

Chromatographic conditions are as follows: by TSK GMPW_XLThe gel column was used for separation at a flow rate of 0.8mg/mL and a sample volume of 20. mu.L, with ultrapure water as the mobile phase, at a column temperature of 25 ℃ and an Evaporative Light Scattering Detector (ELSD).

The experimental method comprises the following steps: accurately weighing 2.0mg of each of homopolysaccharide and dextran series standard, preparing a solution of 2.0mg/mL with ultrapure water, filtering with a 0.45-micrometer microporous filter membrane before sample introduction, detecting, recording retention time, drawing a standard curve by taking a logarithmic value (Lg) of the molecular weight of the standard polysaccharide as a vertical coordinate and the retention time as a horizontal coordinate, obtaining a corresponding linear regression equation, and calculating the relative molecular weight of the homopolysaccharide. The relative molecular weights of MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are 30.2-274.1kDa, 269.3kDa, 46.5kDa, 80.1kDa, 56.4kDa, 153.6kDa and 34.4kDa, respectively.

2) Content determination of total sugar, uronic acid, protein, sulfate and flavone

The total sugar content of MBAP90 determined by a sulfuric acid-phenol method is 64.36%; the total sugar content of MBAP-1 is 86.06%; the total sugar content of MBAP-2 is 82.03%; the total sugar content of MBAP-3 is 89.01 percent; the total sugar content of MBAP-4 is 88.66%; the total sugar content of MBAP-5 was 83.98% and the total sugar content of MBAP-6 was 88.69%.

The content of uronic acid is detected by an m-hydroxy biphenyl method, and the content of uronic acid of MBAP-1 is 9.64%; the uronic acid content of MBAP-3 is 10.51%; the uronic acid content of MBAP-4 is 11.02%; the uronic acid content of MBAP-5 is 24.86%; the uronic acid content of MBAP-6 was 9.51%.

Protein content determination by Coomassie Brilliant blue method: the protein content of MBAP90 was 3.53%; the protein content of MBAP-1 is 1.90%; the protein content of MBAP-2 is 2.06%; the protein content of MBAP-3 is 1.55%; the protein content of MBAP-4 is 1.49%; the protein content of MBAP-5 is 1.07%; the protein content of MBAP-6 is 0.91%.

BaCl₂MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP90-6 do not contain sulfate groups as measured by turbidimetry.

Measuring flavone content by an aluminum nitrate color development method: the total flavone content of MBAP90 is 12.18%; the quercetin content of MBAP-1 is 12.03%; the quercetin content of MBAP-2 is 15.96%; the myricetin content of MBAP-3 is 9.43%; MBAP-4 has myricetin content of 9.84%; the quercetin content of MBAP-5 is 14.94%; the quercetin content of MBAP-6 is 10.42%.

3) Monosaccharide composition analysis

MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are respectively subjected to 2mol/L TFA and total hydrolysis at 110 ℃ to obtain products, and then PMP derivatization is carried out, and then liquid phase analysis is carried out.

MBAP90 is a polysaccharide composed of six monosaccharides, D-glucose: d-galactose: l-arabinose: d-mannose: d-glucuronic acid: d-galacturonic acid (21.2: 8.12:9.31:4.14:2.07: 1.12).

MBAP-1 is a polysaccharide composed of five monosaccharides, D-glucose: d-galactose: l-arabinose: d-glucuronic acid: d-galacturonic acid 54.54:4.21:18.18:4.87: 4.21.

MBAP-2 is composed of D-glucose.

MBAP-3 is a polysaccharide composed of five monosaccharides, D-mannose: d-glucuronic acid: d-glucose: d-galactose: l-arabinose 11.87:10.51:63.21:5.04: 10.21.

MBAP-4 is a polysaccharide composed of three monosaccharides, D-glucuronic acid: d-glucose: d-galacturonic acid 6.35:87.86: 5.79.

MBAP-5 is a polysaccharide consisting of four monosaccharides, D-glucose: d-glucuronic acid: d-galactose: l-arabinose 50.14:24.86:15.42: 9.58.

MBAP-6 is a polysaccharide composed of two monosaccharides, D-glucose: d-glucuronic acid 82.4: 17.6.

4) Methylation analysis

Polysaccharide separation by modified Hakomori methodMethylation (MBAP-1, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 with uronic acid polysaccharides₄Reduced and then methylated), the methylated product was perhydrolyzed with 2mol/L TFA, NaBD₄Reduction and acetylation of acetic anhydride to produce a partially methylated alditol acetate derivative, followed by GC-MS analysis.

The MBAP-1 structure comprises: 1, 4-linked glucose, 1,3,4, 6-linked glucose, 1,2, 3-linked glucose, end-linked glucuronic acid, 1, 5-linked arabinose, end-linked arabinose, 1, 4-linked galactose and end-linked galacturonic acid in a molar ratio of 7.89:2.01:1:0.95:1.1:1.95:2.24:0.99: 1.12.

The MBAP-2 structure comprises: 1, 4-linked glucose, 1,3, 6-linked glucose, terminal linked glucose, 1, 6-linked glucose, 1,3, 4-linked glucose, in a molar ratio of 0.44:0.35:1:0.9: 0.63.

The MBAP-3 structure comprises: the molar ratio of terminal-linked arabinose to terminal-linked glucuronic acid to 1, 4-linked galactose to 1, 4-linked glucose to 1,3, 4-linked glucose to 1,4, 6-linked mannose to 1,3,4, 6-linked glucose is 2.01:1.89: 1.42: 2.11:1.16: 0.79.

The MBAP-4 structure comprises: the molar ratio of the end group connected glucuronic acid to the end group connected galacturonic acid to the end group connected glucose, 1, 4-connected glucose, 1,3, 4-connected glucose to the end group connected galacturonic acid to the end group connected glucose is 1:1.01:14.93:0.79: 2.33.

The MBAP-5 structure comprises: 1, 4-linked glucuronic acid, 1, 6-linked glucose, end-linked arabinose, 1,4, 6-linked glucose, end-linked galactose, 1,3, 4-linked glucose and 1,3,4, 6-linked glucose in a molar ratio of 4.86:5.06:1.79:2.24:3.11:1.96: 0.89.

The MBAP-6 structure contains: 1, 4-linked glucuronic acid, 1, 6-linked glucose, 1,3,4, 6-linked glucose, 1,3, 6-linked glucose and end-group linked glucose in a molar ratio of 4.15:3.67:0.92:3.13:2.15: 5.93.

5) Confirmation of flavone structure and linkage mode in homogeneous polysaccharide

MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 and MBAP-6 are respectively subjected to total hydrolysis by 2mol/L TFA at 110 ℃, and the obtained product is spun dry and then dissolved by adding a proper amount of methanol for LC-MS analysis. After the primary analysis is carried out according to the fragments, LC-MS further confirmation is carried out by respectively taking myricetin and quercetin as controls.

LC-MS analysis results show that structures of MBAP-1, MBAP-2, MBAP-5 and MBAP-6 contain quercetin; the structures of MBAP-3 and MBAP-4 contain myricetin.

The nuclear magnetism HMBC related signal confirms that the quercetin in the MBAP-1 is connected to 6 sites of 1,3,4, 6-connection glucose; in MBAP-2, the 4' position of quercetin is connected with the 6 position of 1,3, 6-linked glucose, and the 5 position of quercetin is linked with the 1 position of 1, 6-linked glucose; in MBAP-3, myricetin is connected with 4 positions of 1, 4-connecting galactose; in MBAP-4, myricetin is connected at the 6-position of 1,4, 6-connecting glucose; in MBAP-5, quercetin is linked to the 4-position of 1,3,4, 6-linked glucose; in MBAP-6 quercetin is attached to position 3 of 1,3,4, 6-linked glucose.

Example 3 classical pathway complement inhibition assay

Serum from 3-month-old guinea pigs was diluted 1: 100 with BBS buffer (barbital buffer, pH 7.4) as a source of complement for the present classical pathway. Diluting rabbit anti-sheep erythrocyte antibody with BBS buffer solution to 1: 1000 as hemolysin; sheep Red Blood Cells (SRBC) were configured as 2% SRBC. Polysaccharide 3mg is precisely weighed, added with BBS buffer solution for dissolution, and diluted into 8 concentrations in a double way. After 200 mul of polysaccharide solution with different concentrations and 200 mul of complement which has been diluted to 1: 100 are preincubated for 10min at 37 ℃, 100 mul of hemolysin (1: 1000) and 100 mul of 2% SRBC are sequentially added, and the mixture is put into a low-temperature high-speed centrifuge after being put into a water bath at 37 ℃ for 30min and centrifuged for 10min at 5000rpm and 4 ℃. mu.L of supernatant was taken from each tube in a 96-well plate and absorbance was measured at 405 nm. The experiment was performed with a polysaccharide control group (200. mu.L of polysaccharide at the corresponding concentration plus 400. mu.BBS buffer), a complement control group (200. mu.LBBS buffer instead of polysaccharide), and a whole blood-lysed group (100. mu.L of 2% SRBC in 500. mu.L of distilled water). And subtracting the absorbance value of the polysaccharide group of each concentration from the absorbance value of the corresponding polysaccharide control group to calculate the hemolysis inhibition rate. The logarithm of the polysaccharide concentration was plotted on the X-axis and the inhibition rate of hemolysis was plotted on the Y-axis, and the resulting fitted curve was used to calculate the 50% inhibitionConcentration of test substance (CH) required for hemolysis₅₀Value). The results of using heparin as a positive control show that 90 alcohol precipitated polysaccharide and six homogeneous polysaccharides all have significant inhibitory activity on classical pathway activation of complement (as shown in table 1).

TABLE 1 inhibition of complement activation by Tamarix chinensis polysaccharides

Tamarix chinensis polysaccharide

MBAP90

MBAP-1

MBAP-2

MBAP-3

MBAP-4

MBAP-5

MBAP-6

CH₅₀(μg/mL)

96±4

33±4

122±5

72±3

67±2

56±2

102±8

CH₅₀The values are expressed as:mean ± SD (n ═ 3); positive control heparin CH₅₀：34±2μg/mL。

Example 4 Effect of Tamarix chinensis polysaccharide MBAP90 on H1N1 induced acute Lung injury mouse Lung index

Balb/c mice were 36 (15-16g) and were randomized into 6 groups by body weight (A, B, C, D, E, F): group A is a normal control group, group B is an H1N1 virus model group, group C is a positive control ribavirin group, group D is a tamarix chinensis polysaccharide MBAP90 low dose group (MBAP90-100, 100mg/kg), group E is a tamarix chinensis polysaccharide MBAP90 medium dose group (MBAP90-200, 200mg/kg), group F is a tamarix chinensis polysaccharide MBAP90 high dose group (MBAP90-400, 400mg/kg), and 6 groups are selected. All animals were anesthetized by sodium pentobarbital intraperitoneal injection and infected with 3LD by nasal drip₅₀H1N1 virus solution 30 μ L, group A nose drop infection 1640 culture solution 30 μ L as control; B-F group is infected with H1N1 virus diluent, and C-F group is infected with corresponding dose of medicine after 2 hours; meanwhile, A, B groups were administered with 0.5% CMC-Na by intragastric administration as normal group and virus control group. The body weight was weighed daily and administered once a day for four consecutive days. Weighing the weight of the animal after 96 hours of H1N1 virus attack, picking eyeballs and taking blood; taking a lung tissue specimen of the experimental mouse according to a conventional method, and weighing and recording; placing in 10% formalin solution, making pathological section, and performing pathological evaluation; the right lung is stored in a refrigerator at-80 ℃ and is used for detecting the index of the pneumonia factor.

(1) Tamarix chinensis polysaccharide MBAP90 has double effects on H1N1 virus infected mice

The weight change is a macroscopic index for evaluating the protective effect of the medicament on mice with viral pneumonia. The change rate of the body weight of the mouse on the day of infection is obtained by dividing the body weight of the mouse on the day of infection, and a body weight change curve is drawn according to the change rate of the body weight of the mouse on the day of infection. The results show that the weight reduction of the mice in the model group is the largest, and the weight reduction of the mice in each group is significantly smaller than that of the mice in the model group, wherein the weight reduction of the tamarix chinensis polysaccharide MBAP90-400(400mg/kg) is slightly better than that of the tamarix chinensis polysaccharide MBAP90-200(200mg/kg) and the weight reduction of the tamarix chinensis polysaccharide MBAP90-100(100mg/kg) on the fourth day, and the results are shown in FIG. 3.

(2) Effect of Tamarix chinensis polysaccharide MBAP90 on H1N1 virus infected rat Lung index

The lung index is the ratio of the lung weight to the body weight of the mouse, and the larger the ratio is, the more serious the lung lesion degree is, which is also an important indicator of the virus pneumonia. The result shows that the lung index of the mice in the model group is obviously increased (P <0.001) compared with that in the normal group; after the administration of the drugs, the lung index of the MBAP90 (MBAP90-200, MBAP90-400) was significantly lower than that of the model group (P <0.05, P <0.01) except for the MBAP90 low dose group. The tamarix chinensis polysaccharide high dose group was slightly better than the medium dose group (as shown in fig. 4).

(3) Effect of tamarix chinensis polysaccharide MBAP90 on TNF-alpha, IL-18, C3C and IL-1 beta of H1N1 virus infected mouse serum

Centrifuging whole blood of the mouse to obtain serum, subpackaging and freezing. Supernatants were assayed using the Elisa method according to the kit instructions for TNF-. alpha.IL-18, C3C and IL-1. beta. The results show that the levels of TNF-alpha, IL-18, C3C and IL-1 beta in the serum of the model group are significantly increased compared with the normal group (P <0.001, P < 0.01); after administration, except for the MBAP90 low dose group, the serum TNF- α, IL-18, C3C and IL-1 β levels were significantly lower in the other polysaccharide administration groups (MBAP90-200, MBAP90-400) than in the model group (TNF- α: P <0.05, P < 0.01; IL-18: P <0.001, P < 0.001; IL-1 β: P <0.05, P < 0.05; C3C: P <0.01, P <0.01), and the Tamarix chinensis polysaccharide MBAP90 high dose group was slightly lower than in the medium dose group (as shown in FIG. 5).

(4) Action of Tamarix chinensis polysaccharide MBAP90 on H1N1 virus infected mouse lung homogenate MCP-1, IL-8, IL-6 and C3C

The homogenate supernatants were assayed using the Elisa method according to the instructions of MCP-1, IL-8, IL-6, C3C kits. Compared with a normal group, the lung homogenate MCP-1, IL-8, IL-6 and C3C levels of the mice in the model group are obviously increased (P <0.001, P <0.001 and P < 0.01); after administration, the levels of MCP-1 and IL-6 in lung homogenate of each polysaccharide administration group are significantly lower than those of a model group (MCP-1: P <0.001, P <0.001P < 0.001; IL-6: P <0.001, P <0.001P < 0.001); meanwhile, after administration, in addition to the MBAP90 low dose group, the levels of IL-8 and C3C were significantly reduced (IL-8: P <0.001, P < 0.001; C3C: P <0.01, P <0.01) in the polysaccharide administration groups (MBAP90-200, MBAP90-400) as compared to the model group, and the results are shown in FIG. 6,

(5) influence of tamarix chinensis polysaccharide MBAP90 on lung pathological changes of H1N1 virus infected mice

The pathological examination result shows that the normal group alveolus has clear outline, complete structure, no bleeding phenomenon and basically no inflammation; the pathological section of the model group shows that the alveolar wall is obviously thickened, the alveoli are atrophied and deformed, a large number of neutrophils and lymphomonocytes are infiltrated into the pulmonary interstitium, and the inflammation is serious. Each administration group can obviously improve pathological injury of the lung, the outline of the pulmonary alveoli is clear, the structure is relatively complete, and the inflammatory cell infiltration condition is obviously relieved. The lung injury was minimal in the tamarix chinensis polysaccharide MBAP90 high dose group (400mg/kg) and medium dose group (200mg/kg), which were substantially similar to the normal group (as shown in FIG. 7).

Example 5 Effect of Tamarix chinensis homopolysaccharides MBAP-2, MBAP-3 and MBAP-5 on H1N1 induced acute Lung injury mouse Lung index

Balb/c mice were 36 (15-16g) and were randomized into 6 groups by body weight (A, B, C, D, E, F): group A is a normal control group, group B is a H1N1 virus model group, group C is a positive control ribavirin group, group D is a homopolysaccharide MBAP-2 group (MBAP-2, 400mg/kg), group E is a homopolysaccharide MBAP-3 group (MBAP-3, 400mg/kg), group F is a homopolysaccharide MBAP-5 group (MBAP-5, 400mg/kg), and 6 groups are selected. All animals were anesthetized by sodium pentobarbital intraperitoneal injection and infected with 3LD by nasal drip₅₀H1N1 virus solution 30 μ L, group A nose drop infection 1640 culture solution 30 μ L as control; B-F group is infected with H1N1 virus diluent, and C-F group is infected with corresponding dose of medicine after 2 hours; meanwhile, A, B groups were given 0.5% CMC-Na as a control group of normal and virus. The body weight was weighed daily and administered once a day for four consecutive days. Weighing the weight of the animal after 96 hours of H1N1 virus attack, picking eyeballs and taking blood; taking a lung tissue specimen of the experimental mouse according to a conventional method, and weighing and recording; placing in 10% formalin solution, making pathological section, and performing pathological evaluation; the right lung is stored in a refrigerator at the temperature of-80 ℃ and is used for detecting the index of the pneumonia factors.

(1) Tamarix chinensis homogeneous polysaccharide MBAP-2, MBAP90-3 and MBAP-5 double-sound effect on H1N1 virus infected mice

The weight change is a macroscopic index for evaluating the protective effect of the medicament on mice with viral pneumonia. The change rate of the body weight of the mouse on the day of infection is obtained by dividing the body weight of the mouse on the day of infection, and a body weight change curve is drawn according to the change rate of the body weight of the mouse on the day of infection. The results showed that the weight loss of the model group mice was the greatest, and the weight loss of each homopolysaccharide-administered group was significantly less than that of the model group (as shown in fig. 8).

(2) Influence of homogenous polysaccharides MBAP-2, MBAP-3 and MBAP-5 of Tamarix chinensis on lung index of H1N1 virus infected mouse

The lung index is the ratio of the lung weight to the body weight of the mouse, and the larger the ratio is, the more serious the lung lesion degree is, which is also an important indicator of the virus pneumonia. The result shows that the lung index of the mice in the model group is obviously increased (P <0.001) compared with that in the normal group; after the administration of the drugs, the lung indices of the homopolysaccharide-administered groups (MBAP-2, MBAP-3, and MBAP-5) were significantly lower than those of the model groups (P <0.05, P <0.01, and P <0.01), and the results are shown in FIG. 9.

(3) Effect of Tamarix chinensis homogeneous polysaccharides MBAP-2, MBAP-3 and MBAP-5 on H1N1 virus infected mouse lung homogenate IL-8, IL-6, IL-18, IL-1 beta, C3C and C5b-9

Lung homogenate supernatants were assayed using the Elisa method according to the kit instructions for IL-8, IL-6, IL-18, IL-1. beta., C3C, and C5 b-9. Compared with a normal group, the levels of IL-8, IL-6, IL-18, IL-1 beta, C3C and C5b-9 in the lung homogenate of the mice in the model group are obviously increased (P <0.001 ); after administration, the levels of IL-8, IL-6, IL-18, IL-1. beta., C3C, and C5b-9 in the lung homogenates of each homopolysaccharide administration group (MBAP-2, MBAP-3, MBAP-5) were significantly lower than those of the model group (IL-8: P <0.001, P < 0.001; IL-6: P <0.001, P < 0.001; IL-18: P <0.001, P < 0.001; IL-1. beta.: P <0.001, P < 0.001; C3C: P <0.001, P < 0.001; C5b-9: P <0.001, P < 0.001); among them, MBAP-5 was slightly superior to MBAP-3, while MBAP-3 was slightly superior to MBAP-2, and the results are shown in FIG. 10.

(4) Influence of homogenous polysaccharides MBAP-2, MBAP-3 and MBAP-5 of Tamarix chinensis on lung pathological changes of H1N1 virus infected mice

The pathological examination result shows that the normal group alveolus has clear outline, complete structure, no bleeding phenomenon and basically no inflammation; the pathological section of the model group shows that the alveolar wall is obviously thickened, the alveoli are atrophied and deformed, a large number of neutrophils and lymphomonocytes are infiltrated into the pulmonary interstitium, and the inflammation is serious. Each administration group can obviously improve pathological damage of the lung, the outline of the alveolus is clear, the structure is relatively complete, and the inflammatory cell infiltration condition is obviously relieved. MBAP-5(400mg/kg) and MBAP-3(400mg/kg) showed minimal lung injury, and were substantially similar to the normal group (see FIG. 11).

Claims

1. Tamarix chinensis polysaccharide MBAP90, MBAP-1, MBAP-2, MBAP-3, MBAP-4, MBAP-5 or MBAP-6, and has the following structural characteristics:

(1) the tamarix chinensis polysaccharide MBAP90 has the structural characteristics that: consists of six monosaccharides with the molecular weight range of 30.2-274.1 kDa; the total sugar content was 64.36%; the protein content was 3.53%; uronic acid content 13.52%; the flavone content is 12.18%; free of sulfate groups, monosaccharide molar ratio D-glucose: d-galactose: l-arabinose: d-mannose: d-glucuronic acid: d-galacturonic acid (21.2: 8.12:9.31:4.14:2.07: 1.12);

(2) the tamarix chinensis polysaccharide MBAP-1 has the structural characteristics that: polysaccharides composed of five monosaccharides and having a molecular weight of about 269.3 kDa; total sugar content 86.06%; the protein content is 1.90%; the uronic acid content is 9.64%; the flavone content is 12.03%, and the flavone does not contain sulfate; monosaccharide molar ratio D-glucose: d-galactose: l-arabinose: d-glucuronic acid: d-galacturonic acid 54.54:4.21:18.18:4.87: 4.21; the connection mode comprises 1, 4-connection glucose, 1,3,4, 6-connection glucose, 1,2, 3-connection glucose, end connection glucuronic acid, 1, 5-connection arabinose, end connection arabinose, 1, 4-connection galactose and end connection galacturonic acid, and the molar ratio is 7.89:2.01:1:0.95:1.1:1.95:2.24:0.99: 1.12; quercetin is connected to the 6-position of 1,3,4, 6-connecting glucose;

(3) the tamarix chinensis polysaccharide MBAP-2 has the structural characteristics that: consists of glucose and has a molecular weight of 46.5 kDa; total sugar content 82.03%; the protein content is 2.06%; the flavone content is 15.96%, and the product does not contain sulfate radical; the connection mode comprises 1, 4-connection glucose, 1,3, 6-connection glucose, terminal connection glucose, 1, 6-connection glucose and 1,3, 4-connection glucose, and the molar ratio is 0.44:0.35:1:0.9: 0.63; quercetin is connected to the 6-position of 1,3, 6-connecting glucose and the 1-position of 1, 6-connecting glucose at the same time;

(4) the tamarix chinensis polysaccharide MBAP-3 has the structural characteristics that: polysaccharides composed of five monosaccharides and having a molecular weight of about 80.1 kDa; the total sugar content was 89.01%; the protein content is 1.55%; the uronic acid content is 10.51%; the content of flavone is 9.43%, and the flavone does not contain sulfate radical; monosaccharide molar ratio D-mannose: d-glucuronic acid: d-glucose: d-galactose: l-arabinose 11.87:10.51:63.21:5.04: 10.21; the connection mode comprises end group connection arabinose, end group connection glucuronic acid, 1, 4-connection galactose, 1, 4-connection glucose, 1,3, 4-connection glucose, 1,4, 6-connection mannose and 1,3,4, 6-connection glucose, wherein the molar ratio is 2.01:1.89:1:10.42:2.11:1.16: 0.79; myricetin is connected with the 4 position of the 1, 4-connecting galactose;

(5) the tamarix chinensis polysaccharide MBAP-4 has the structural characteristics that: polysaccharides composed of three monosaccharides, with a molecular weight of 56.4 kDa; the total sugar content was 88.66%; the protein content is 1.49%; the uronic acid content is 11.02%; the content of flavone is 9.84%, and the flavone does not contain sulfate radical; monosaccharide molar ratio D-glucuronic acid: d-glucose: d-galacturonic acid 6.35:87.86: 5.79; the connection mode comprises a terminal group connected glucuronic acid, a terminal group connected galacturonic acid, 1, 4-connected glucose, 1,3, 4-connected glucose and 1,4, 6-connected glucose, and the molar ratio is 1:1.01:14.93:0.79: 2.33; the 6-position of the 1,4, 6-connecting glucose is connected with myricetin;

(6) the tamarix chinensis polysaccharide MBAP-5 has the structural characteristics that: polysaccharides composed of four monosaccharides and having a molecular weight of 153.6 kDa; the total sugar content was 83.98%; the protein content is 1.07%; the uronic acid content is 24.86%; the content of flavone is 14.94%, and the flavone does not contain sulfate; monosaccharide molar ratio D-glucose: d-glucuronic acid: d-galactose: l-arabinose 50.14:24.86:15.42: 9.58; the linkage mode comprises 1, 4-linked glucuronic acid, 1, 6-linked glucose, end-linked arabinose, 1,4, 6-linked glucose, end-linked galactose, 1,3, 4-linked glucose and 1,3,4, 6-linked glucose, and the molar ratio is 4.86:5.06:1.79:2.24:3.11:1.96: 0.89; quercetin is connected to the 4-position of 1,3,4, 6-linked glucose.

(7) The tamarix chinensis polysaccharide MBAP-6 has the structural characteristics that: polysaccharide composed of two monosaccharides and having a molecular weight of 34.4 kDa; total sugar content 88.69%; the protein content is 0.91%; the uronic acid content is 9.51%; the flavone content is 10.42%, and the product does not contain sulfate radical; monosaccharide molar ratio D-glucose: d-glucuronic acid 82.4: 17.6; the linking means comprises 1, 4-linked glucuronic acid, 1, 6-linked glucose, 1,3,4, 6-linked glucose, 1,3, 6-linked glucose and terminal linked glucose in a molar ratio of 4.15:3.67:0.92:3.13:2.15: 5.93; quercetin is attached to the 3-position of the 1,3,4, 6-linked glucose.

2. Use of tamarix chinensis polysaccharides according to claim 1 for the preparation of a medicament for the preparation of a complement inhibitor.

3. Use of the tamarix chinensis polysaccharide of claim 1 in the preparation of a medicament for the control of viral pneumonia.

4. The tamarix chinensis polysaccharide of claim 1, prepared by the process of:

extracting the branches and leaves of the tamarix chinensis by using ethanol, filtering, extracting dregs of the decoction by using hot water, filtering, concentrating, centrifuging, adding a proper amount of ethanol into supernate, wherein the final concentration of the ethanol is 70-80%, standing, centrifuging, adding a proper amount of ethanol into the supernate, wherein the final concentration of the ethanol is 88-93%, redissolving the precipitate by using water, and removing free protein to obtain crude polysaccharide MBAP 90; dissolving MBAP90 in water, performing primary separation by DEAE-cellulose chromatography, eluting with distilled water and NaCl solution of 0.2, 0.4, 0.8 and 2.0mol/L, and mixing the same polysaccharide components according to the effluent solution of sugar color reaction and ultraviolet detection result; the 3 polysaccharide fractions eluted with 0.2, 0.8 and 2.0mol/L NaCl solution were further purified by Sephacryl S-200 gel chromatography, eluted with distilled water, pooled according to the results of glycochromogen reaction and HPGPC detection, and assayed for anticomplementary activity and homogeneity to give homogeneous polysaccharides having anticomplementary activity.