CN113185619B

CN113185619B - Traditional Chinese medicine polysaccharide with anti-osteoporosis activity and preparation method and application thereof

Info

Publication number: CN113185619B
Application number: CN202110466927.4A
Authority: CN
Inventors: 王娜妮; 张岩; 许平翠
Original assignee: Zhejiang Academy of Traditional Chinese Medicine; Longhua Hospital Affiliated to Shanghai University of TCM
Current assignee: Zhejiang Academy of Traditional Chinese Medicine; Longhua Hospital Affiliated to Shanghai University of TCM
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2022-04-26
Anticipated expiration: 2041-04-28
Also published as: CN113185619A

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine polysaccharide with anti-osteoporosis activity and a preparation method and application thereof. The phellodendron polysaccharide in the invention can be prepared by the following steps: step S1, preparing crude cortex Phellodendri polysaccharide (PCP), step S2, preparing neutral cortex Phellodendri polysaccharide (PCP-1), and step S3, preparing cortex Phellodendri neutral homogeneous polysaccharide (PPCP-1). The invention can be better suitable for industrial production and has better effect of treating diabetes and osteoporosis complicating the diabetes.

Description

Traditional Chinese medicine polysaccharide with anti-osteoporosis activity and preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine polysaccharide with anti-osteoporosis activity and a preparation method and application thereof.

Background

The diabetic osteoporosis is a serious diabetic complication, has high morbidity, has the characteristics of reduced bone mass, damaged bone microstructure, easy fracture and the like, is clinically manifested by pain, deformity and fracture, and seriously affects the life quality of patients. The pathogenesis of the diabetic osteoporosis is mainly that end-stage glycosylation end products accumulate on bone tissues, the bone homeostasis is damaged, the bone loss is accelerated, at present, the treatment mainly depends on a hypoglycemic medicament and an anti-osteoporosis medicament, but the problems of large adverse reaction, poor tolerance of patients, uncertain curative effect and the like exist. Therefore, there is a need to find new drugs that are safe and effective.

Cortex Phellodendri is bark of cortex Phellodendri belonging to family Rutaceae and arbor, and has remarkable effect on diabetes and its complications. The cortex Phellodendri is rich in polysaccharide component, and its content is about 3%. At present, researches on antidiabetic drug substances of phellodendron amurense are mostly focused on alkaloids and sterols, and whether phellodendron amurense polysaccharide has activity is not clear. A few documents research the extraction process of crude phellodendron amurense polysaccharides, but no report is found on the study of homogeneous phellodendron amurense polysaccharides.

Chinese patent application No. 201110077860.1 discloses a cortex Phellodendri polysaccharide extract, and its preparation method and medicinal application. The method comprises extracting with water under reflux, precipitating with ethanol, removing pigment and protein from total polysaccharide with weak acid and weak base type ion exchange resin, dialyzing with dialysis bag with molecular weight of 3000-12000, and precipitating with 80% ethanol to obtain cortex Phellodendri polysaccharide with content of 70% calculated on glucose, and making into infusion solution or tablet. Experimental study: the cortex Phellodendri polysaccharide has antiinflammatory, antipyretic, immunosuppressive, carbon clearing away, and chronic nephritis resisting effects. However, the phellodendron amurense polysaccharide in the invention is crude polysaccharide, a gel chromatography purification step is not used, structural characterization steps such as functional group analysis, monosaccharide composition analysis, main chain structure analysis, molecular weight analysis and the like are not performed, uniform phellodendron amurense polysaccharide is not obtained, and the fine structure of the phellodendron amurense polysaccharide is not clarified. In the pharmacodynamic activity research experiment, the influence of the phellodendron polysaccharide on diabetes is not involved, and the influence of the phellodendron polysaccharide on bone microstructure is not involved.

Disclosure of Invention

The present invention provides a traditional Chinese medicine polysaccharide which can overcome some or some of the defects of the prior art.

The traditional Chinese medicine polysaccharide has a structural formula as follows:

wherein, alpha-L-Araf is arabinose with alpha and L configuration, beta-D-Galp is galactose with beta and D configuration, alpha-D-Glcp is glucose with alpha and D configuration, alpha-D-Manp is mannose with alpha and D configuration, and n is 6-7.

In addition, the invention also provides a preparation process of the traditional Chinese medicine polysaccharide, which comprises the following steps:

step S1, preparing crude polysaccharide of phellodendron (PCP)

In the step, phellodendron amurense is crushed and then added with ethanol for extraction, the extracted residues are extracted by adopting water so as to obtain an extracting solution, the obtained extracting solution is added with ethanol for precipitation, and the obtained precipitate is phellodendron amurense crude polysaccharide (PCP);

step S2, preparing cortex Phellodendri neutral polysaccharide (PCP-1)

Dissolving the crude phellodendron amurense polysaccharide (PCP) obtained in the step S1 by using water, removing protein in a dissolving solution by using trichloroacetic acid, collecting supernatant, dialyzing the supernatant, carrying out chromatographic purification on the obtained dialysate by using a DEAE anion exchange chromatography column, eluting by using water, and collecting eluent, wherein the obtained eluent is phellodendron amurense neutral polysaccharide (PCP-1) of a water-washing component;

step S3, preparing neutral homogeneous polysaccharide of phellodendron bark (PPCP-1)

In the step, the phellodendron neutral polysaccharide (PCP-1) obtained in the step S2 is purified by adopting a gel chromatographic column, water is adopted for elution, an eluent is collected, and the obtained eluent is dried to obtain phellodendron neutral homogeneous polysaccharide (PPCP-1), wherein the phellodendron neutral homogeneous polysaccharide (PPCP-1) is the traditional Chinese medicine polysaccharide.

Preferably, in step S1, when ethanol is added to the crushed phellodendron amurense for extraction, the ethanol is 70-90% ethanol solution, and the weight of the ethanol is 5-10 times of the crushed phellodendron amurense; and extracting for 2-4 times by reflux extraction, each time for 1-3 hours.

Preferably, in the step S1, when the residue is extracted, the residue is extracted by refluxing for 1 to 3 hours for 2 to 4 times with 10 to 20 times by weight of distilled water.

Preferably, in step S2, protein is removed by using trichloroacetic acid solution with concentration of 5-15% or mixed solution of trichloromethane and n-butanol.

Preferably, in step S2, the cut-off molecular weight of the dialysis bag is set to 3500Da when the solution of Phellodendron Crude Polysaccharide (PCP) is dialyzed.

Preferably, in step S2, the exchanger in the DEAE anion exchange chromatography column includes at least one of DEAE-cellulose, DEAE-dextran and DEAE-agarose.

Preferably, in step S3, the gel chromatography column used includes at least one of sephadex, polyacrylamide sephadex, sepharose and polyacrylamide gel.

The invention also provides application of the traditional Chinese medicine polysaccharide in preparing a medicine for reducing blood sugar of a diabetic patient and a medicine for treating osteoporosis caused by diabetes.

The invention has the following beneficial effects:

1. the traditional Chinese medicine polysaccharide is phellodendron neutral homogeneous polysaccharide (PPCP-1), which is homogeneous polysaccharide, has the effect of reducing blood sugar obviously superior to phellodendron neutral polysaccharide (PCP-1) and Phellodendron Crude Polysaccharide (PCP), and has the effect of reducing blood sugar equivalent to that of a positive medicine metformin; meanwhile, the bone mass, the number of trabeculae and the thickness of trabeculae of the diabetic osteoporosis rat can be obviously improved, and the trabecular bone separation degree of the diabetic osteoporosis rat can be effectively reduced;

2. the phellodendron neutral homogeneous polysaccharide can effectively reduce the content of advanced glycosylation end products of femoral tissues of diabetic rats, lower the receptor expression of the advanced glycosylation end products and reduce the damage of the advanced glycosylation end products to bone tissues;

3. the preparation method of the neutral uniform polysaccharide of the golden cypress provided by the invention has good repeatability and can be preferably used for industrial production.

Drawings

FIG. 1 is a gel chromatogram of crude phellodendron amurense polysaccharide (PCP), neutral phellodendron amurense polysaccharide (PCP-1) and neutral phellodendron amurense homopolysaccharide (PPCP-1) in example 1, wherein A is a gel chromatogram of crude phellodendron amurense polysaccharide (PCP), B is a gel chromatogram of neutral phellodendron amurense polysaccharide (PCP-1), and C is a gel chromatogram of neutral phellodendron amurense homopolysaccharide (PPCP-1);

FIG. 2 is a UV spectrum of the neutral homopolysaccharide (PPCP-1) of phellodendron amurense of example 1;

FIG. 3 is an infrared spectrum of a neutral homopolysaccharide (PPCP-1) of phellodendron amurense obtained in example 1;

FIG. 4 shows the preparation of neutral homopolysaccharide (PPCP-1) from Phellodendri cortex in example 1¹³A CNMR spectrum;

FIG. 5 shows the preparation of the neutral homopolysaccharide (PPCP-1) of Phellodendri cortex in example 1¹HNMR spectra;

FIG. 6 is a graph showing the effect of phellodendron neutral homopolysaccharide (PPCP-1) in example 1 on the content of advanced glycation end products in femoral tissues of diabetic rats; wherein A is blank group, B is diabetes rat model group, C is cortex Phellodendri neutral homogeneous polysaccharide (PPCP-1) treatment group, and represents P < 0.01;

FIG. 7 is a graph of the effect of phellodendron neutral homopolysaccharide (PPCP-1) in example 1 on the expression of advanced glycation end product receptors in femoral tissues of diabetic rats; wherein A is blank group, B is diabetes rat model group, C is cortex Phellodendri neutral homogeneous polysaccharide (PPCP-1) treatment group, and represents P < 0.01.

Detailed Description

For a further understanding of the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings and examples. It is to be understood that the examples are illustrative of the invention and not limiting.

Example 1

The embodiment provides a preparation method of traditional Chinese medicine polysaccharide, which comprises the following steps:

step S1, preparing crude polysaccharide of phellodendron (PCP)

step S2, preparing cortex Phellodendri neutral polysaccharide (PCP-1)

In the step, the phellodendron neutral polysaccharide (PCP-1) obtained in the step S2 is purified by using a gel chromatographic column, water is used for elution, an eluent is collected, and the obtained eluent is dried to obtain the phellodendron neutral homogeneous polysaccharide (PPCP-1).

Through the above steps S1-S3, the active ingredient with higher purity, i.e., the phellodendron neutral homopolysaccharide (PPCP-1), can be extracted from phellodendron amurense, and the weight ratio of the phellodendron neutral homopolysaccharide (PPCP-1) extracted by the method in this example to the extract can be 40% or more.

In step S1 of this example, when ethanol is added to the crushed phellodendron amurense for extraction, the ethanol used is 70-90% ethanol solution, and the weight of the ethanol used is 5-10 times of the crushed phellodendron amurense; and extracting for 2-4 times by reflux extraction, each time for 1-3 hours.

In step S1 of this example, the residue was extracted by refluxing with 10 to 20 times by weight of distilled water for 1 to 3 hours each time for 2 to 4 times.

In step S2 of this example, a trichloroacetic acid solution with a concentration of 5-15% or a mixed solution of chloroform and n-butanol is used to remove proteins.

In step S2 of this example, the cut-off molecular weight of the dialysis bag was set to 3500Da when dialyzing a solution of crude phellodendron polysaccharide (PCP).

In step S2 of this example, the exchanger in the DEAE anion exchange chromatography column includes at least one of DEAE-cellulose, DEAE-dextran, and DEAE-agarose.

In step S3 of the present embodiment, the gel chromatography column used includes at least one of dextran gel, polyacrylamide dextran gel, agarose gel, and polyacrylamide gel.

To further illustrate the method of the present invention, this embodiment provides a preferred extraction example, which specifically operates as follows:

step S1, preparing crude polysaccharide of phellodendron (PCP)

In the step, 10Kg of phellodendron bark is crushed and then added with 60Kg of 95 percent ethanol solution to extract for 3 times by a reflux extraction method, and the extraction lasts for 2 hours each time; then adding 15 weight times of water into the residue, carrying out reflux extraction for 3 hours at the temperature of 100 ℃, collecting filtrate, adding 10 weight times of water into the residue, carrying out reflux extraction for 1 hour at the temperature of 100 ℃, and collecting the filtrate; then combining the two filtrates, concentrating to 1/10, adding 4 weight times of anhydrous ethanol solution, standing at 4 deg.C for 20 hr, collecting precipitate, and freeze drying to obtain cortex Phellodendri Crude Polysaccharide (PCP);

step S2, preparing cortex Phellodendri neutral polysaccharide (PCP-1)

In the step, adding water into crude phellodendron polysaccharides (PCP) to dissolve until the concentration is 0.5g/mL, then adding trichloroacetic acid until the concentration of trichloroacetic acid is 5%, standing for 20h at 4 ℃ to collect supernatant, and concentrating the supernatant until the volume of the supernatant is 1/10; then dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, concentrating dialyzate obtained by dialysis until the polysaccharide content is 0.03g/mL, then carrying out chromatography by using DEAE anion exchange chromatography column and eluting by using water to obtain eluent, detecting the sugar content in the eluent by using a phenol-sulfuric acid method until the sugar content flows out, and then carrying out freeze drying on the eluent to obtain phellodendron neutral polysaccharide (PCP-1);

Purifying the eluent obtained in the step S2 by using a gel chromatographic column, eluting by using water, collecting the eluent, detecting the sugar content in the eluent by using a phenol-sulfuric acid method until the sugar content does not flow out, and freeze-drying the eluent to obtain the neutral uniform polysaccharide (PPCP-1) of the phellodendron.

In order to analyze the fine structure of the neutral homopolysaccharide (PPCP-1) of phellodendron amurense obtained in this example, the structure thereof was analyzed by the following method in this example.

In this example, the molecular weight and purity of the crude phellodendron amurense polysaccharide (PCP) obtained in step S1, the neutral phellodendron amurense polysaccharide (PCP-1) obtained in step S2, and the neutral homogeneous phellodendron amurense polysaccharide (PPCP-1) obtained in step S3 were analyzed by HPLC. The chromatographic column adopted by the method is tsk-gel3000pwxl, the mobile phase is water, the column temperature is 30 ℃, the flow rate is 0.8mL/min, and the sample injection volume is 10 muL.

Referring to fig. 1, the traditional Chinese medicine polysaccharide (PPCP-1) in this example is a single symmetrical peak, indicating that it is a homogeneous polysaccharide. In this example, the calibration curve equation of logMw (weight average molecular weight) -Rt (retention time) of the traditional Chinese medicine polysaccharide (PPCP-1) in this example was obtained with dextrans of different relative molecular weights as controls: y ═ 0.558x +9.142, R²0.9976; according to the calibration curve equation, the weight average molecular weight of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment is calculated to be 10.8 KDa.

Referring to fig. 2, the absorbance of the traditional Chinese medicine polysaccharide (PPCP-1) solution in this embodiment is measured by an ultraviolet spectrophotometer, and no characteristic absorption peak is found at 280nm and 260nm, so the traditional Chinese medicine polysaccharide (PPCP-1) in this embodiment has no typical peptide chain structure, i.e., it does not belong to protein substances and nucleic acids.

As shown in FIG. 3, when the functional group analysis was performed on the polysaccharide (PPCP-1) of the present example, it was 3282cm^-1The characteristic absorption peak of the compound is attributed to-OH stretching vibration and is 2936cm^-1The absorption peak of (A) is attributed to C-H stretching vibration at 1604cm^-1The absorption peak of (2) is attributed to O-H bending vibration at 1418cm^-1The absorption peak of (2) is attributed to bending vibration of methylene, at 1032cm^-1、592cm^-1The absorption peak of (A) was attributed to C-O stretching vibration at 893cm^-1、770cm^-1The absorption peak of (a) is ascribed to the pyranose ring.

In order to analyze monosaccharide composition of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment, 10mg of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment is taken, and then trifluoroacetic acid is added, and hydrolysis is carried out for 3h in an environment of 120 ℃; then absorbing the acid hydrolysis solution, transferring the acid hydrolysis solution into a test tube, blowing the test tube to dry the test tube by using nitrogen, adding 5ml of water, whirling and uniformly mixing the mixture, absorbing 100uL of mixed solution, adding 900uL of deionized water, centrifuging the mixture for 5min at the rotating speed of 12000rpm, and then taking the supernatant to perform ion chromatography analysis; wherein the column was Dionex CarbopacTMPA20 (3X 150), the mobile phase was 15mmol/L sodium hydroxide and 100mmol/L sodium acetate, the flow rate was set to 0.3mL/min, the amount of sample was set to 5. mu.L, and the column temperature was set to 30 ℃. The detection of the electrochemical detector shows that the monosaccharide components of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment comprise arabinose, galactose, glucose and mannose, which are specifically shown in Table 1.

TABLE 1 monosaccharide composition of the Chinese medicine polysaccharide (PPCP-1) in this example

Monosaccharide name	Retention time (min)	Molar ratio of
			Arabinose	12.8	0.4972
Galactose	16.1	0.1871
			Glucose	18.4	0.1953
Mannose	22.9	0.1204

When methylation analysis is performed on the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment, after methylation, hydrolysis and acetylation are performed on a sample in sequence, the result of GC-MS measurement is compared with a standard mass spectrum library. The conditions for GC-MS measurement in this example were: an RXI-5 SIL MS type chromatographic column is adopted, the specification is 30m 0.25mm 0.25um, the programmed heating condition is set to be 120 ℃, the temperature is increased to 250 ℃/min at 3 ℃/min, the temperature is kept for 5min at times, the temperature of a sample inlet is 250 ℃, the temperature of a detector is 250 ℃/min, carrier gas is helium, and the flow rate is 1 mL/min.

Through methylation analysis, the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment has various glycosidic bonds, as shown in Table 2.

TABLE 2 analysis results of acetyl esters of methylated sugar alcohols of the neutral homopolysaccharide PPCP-1 of phellodendron amurense

Based on the above, it can be seen from the methylation analysis that the traditional Chinese medicine polysaccharide (PPCP-1) in the present embodiment has a repeatable unit structure, and the backbone skeleton thereof is "→ 3) - β -D-galactose- (1 → 4) - α -D-glucose- (1 → 4) - α -D-mannose- (1 → 4) -," α -L-arabinose- (1 → 5) - α -L-arabinose- (1 → ″ branch chain on the 6-position carbon atom of one of the galactose atoms of the backbone, the 3-position of one of the branched arabinose has an 'alpha-L-arabinose- (1 →' meaning that the structure of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment contains a beta-type pyran ring and an alpha-type pyran ring.

Therefore, the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment has the structure as follows:

The traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment is homogeneous polysaccharide, and the relative molecular mass is 10.8 KDa. It is mainly composed of 4 monosaccharides, which are arabinose, galactose, glucose and mannose, respectively.

As shown by the IR spectrum in FIG. 3, the traditional Chinese medicine polysaccharide (PPCP-1) in the present example is a typical polysaccharide absorption peak, including 3282cm^-1Absorption peak of hydroxyl group of (2), 893cm^-1And 770cm^-1And a sugar ring absorption peak of 1700cm^-1No carbonyl absorption peak, which is consistent with no absorption peak at the wavelength of 280nm of the ultraviolet spectrum; as shown by GC-MS analysis results, the traditional Chinese medicine polysaccharide (PPCP-1) mainly comprises arabinose, galactose, glucose and mannose.

When the nuclear magnetic resonance spectrum analysis is carried out on the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment, 50mg of the sample is weighed, dissolved in 0.5ml of heavy water and freeze-dried; then dissolving the freeze-dried powder in 0.5ml of heavy water, continuously freezing and drying, and repeating the processes to fully exchange active hydrogen; the sample was then dissolved in 0.5ml of heavy water and examined by NMR at 600MHz at 25 ℃ at room temperature.

As shown in connection with figure 4 of the drawings,¹³the C NMR spectrum shows seven terminal carbon signals, namely delta 108.60, delta 108.46, delta 108.77, delta 104.48, delta 104.69, delta 101.49 and delta 101.05 ppm; as shown in connection with figure 5 of the drawings,¹the H NMR spectrum gave δ 5.01, δ 5.09, δ 4.96, δ 4.40, δ 4.47, δ 4.67 and δ 5.31ppm of hydrogen as the main terminal group.

The structure of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment can be preferably verified through data of nuclear magnetic resonance spectroscopy.

Meanwhile, the embodiment also provides specific application of the traditional Chinese medicine polysaccharide (PPCP-1) in the embodiment, in particular to preparation of a medicine for reducing blood sugar of a diabetic patient and preparation of a medicine for treating osteoporosis caused by diabetes.

To verify the effect of the traditional Chinese medicine polysaccharide (PPCP-1) in this example on lowering blood sugar in diabetic rats and treating osteoporosis due to diabetes, the following comparative tests were set up in this example.

In this example, a male SD rat was selected to construct a test model, specifically: randomly extracting 6 rats as blank groups, injecting 35mg/kg streptozotocin into abdominal cavities of the other rats, detecting fasting blood glucose, and taking the fasting blood glucose of more than or equal to 16.7mmol/L as a judgment standard for success of the model. Male SD rats with fasting plasma glucose not less than 16.7mmol/L are divided into 5 groups and respectively used as a model group, a PCP-1 group, a PPCP-1 group and a metformin group.

Feeding the blank group with common feed;

in the model group, the normal saline is administered by gavage for 4 weeks 1 time a day;

in the PCP group, 100mg/kg/d of Phellodendron Crude Polysaccharide (PCP) prepared in the step S1 is administered by intragastric administration for 4 weeks 1 time a day;

in the PCP-1 group, 100mg/kg/d of the phellodendron neutral polysaccharide (PCP-1) prepared in the step S2 is administered by intragastric administration, 1 time per day for 4 weeks;

in the PPCP-1 group, 100mg/kg/d of the cortex Phellodendri neutral homogeneous polysaccharide (PPCP-1) prepared in step S3 is administered by intragastric administration, 1 time per day for 4 weeks;

in the metformin group, metformin was administered at 300mg/kg/d by gavage for 4 weeks 1 time per day.

After 4 weeks, the average blood glucose level of male SD rats of each test group was measured, and the measurement results are shown in table 3.

TABLE 3 effects of PCP, PCP-1, PPCP-1 on fasting plasma glucose in diabetic rats

As can be seen from Table 3, the effect of the phellodendron neutral homopolysaccharide (PPCP-1) is superior to that of the phellodendron neutral polysaccharide (PCP-1) and Phellodendron Crude Polysaccharide (PCP).

Meanwhile, 6 male SD rats fed with common feed are selected to construct a blank group of the anti-diabetic osteoporosis experiment, 6 male SD rats with fasting blood sugar more than or equal to 16.7mmol/L are selected to construct a model group of the anti-diabetic osteoporosis experiment, and 6 male SD rats with fasting blood sugar more than or equal to 16.7mmol/L are selected to construct a PPCP-1 group of the anti-diabetic osteoporosis experiment.

Feeding the blank group with common feed;

the model group is administered with normal saline by gavage for 12 weeks 1 time a day;

in the PPCP-1 group, 100mg/kg/d of the phellodendron neutral homopolysaccharide (PPCP-1) prepared in step S3 was administered by gavage for 12 weeks 1 time per day.

After 12 weeks, bone microstructure of male SD rats of each test group was measured, and the measurement results are shown in table 4.

TABLE 4 Effect of PPCP-1 on bone microstructure in diabetic rats

As can be seen from Table 4, the cortex Phellodendri neutral homopolysaccharide (PPCP-1) can effectively improve the bone mass, the number of trabeculae and the thickness of the trabeculae of the diabetic rat femur and reduce the trabecular bone separation degree of the diabetic rat femur.

After 12 weeks, the content of advanced glycosylation end products and the receptor expression of advanced glycosylation end products at the cancellous bone part of femur of the male SD rats of each test group were measured by immunohistochemical technique, and the measurement results are shown in fig. 6 and 7.

As can be seen from the combination of FIG. 6 and FIG. 7, the phellodendron neutral homopolysaccharide (PPCP-1) can effectively reduce the accumulation degree of advanced glycosylation end products of femurs of diabetic rats and the receptor expression thereof, and reduce the damage of the advanced glycosylation end products to bone tissues.

In conclusion, compared with the existing extracted cortex phellodendri neutral polysaccharide (PCP-1) and cortex Phellodendri Crude Polysaccharide (PCP), the cortex phellodendri neutral homogeneous polysaccharide (PPCP-1) in the embodiment is a homogeneous polysaccharide, and has a significantly better effect on reducing blood sugar than the cortex phellodendri neutral polysaccharide (PCP-1) and cortex Phellodendri Crude Polysaccharide (PCP), and the blood sugar reducing effect is equivalent to that of the positive drug metformin; meanwhile, the bone mass, the number of trabeculae and the thickness of trabeculae of a diabetic rat can be remarkably improved, the trabeculae separation degree of the diabetic rat can be effectively reduced, the content of advanced glycosylation end products in femoral tissues of the diabetic rat and the receptor expression of the advanced glycosylation end products can be effectively reduced by the PPCP-1, the damage of the advanced glycosylation end products to the bone tissues is reduced, and the drug effect of resisting diabetes and osteoporosis is exerted.

Therefore, the phellodendron neutral homopolysaccharide (PPCP-1) in the embodiment can be better used as a natural raw material of a new medicine and a functional health food for treating diabetes and osteoporosis complicated with the diabetes, and the preparation method of the phellodendron neutral homopolysaccharide provided in the embodiment has good repeatability, and can be better used for industrial production.

The present invention and its embodiments have been described above schematically, without limitation, and what is shown in the drawings is only one of the embodiments of the present invention, and the actual structure is not limited thereto. Therefore, if the person skilled in the art receives the teaching, without departing from the spirit of the invention, the person skilled in the art shall not inventively design the similar structural modes and embodiments to the technical solution, but shall fall within the scope of the invention.

Claims

1. A traditional Chinese medicine polysaccharide has a structural formula as follows:

2. The method for preparing a Chinese medicinal polysaccharide as claimed in claim 1, which comprises the following steps:

step S1, preparing crude polysaccharide of phellodendron (PCP)

step S2, preparing neutral phellodendron polysaccharide (PCP-1)

Dissolving the crude phellodendron amurense polysaccharide (PCP) obtained in the step S1 by using water, removing protein in a dissolving solution by using trichloroacetic acid, collecting supernatant, dialyzing the supernatant, carrying out chromatographic purification on the obtained dialysate by using a DEAE anion exchange chromatography column, eluting by using water, and collecting eluent, wherein the obtained eluent is neutral phellodendron amurense polysaccharide (PCP-1) of a water-washing component;

Purifying the neutral phellodendron amurense polysaccharide (PCP-1) obtained in the step S2 by using a gel chromatographic column, eluting by using water, collecting eluent, and drying the obtained eluent to obtain phellodendron amurense neutral homogeneous polysaccharide (PPCP-1); the phellodendron neutral homopolysaccharide (PPCP-1) is the traditional Chinese medicine polysaccharide in claim 1.

3. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S1, when ethanol is added to the crushed phellodendron amurense for extraction, the ethanol is 70-90% ethanol solution, and the weight of the ethanol is 5-10 times of the crushed phellodendron amurense; and extracting for 2-4 times by reflux extraction, each time for 1-3 hours.

4. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S1, when the residue is extracted, 10-20 times of distilled water by weight is used for reflux extraction for 2-4 times, and each time lasts for 1-3 hours.

5. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S2, trichloroacetic acid solution with concentration of 5-15% is used for protein removal.

6. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S2, when the solution of Phellodendron Crude Polysaccharide (PCP) is dialyzed, the cut-off molecular weight of the dialysis bag is set to 3500 Da.

7. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S2, the exchanger in the DEAE anion exchange chromatography column includes at least one of DEAE-cellulose, DEAE-dextran, and DEAE-agarose.

8. The preparation method of the traditional Chinese medicine polysaccharide according to claim 2, which is characterized in that: in step S3, the gel chromatography column used includes at least one of sephadex, polyacrylamide sephadex, sepharose and polyacrylamide gel.

9. The use of a polysaccharide of a Chinese medicinal material as claimed in claim 1 for the manufacture of a medicament for lowering blood glucose in a diabetic patient and for the manufacture of a medicament for the treatment of osteoporosis due to diabetes.