Croton bean polysaccharide capable of relieving cough, reducing sputum and improving immunity as well as preparation method and application of carob bean polysaccharide

Abstract

The invention relates to carob bean polysaccharide for relieving cough, reducing sputum and improving immunity as well as a preparation method and application thereof. The carob polysaccharide is composed of two monosaccharides, namely galactose and glucose, the molar ratio of the galactose to the glucose is (88-92): (0.8-1.2), and the carob polysaccharide is formed by connecting and combining glycosidic bonds (1-> 4)-Glu, (1-> 3, 4, 6)-Glu, (1-> 3)-Gal, (3-> 6)-Gal, (2-> 4, 6)-Gal, (2-> 6)-Gal and (1->)-Gal. The carob bean polysaccharide can improve the respiratory tract, relieve cough, reduce sputum and enhance the immunity.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and health food, and particularly relates to a carob polysaccharide for relieving cough and reducing phlegm and enhancing immunity, a preparation method thereof, and uses thereof. Background Art

[0002] Carob trees are distributed along the Mediterranean coast, in California of the United States, and in Australia, etc., and are mainly planted in Guangxi, Sichuan, Yunnan, and Guangdong in China. Carob is low in calories, fat-free, and rich in natural polysaccharides, various vitamins, proteins, and trace elements. Currently, carob extracts are mainly applied in the fields of edible spices, natural food thickeners, and stabilizers. Carob not only has very high nutritional value but also has extremely high medicinal value. The syrup made from carob pods can play a good role in relieving colds, coughs, and sore throats. The main component of carob pods is carbohydrates.

[0003] Respiratory diseases are one of the common diseases, usually with symptoms such as cough, expectoration, and even coughing up blood, which often cause a decline in human immunity. Currently, there are a wide variety of cough-relieving and phlegm-reducing drugs on the market. Western medicine treatment can neither effectively eradicate the disease, nor is it easy to produce drug resistance, and it has large side effects. Traditional Chinese medicine products have a slow treatment effect and an unacceptable smell. However, products with good taste and smell are difficult to take into account the curative effect. Therefore, it is very important to develop a product with a quick effect, good curative effect, and at the same time can enhance immunity. Summary of the Invention

[0004] An embodiment of the present invention provides a carob polysaccharide, which can improve cough and reduce phlegm in the respiratory tract and can also enhance immunity. The carob polysaccharide in the embodiment of the present invention has a wide application range, a short course of treatment, a quick effect, and no observed toxic and side effects.

[0005] According to one aspect of the present invention, there is provided a carob polysaccharide, which is composed of two monosaccharides, galactose and glucose, and the molar ratio of galactose to glucose is (88 - 92):(0.8 - 1.2). The carob polysaccharide is combined by glycosidic bonds (1→4)-Glu, (1→3,4,6)-Glu, (1→3)-Gal, (3→6)-Gal, (2→4,6)-Gal, (2→6)-Gal, (1→)-Gal.

[0006] In some embodiments of the present invention, the molar ratio of galactose to glucose in the carob polysaccharide is 89.666:1.068.

[0007] Herein, Glu represents glucose and Gal represents galactose.

[0008] In some embodiments of the present invention, the molecular weight of the carob polysaccharide is (1 - 6)×10 6 . Optionally, it is (4.8 - 5)×10 6 .

[0009] In some embodiments of the present invention, the molecular weight of the carob polysaccharide is (1-2)×10 6 .

[0010] In some embodiments of the present invention, the molecular weight of the carob polysaccharide is (2-3)×10 6 .

[0011] In some embodiments of the present invention, the molecular weight of the carob polysaccharide is (3-4)×10 6 .

[0012] In some embodiments of the present invention, the average molecular weight of the carob polysaccharide is (4.8-5)×10 6 .

[0013] In some embodiments of the present invention, the molecular weight distribution width (Mw / Mn) of the carob polysaccharide is 1.5-1.6, and may be 1.574.

[0014] In some embodiments of the present invention, the carob polysaccharide is isolated from carob tree pods.

[0015] In some embodiments of the present invention, the carob polysaccharide has at least the effects of relieving cough and reducing phlegm and enhancing immunity.

[0016] On the other hand, according to the present invention, there is provided the use of the carob polysaccharide described in the present invention as a drug or a health product.

[0017] On the other hand, according to the present invention, there is provided the use of the carob polysaccharide described in the present invention in the preparation of a drug for relieving cough and reducing phlegm.

[0018] On the other hand, according to the present invention, there is provided the use of the carob polysaccharide described in the present invention in the preparation of a drug for enhancing immunity.

[0019] On the other hand, according to the present invention, there is also provided an effective dose of the above carob polysaccharide for treating diseases.

[0020] Wherein, the diseases include cough, expectoration, and low immunity.

[0021] On the other hand, according to the present invention, there is provided a pharmaceutical composition which comprises the carob polysaccharide described in the present invention and further comprises a pharmaceutically acceptable carrier.

[0022] On the other hand, according to the present invention, there is provided a food which comprises the carob polysaccharide described in the present invention and further comprises food raw materials and auxiliary materials.

[0023] In some embodiments of the present invention, the food includes functional foods and dietary supplements.

[0024] In some embodiments of the present invention, the food may be one or more of beverages, candies, syrups, sweeteners, spices, etc.

[0025] According to another aspect of the present invention, there is also provided a method for treating a disease, which includes administering an effective dose of the above-mentioned carob polysaccharide or the above-mentioned pharmaceutical composition or the above-mentioned food to a subject or patient in need thereof through an oral or non-oral route. Among them, the diseases include cough, expectoration, and low immunity.

[0026] According to another aspect of the present invention, there is also provided a preparation method of carob polysaccharide. By using this method, the carob polysaccharide described in the present invention can be prepared, and the purity can reach ≥93%.

[0027] A preparation method of carob polysaccharide includes: taking the seedless carob tree pods after being crushed, degreasing with 90-95% ethanol (1-5 times the weight), and drying the degreased fine powder; extracting with water; centrifuging the extract, concentrating the supernatant, adding ethanol to make the mass fraction of ethanol 55-60%, centrifuging to collect the precipitate; dispersing the obtained precipitate with water, removing proteins by the Sevage method, adsorbing with macroporous adsorption resin D101, eluting with distilled water, collecting the mobile phase components, subjecting to DEAE-52 cellulose column chromatography, eluting with distilled water, subjecting to G-100 Sephadex gel column chromatography with deionized water as the mobile phase, collecting the eluate, then subjecting to G-150 Sephadex gel column chromatography with deionized water as the mobile phase, collecting the eluate, combining the same elution peak, concentrating under reduced pressure, and freeze-drying to obtain carob polysaccharide.

[0028] In some embodiments of the present invention, the carob tree pods are crushed into fine powder of 80-300 mesh, breaking the cell structure of the pods is beneficial to the dissolution of intracellular substances, and at the same time increasing the contact area between the pod powder and the extract to improve the extraction rate. In some embodiments of the present invention, ultrafine wall-breaking crushing technology can be used for crushing.

[0029] In some embodiments of the present invention, the ethanol used for degreasing is 2-5 times the weight of the seedless carob tree pods.

[0030] In some embodiments of the present invention, the extraction temperature is 40-50 °C, such as 45 °C. Within this temperature range, it is beneficial to fully dissolve carob polysaccharide.

[0031] In some embodiments of the present invention, extraction is carried out 2-4 times, and the extraction time for each time is 1-5 h.

[0032] In some embodiments of the present invention, the filtrate (extract) is centrifuged at 5000 rpm for 10 min, and the supernatant is combined.

[0033] In some embodiments of the present invention, the supernatant is concentrated under reduced pressure to a viscous concentrated solution at 60 °C and 100 Pa.

[0034] In some embodiments of the present invention, the carob pods are dried pods of the carob tree (Latin name: Ceratonia siliqua Linn.) of the genus Vigna in the legume family.

[0035] Experimental results show that when mice were placed in a large beaker with concentrated ammonia water to induce coughing, by measuring the number of coughs in the Hocacy group, positive group, and blank control group of carob polysaccharide at different concentrations by gavage, and analyzing the phenol red excretion in the trachea of the mice, the results indicate that the carob polysaccharide Hocacy in the embodiments of the present invention has good cough-relieving and phlegm-dissolving effects. Through the experiment of "improving the effect of reducing T cells in transgenic zebrafish emitting red fluorescence", it is shown that the carob polysaccharide Hocacy in the embodiments of the present invention has the effect of enhancing immunity. Description of the Drawings

[0036] Figure 1 It is the detection spectrum of the molecular weight of the carob polysaccharide Hocacy in the embodiments of the present invention.

[0037] Figure 2 It is the infrared detection spectrum of the carob polysaccharide Hocacy in the embodiments of the present invention.

[0038] Figure 3 It is the total ion analysis spectrum of GC-MS of the carob polysaccharide Hocacy in the embodiments of the present invention.

[0039] Figure 4 It is the HPLC chart of the carob polysaccharide Hocacy.

[0040] Figure 5 It is the ion fragment identification spectrum of the monosaccharide composition - galactose of the carob polysaccharide Hocacy.

[0041] Figure 6 It is the ion fragment identification spectrum of the monosaccharide composition - galactose of the carob polysaccharide Hocacy.

[0042] Figure 7 It is the ion fragment identification spectrum of the monosaccharide composition - galactose of the carob polysaccharide Hocacy.

[0043] Figure 8 It is the ion fragment identification spectrum of the monosaccharide composition - glucose of the carob polysaccharide Hocacy.

[0044] Figure 9 It is the ion fragment identification spectrum of the monosaccharide composition - galactose of the carob polysaccharide Hocacy.

[0045] Figure 10 It is the ion fragment identification spectrum of the monosaccharide composition - glucose of the carob polysaccharide Hocacy.

[0046] Figure 11It is the identification spectrum of ionic fragments of monosaccharide composition - glucose of carob polysaccharide Hocacy.

[0047] Figure 12 This is the methylation infrared spectrum of carob polysaccharide Hocacy in the embodiment of the present invention.

[0048] Figure 13 It is the intuitive graph of the influence of carob polysaccharide Hocacy on the cough latency of mice.

[0049] Figure 14 It is the intuitive graph of the number of coughs within 2 minutes of the influence of carob polysaccharide Hocacy on the cough of mice.

[0050] Figure 15 It is the intuitive graph of the influence of carob polysaccharide Hocacy on the phenol red secretion volume of mice.

[0051] Figure 16 It is the intuitive graph of the influence of carob polysaccharide Hocacy on the daily body weight change of mice.

[0052] Figure 17 It is the comparison graph of the fluorescence intensity of T cells of carob polysaccharide Hocacy with the model control group in zebrafish. Detailed implementation mode

[0053] The present invention will be further described below in conjunction with embodiments, but it does not limit the scope of the present invention.

[0054] The following carob tree pods are the dried pods of the carob tree (Latin name: Ceratonia siliqua Linn.) of the leguminous genus Vigna, provided by Shenzhen Aimin Medical Technology Co., Ltd.

[0055] The following D101 macroporous resin, dextran gel G-150 and G-100, and DEAE-52 cellulose are all commercially available.

[0056] Example 1 Extraction and separation of carob polysaccharide Hocacy

[0057] 8 kg of carob tree pods without seeds were taken, crushed, defatted with 3 times the weight of 95% ethanol, and dried in the air; deionized water was added according to the solid-liquid ratio of 1:2.3 (g / mL), and extraction was carried out 4 times, with each extraction lasting 2 hours at an extraction temperature of 45 °C. The extraction solutions were combined; the combined extraction solution was centrifuged (5000 rpm, 10 min), and the supernatant was concentrated under reduced pressure to a viscous state at 60 °C and 100 Pa. Ethanol was slowly added to the obtained concentrated solution until the mass fraction of ethanol reached 60%, and the precipitate was collected by centrifugation to obtain crude polysaccharide. The crude polysaccharide was dispersed with 20 times the weight of purified water, and the protein was removed by the Sevage method, with the operation repeated 6 times. The protein-free polysaccharide was dispersed with 10 times the weight of water, adsorbed by macroporous adsorption resin D101, eluted with distilled water for 2 column volumes, and the mobile phase components were collected. After column chromatography on a DEAE-52 cellulose column and elution with 2 column volumes of distilled water, it was successively subjected to column chromatography on a Sephadex G-100 column and a Sephadex G-150 column, with deionized water as the mobile phase. The eluates were collected, the elution peaks were combined, concentrated under reduced pressure, and freeze-dried to obtain carob polysaccharide Hocacy, with a polysaccharide content of 93%.

[0058] The content detection method of carob polysaccharide Hocacy in this example is as follows:

[0059] Precisely weigh 9.6 mg of carob polysaccharide Hocacy, dissolve it in pure water to prepare a solution with a concentration of 0.25 mg / mL. Take 1.0 mL of the test solution and draw a glucose standard curve by the phenol-sulfuric acid method, and calculate the mass concentration of polysaccharide in the test solution through the regression equation.

[0060] The standard curve of glucose is: y = 1.3421x + 0.2255 (R 2 = 0.9992)

[0061] Among them, x is the polysaccharide concentration (mg / ml), and y is the absorbance.

[0062] Polysaccharide content (%) = (mass concentration (μg / mL) × dilution factor × volume (mL)) / (dry weight of polysaccharide (g) × 1000)

[0063] Experimental Example 1 Structural analysis of carob polysaccharide Hocacy (prepared in Example 1)

[0064] 1) Determination of the molecular weight of carob polysaccharide Hocacy:

[0065] Precisely weigh 10.0 mg of carob polysaccharide Hocacy and prepare a solution with a concentration of 2 mg / mL. It was detected by a differential refractive index detector and a light scattering detector of a gel permeation chromatography (GPC) instrument, with 0.1 mol / L NaNO3 solution as the mobile phase and a flow rate of 0.7 mL / min.

[0066] The measurement results of the molecular weight of Hocacy, a carob polysaccharide, are as Figure 1 shown (GPC). Through analysis and calculation, the average molecular weight is 4.895×10 6 , the distribution width (Mw / Mn) is 1.574, and the distribution is relatively narrow, proving that the purity of Hocacy, a carob polysaccharide, is relatively high. Figure 1 In it, the abscissa represents the retention time, and the ordinate represents the refractive index; GPC chromatography represents the GPC chromatogram, Refractive Index (mV) represents the refractive index, Right Angle Light Scattering (mV) represents the right-angle light scattering, and Low Angle Light Scattering (mV) represents the low-angle light scattering.

[0067] 2) Infrared spectroscopy (IR) analysis of Hocacy, a carob polysaccharide:

[0068] Weigh 5 mg of the Hocacy, a carob polysaccharide, sample, add an appropriate amount of dry KBr, grind it to make a uniform and transparent tablet, and scan and analyze it with a Fourier transform infrared spectrometer in the range of 4000 cm -1 ~400 cm -1 .

[0069] The infrared spectrogram of Hocacy, a carob polysaccharide, is as Figure 2 shown. From Figure 2 it can be seen that the stretching vibration peak of -OH is at 3385 cm -1 , and the stretching vibration peak of -CH2 is at 2927 cm -1 ; there is a strong absorption peak at 1020 cm -1 , representing the stretching vibration peak of C-O. Figure 2 The ordinate is the transmittance (%).

[0070] 3) Monosaccharide composition analysis of Hocacy, a carob polysaccharide:

[0071] Weigh 5.5 mg of the Hocacy, a carob polysaccharide, sample, add 1 mL of 2 mol / L trifluoroacetic acid (TFA), seal the tube with nitrogen, and heat it at 110 °C for 120 min for hydrolysis. Dissolve the decomposed sample, add 90 mg of NaBH4 for reduction reaction for 4 h. Dry the reduced sample in an oven, add 5 mL of acetic anhydride, and carry out acetylation at 110 °C. Dissolve the final acetylated monosaccharide in ethyl acetate, filter it through a membrane, and carry out GC-MS structure identification.

[0072] After hydrolysis and acetylation of Hocacy, a carob polysaccharide, GC-MS identification is carried out, and the total ion chromatogram is as Figure 3 shown. Figure 3Among them, the abscissa Time represents the peak time, and the ordinate Relative abundance represents the relative abundance.

[0073] By HPLC method and through spectral library analysis, the monosaccharide composition (molar ratio) of locust bean polysaccharide Hocacy is: galactose: glucose = 89.666:1.068. Fructose was not detected in locust bean polysaccharide Hocacy by HPLC-ELSD method. The HPLC chart is as Figure 4 shown. Figure 4 Among them, the abscissa represents time, the ordinate Absorbance (mAU) represents absorbance, solvent represents the solvent peak, PMP represents the derivatizing reagent (1-phenyl-3-methyl-5-pyrazolone), Gal represents galactose, and Glu represents glucose.

[0074] The following conclusions were drawn through spectral library analysis:

[0075] The analysis results of the monosaccharide composition of locust bean polysaccharide Hocacy are as Figures 5 to 11 shown.

[0076] Figure 5 It is the ion fragment identification map of the monosaccharide composition - galactose of locust bean polysaccharide Hocacy, where t = 17.03. Figure 6 It is the ion fragment identification map of the monosaccharide composition - galactose of locust bean polysaccharide Hocacy, where t = 18.78. Figure 7 It is the ion fragment identification map of the monosaccharide composition - galactose of locust bean polysaccharide Hocacy, where t = 20.88. Figure 8 It is the ion fragment identification map of the monosaccharide composition - glucose of locust bean polysaccharide Hocacy, where t = 30.21. Figure 9 It is the ion fragment identification map of the monosaccharide composition - galactose of locust bean polysaccharide Hocacy, where t = 30.51. Figure 10 It is the ion fragment identification map of the monosaccharide composition - glucose of locust bean polysaccharide Hocacy, where t = 38.83. Figure 11 It is the ion fragment identification map of 2-methyl-glucose of the monosaccharide composition - glucose of locust bean polysaccharide Hocacy, where t = 61.04.

[0077] Figure 5 In (mainlib), 1,5-Anhydro-3-O-acetyl-2,4,6-tri-O-methyl-D-Galactitol represents (main spectral library) 1,5-anhydride-3-O-acetyl-2,4,6-tri-O-methyl-D-galactose. Figure 6In (mainlib), D-Galactitol, 3-6-anhydro-1,2,4,5-tetra-O-methyl- represents (main library) 3-6-anhydro-1,2,4,5-tetra-O-methyl-D-galactose. Figure 7 In (mainlib), Galactitol, 1,3,5-tri-O-methyl-, triacetate represents (main library) 1,3,5-tri-O-methylgalactose, triacetate. Figure 8 In (mainlib), D-Glucose, 2,3,4,6-tetra-O-methyl- represents (main library) 2,3,4,6-tetra-O-methyl-D-glucose. Figure 9 In (mainlib), D-Galactitol, 1,3,4,5-tetra-O-methyl-, diacetate represents (main library) 1,3,4,5-tetra-O-methyl-D-galactose, diacetate. Figure 10 In (mainlib), 5-O-Acetyl-2,3,4,6-tetra-O-methyl-galactonitrile represents (main library) 2,3,4,6-tetra-O-methylgalactonitrile. Figure 11 In (mainlib), 2-methyl-D-glucose represents (main library) 2-methyl-D-glucose.

[0078] 4) Measurement of the monosaccharide linkage sites of carob polysaccharide Hocacy:

[0079] Weigh about 20 mg of the carob polysaccharide Hocacy sample and dissolve it in 6 mL of dimethyl sulfoxide (DMSO). Stir magnetically at 40 °C for 15 hours until the sugar sample is fully dissolved. After the carob polysaccharide Hocacy is methylated, hydrolyzed, and acetylated, the total ion chromatogram identified by GC-MS is as Figure 3 shown. Figure 3 In it, the abscissa Time represents the peak time, and the ordinate Relative abundance represents the relative abundance. The infrared spectrum of carob polysaccharide Hocacy after methylation is as Figure 12 shown.

[0080] By analyzing the ion fragmentation pattern of the mass spectrum, it can be seen that the glycosidic bonds of carob polysaccharide Hocacy are (1→4)-Glu, (1→3,4,6), (1→3)-Gal, (3→6)-Gal, (2→4,6)-Gal, (2→6)-Gal, (1→)-Gal. The analysis results are shown in Table 1. Among them, Glu represents glucose and Gal represents galactose.

[0081] Table 1 Hocacy Methylation Analysis of Ceratonia siliqua Polysaccharide

[0082]

[0083] Experimental Example 2 Experiment on the Effect of Hocacy of Ceratonia siliqua Polysaccharide (Prepared in Example 1) on the Respiratory Function of Mice Induced by Ammonia Water Cough Stimulation

[0084] Put the white mice into a large beaker containing concentrated ammonia water to induce coughing in the white mice.

[0085] 1. Effect of Drug Administration on the Coughing Frequency of Coughing Mice

[0086] The experiment was divided into 5 groups as follows: Group 1, the blank group given normal saline; Group 2, the positive group given the drug of benproperine phosphate (Tonghua Great Wall Pharmaceutical Co., Ltd., batch number H22020149) (60 mg / kg); Groups 3 - 5, the administration groups given low-dose (100 mg / kg), medium-dose (200 mg / kg), and high-dose (400 mg / kg) Hocacy of Ceratonia siliqua polysaccharide; continuous intragastric administration for 14 days. 1 hour after the last intragastric administration, invert a 1000 mL large beaker on the experimental table, continuously spray 25% concentrated ammonia water into it for 10 s with a sprayer, then put the white mice into the large beaker and start timing. Taking abdominal muscle contraction, opening the mouth to breathe, and the appearance of coughing sounds as indicators, record the time of the first cough (latency period) of the mice in the beaker and the coughing frequency within 2 min as shown in Table 2. It shows that the coughing degrees of mice in each group are different after ammonia water-induced cough stimulation. High, medium, and low doses of Hocacy of Ceratonia siliqua polysaccharide can significantly reduce the coughing frequency of mice caused by ammonia water and prolong the latency period, showing significant or very significant effects on the blank group (****p < 0.0001 ***p < 0.001,

[0087] **p < 0.05), Hocacy of Ceratonia siliqua polysaccharide has a certain antitussive effect on the cough of mice caused by ammonia water and shows a dose-dependent relationship. The high-dose group (400 mg / kg) has the most obvious antitussive effect. The first cough time of the high-dose and medium-dose groups is significantly longer than that of the positive group (p < 0.01, p < 0.05).

[0088] There is a significant difference in the coughing frequency within 2 min between the high-dose group and the positive group (p < 0.01). Therefore, Hocacy of Ceratonia siliqua polysaccharide has strong antitussive potential. The intuitive diagram of the latency period of the effect of Hocacy of Ceratonia siliqua polysaccharide on mouse cough is as Figure 13 shown, and the intuitive diagram of the coughing frequency within 2 min is as Figure 14 shown

[0089] Table 2

[0090]

[0091] Note: Compared with the blank group, ****p < 0.0001, ***p < 0.001, **p < 0.01; compared with the positive group

[0092] &&p < 0.01, &p < 0.05.

[0093] 2. Effect of drug administration on expectoration in coughing mice.

[0094] Each group of mice was continuously administered the drug for 2 days. 30 minutes after the last drug administration on the 16th day, 0.5 mL / only of 5% phenol red saline was intraperitoneally injected. 30 minutes after the injection, the mice were sacrificed by cervical dislocation. A section of the trachea from the thyroid cartilage to the tracheal bifurcation was cut off and ultrasonically cleaned with 3 mL of normal saline and 0.1 mL of 1 moL / L sodium hydroxide solution for 10 minutes to completely release the phenol red in the trachea of the mice. The absorbance value of the solution was measured at 546 nm, and the phenol red content was calculated according to the regression equation of the standard curve as shown in Table 3. It shows that Hocacy of carob polysaccharide can significantly increase the phenol red excretion volume in the trachea of mice and has significant differences compared with normal saline (***p < 0.001), indicating that Hocacy of carob polysaccharide has a certain expectorant effect. The high dose shows a superior difference compared with the positive drug group (p < 0.05) and shows a dose correlation. The intuitive diagram of the effect of Hocacy of carob polysaccharide on the phenol red secretion volume in mice is as Figure 15 shown.

[0095] Table 3

[0096] Group Dose Phenol red secretion amount (μg / mL) Blank group Normal saline 0.5267±0.6671 Positive group (ammonium chloride) 500 mg / kg (by gavage) 2.5741±0.7873*** High-dose group 400 mg / kg (by gavage) 3.1510±0.8456***& Medium-dose group 200 mg / kg (by gavage) 2.7543±0.0944*** Low-dose group 100 mg / kg (by gavage) 2.4449±1.022***

[0097] Note: Compared with the blank group, ***p < 0.001; compared with the positive group, &p < 0.05

[0098] In addition, by tracking and recording the daily body weight changes of mice, it can be found that the mice in the groups given Hocacy of carob polysaccharide (high, medium, and low doses) lost less body weight than the positive groups (ammonium chloride, benproperine phosphate), showing a steady growth trend in body weight, second only to the blank group. However, the body weights of the two positive drug groups of mice remained basically unchanged, which was significantly different from the polysaccharide group and the body weights were lighter, indicating the non-toxic side effects and good adaptability of carob fruit pod polysaccharide from the side. The body weight changes are as Figure 16 shown.

[0099] In summary, this study shows that Hocacy of carob polysaccharide has obvious antitussive and expectorant effects and has no obvious toxic side effects. This study lays a certain foundation for the new drug development of carob fruit pod polysaccharide.

[0100] Experimental Example 3 Improvement effect of Hocacy of carob polysaccharide (prepared in Example 1) on the reduction of zebrafish T cells

[0101] The transgenic T cell red fluorescent zebrafish are propagated by natural paired mating. Zebrafish at 3 days post-fertilization (3 dpf) are all reared in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; the pH is 6.5 - 8.5; the hardness is 50 - 100 mg / L CaCO3), and are provided by the fish-raising center of Beijing Huante Zhiyu Youjian Biotechnology Co., Ltd.

[0102] Randomly select 3 dpf transgenic T cell red fluorescent zebrafish into 6-well plates, with 30 zebrafish in each well (experimental group). Hocacy prepared into 20.0 mg / mL with standard dilution water is administered by water solution (the concentration is shown in Table 4), and the positive control is 15.0 μg / mL concentration of Bailing Capsule (Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., batch number 2111064). At the same time, a normal control group and a model control group are set up, and the volume of each well is 3 mL. Except for the normal control group, vinorelbine tartrate injection is intravenously injected into the rest of the experimental groups to establish an immunosuppressed zebrafish model. Treat at 28 °C until 5 dpf, randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope for photographing. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, analyze the fluorescence intensity of zebrafish T cells, and evaluate the efficacy of Hocacy in improving T cell reduction based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis, and p < 0.05 indicates that the difference is statistically significant. The experimental results of the efficacy of Hocacy in improving T cell reduction are shown in Table 5. The fluorescence intensity of zebrafish T cells after Hocacy treatment compared with the model control group is as Figure 17 shown, indicating that Hocacy has a good efficacy in improving T cell reduction and does not show an obvious dose-dependence. Therefore, carob polysaccharide Hocacy has the potential to enhance immunity.

[0103] Table 4. Results of the experiment on exploring the concentration for the efficacy of Hocacy in improving T cell reduction (n = 30)

[0104]

[0105] Table 5. Experimental results of the efficacy of Hocacy in improving T cell reduction (n = 10)

[0106]

[0107] Note: Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001

[0108] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A carob polysaccharide, characterized in that, It is composed of two kinds of monosaccharides, galactose and glucose, and the molar ratio of galactose to glucose is (88 - 92):(0.8 - 1.2). The carob polysaccharide is combined through glycosidic bonds (1→4)-Glu, (1→3,4,6)-Glu, (1→3)-Gal, (3→6)-Gal, (2→4,6)-Gal, (2→6)-Gal, (1→)-Gal.

2. The carob polysaccharide according to claim 1, characterized in that, The molar ratio of galactose to glucose in the carob polysaccharide is 89.666:1.

068.

3. The carob polysaccharide according to claim 1 or 2, characterized in that, The molecular weight of the carob polysaccharide is (1-6)×10 6 , and can be optionally (4.8-5)×10 6 , and can be further optionally 4.895×10 6 ; and / or, The molecular weight distribution width (Mw / Mn) of the carob polysaccharide is 1.5 - 1.6, and it can be optionally 1.

574.

4. A preparation method of carob polysaccharide, characterized in that, It includes: Take the crushed carob tree pods without seeds, degrease them with 90 - 95% ethanol, and dry the defatted fine powder; extract with water; Centrifuge the extract, concentrate the supernatant, add ethanol to make the mass fraction of ethanol 55 - 60%, centrifuge to collect the precipitate; disperse the obtained precipitate with water, remove proteins by the Sevage method, adsorb with macroporous adsorption resin D101, elute with distilled water, collect the mobile phase components, perform column chromatography on a DEAE - 52 cellulose column, elute with distilled water, perform column chromatography on a G - 100 Sephadex gel column with deionized water as the mobile phase, collect the eluate, then perform column chromatography on a G - 150 Sephadex gel column with deionized water as the mobile phase, collect the eluate, combine the same elution peak, concentrate under reduced pressure, and freeze - dry to obtain carob polysaccharide.

5. The preparation method of the carob polysaccharide according to claim 4, characterized in that, The carob tree pods are crushed to 80 - 300 meshes; and / or, Calculated by g / mL, the solid - liquid ratio during extraction is 1:(2 - 5); and / or, The extraction temperature is 40 - 50 °C.

6. A carob polysaccharide, characterized in that, It is prepared by the method described in claim 4 or 5.

7. Use of the carob polysaccharide according to any one of claims 1 - 3, 6 as a drug; Optionally, use of the carob polysaccharide in the preparation of a drug for relieving cough and reducing phlegm and / or enhancing immunity.

8. A pharmaceutical composition, characterized in that, It contains the carob polysaccharide according to any one of claims 1 - 3, 6 and also contains a pharmaceutically acceptable carrier.

9. A food product, characterized in that, It contains the carob polysaccharide according to any one of claims 1 - 3, 6 and further includes food raw and auxiliary materials.

10. A method for treating a disease, characterized in that, It includes administering an effective dose of the carob polysaccharide according to any one of claims 1 - 3, 6 or the pharmaceutical composition according to claim 8 or the food according to claim 9 to a subject or patient in need through oral or non - oral routes; Optionally, the diseases include cough, expectoration, and low immunity.

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