Myrtle polysaccharide P1, the separation method thereof and the use in preparing hypolipidemic drugs therefor

Abstract

The invention discloses a myrtle polysaccharide P1, the separation method thereof and the use in preparing hypolipidemic drugs therefor, wherein the P1 contains 6.74% of ribose, 1.73% of rhamnose, 60.06% of arabinose, 3.54% of xylose, 5.64% of mannose, 13.16% of glucose, and 9.13% of galactose. The experiment result shows that the myrtle polysaccharide P1 has a certain ability to bind cholate in vitro. Taking cholestyramine as a positive control and the binding rate of cholestyramine to each cholate as 100%, the relative binding rate of the myrtle polysaccharide P1 to sodium taurocholate, sodium glycocholate and sodium cholate was 25.28%, 44.56%, and 50.10%, respectively.

Claims

1. A method for separating and purifying myrtle polysaccharide P1, comprising the following steps: (1) drying and pulverizing the myrtle fruit, filing the obtained dry powder and distilled water to a reflux device to boil for extraction for several times; then combining the extracts, concentrating under reduced pressure, adding 95% (V/V) ethanol having a volume of several times of the concentrate to the concentrate, and stirring to let the polysaccharide precipitate uniformly; allowing the mixture to stand at 4° C. for more than 12 hours, collecting the precipitate by centrifugation and drying to obtain the crude myrtle fruit polysaccharide; (2) decolorizing the crude myrtle fruit polysaccharide by adding 30% H.sub.2O.sub.2 solution; (3) adding the decolorized polysaccharide to a Sevag reagent for deproteinizing to obtain a preliminary purified myrtle fruit polysaccharide; (4) packing a column with pretreated DEAE-Sepharose filler, dissolving the preliminary purified myrtle fruit polysaccharide in step (3) in deionized water, filtering with a 0.45 μm microporous membrane, and then applying to the column; eluting by distilled water, collecting the eluate, detecting the absorbance of the eluate at a wavelength of 490 nm by phenol-sulfuric acid method, stopping the collection when the absorbance is near zero, combining the obtained fractions, conducting concentration, dialysis, and lyophilizing to obtain the polysaccharide P1; wherein the polysaccharide P1 comprises the following monosaccharides by molar percentage: 6.74% of ribose, 1.73% of rhamnose, 60.06% of arabinose, 3.54% of xylose, 5.64% of mannose, 13.16% of glucose, and 9.13% of galactose; wherein the polysaccharide P1 has a number average molecular weight of 6.44×10.sup.3 Da and a weight average molecular weight of 2.04×10.sup.4 Da; and wherein the polysaccharide P1 has an ability to bind cholate in vitro and has a hypolipidemic effect, and when treating a binding rate of cholestyramine to each choate as 100%, a relative binding rate of the polysaccharide P1 to sodium taurocholate, sodium glycocholate and sodium cholate is 25.28%, 44.56%, and 50.10%, respectively.

2. The separation and purification method according to claim 1, wherein step (2) comprises the following steps: dissolving the crude myrtle fruit polysaccharide and adding distilled water, adjusting the pH to 8.0, and then adding 30% H.sub.2O.sub.2 solution with stirring until the color of the solution becomes lighter; and maintaining the temperature at 50° C. for 2 h in a water bath.

3. The separation and purification method according to claim 1, wherein step (3) comprises the following steps: adding the Sevag reagent to the decolorized polysaccharide solution with a volume ratio of 1:5, shaking and culturing for 20 minutes; standing for 10 minutes followed by centrifugation, removing the lower organic phase and the gel formed by the protein in intermediate layer, chloroform, and n-butanol, and repeating the centrifugation and the removal steps for several times; then, removing the remaining Sevag reagent by rotary evaporation to obtain a deproteinized myrtle fruit polysaccharide solution; collecting precipitate by adding ethanol according to step (1), and drying to obtain a preliminary purified myrtle fruit polysaccharide.

4. The separation and purification method according to claim 1, wherein the flow rate of the loading and elution in step (4) is 0.5 mL/min.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the elution profile of the myrtle fruit polysaccharide.

(2) FIG. 2 is a UV spectrum of polysaccharide P1.

(3) FIG. 3 shows the ability of myrtle fruit polysaccharide to bind cholate in vitro.

(4) FIG. 4 is an infrared spectrum of polysaccharide P1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(5) The present invention will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

(6) In the examples, the analysis of the myrtle fruit polysaccharide P1 was carried out by the Guangzhou Analytical Testing Center, China, and the report number was 2017011500-2.

Example 1

(7) A method for separating and purifying the myrtle polysaccharide P1 comprises the following steps:

(8) (1) extraction of crude myrtle fruit polysaccharide: pulverizing 100 g of dry myrtle fruit, sieving by a 40 mesh sieve and weighing 100 g of the sieved dry powder; adding the obtained dry powder and 1000 mL of distilled water to a reflux device to boil for 4 h; then combining the extracts, concentrating under reduced pressure, adding 95% (VN) ethanol having a volume of 4 times of the concentrate to the concentrate, and stirring with a glass rod to let the polysaccharide precipitate uniformly; allowing the mixture to stand at 4° C. for 12 hours, collecting the precipitate by centrifugation at a speed of 5000 r/min for 15 min and drying to obtain the crude myrtle fruit polysaccharide;

(9) (2) decolorizing the crude myrtle fruit polysaccharide by H.sub.2O.sub.2 solution: dissolving the crude myrtle fruit polysaccharide in 100 mL of distilled water, adjusting the pH to 8.0 by 1% NaOH solution, and then adding 30% H.sub.2O.sub.2 solution with stirring until the color of the solution becomes lighter; and maintaining the temperature at 50° C. for 2 h in a water bath;

(10) (3) deproteinizing by a Sevag reagent: adding the Sevag reagent consisting of chloroform and n-butanol with a volume ratio of 5:1 to the decolorized polysaccharide solution with a volume ratio of 1:5, shaking at a speed of 150 r/min in a shaker for 20 minutes; standing for 10 minutes followed by centrifugation, removing the lower organic phase and the gel formed by the protein in intermediate layer, chloroform, and n-butanol, and repeating the centrifugation and the removal steps for 5 times; then, removing the remaining Sevag reagent by rotary evaporation after centrifuging for the last time at a speed of 4000 r/min for 10 min to obtain a deproteinized myrtle fruit polysaccharide solution; collecting precipitate by adding ethanol according to step (1), and drying to obtain a preliminary purified myrtle fruit polysaccharide;

(11) (4) separation of the myrtle polysaccharide P1 by DEAE-Sepharose fast flow ion exchange column chromatography:

(12) 4.1 pretreatment of the filler: in order to remove a 20% ethanol protection solution, rinsing with distilled water during suction filtration; and since bubbles are generated during the rinsing process, removing the bubbles by ultrasonic wave and allowing to stand;

(13) 4.2 packing: slowly adding the filler to a column (column size is 1.5 cm×20 cm), the bed volume of which is about 20 mL; washing the column with distilled water for 1 h, then washing with 1 mol/L NaCl solution for 1 h, and finally washing with distilled water for 2 h to prepare for loading;

(14) 4.3 loading: dissolving 0.1 g of polysaccharide sample in 2 mL of distilled water and preparing a 50 mg/mL solution; after filtering with a 0.45 μm microporous membrane, loading the sample at 0.5 mL/min;

(15) 4.4 elution: eluting with distilled water at a flow rate of 0.5 mL/min, collecting one tube of eluate per 5 mL, determining the polysaccharide content of each tube by phenol-sulfuric acid method, and stopping the collection until no polysaccharide is detected. Plotting the absorbance at a wavelength of 490 nm against the order of tubes, such that an elution curve is obtained and shown in FIG. 1;

(16) The specific operation of the phenol-sulfuric acid method for detecting the polysaccharide content is as follows: accurately filling 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 0.1 mg/mL anhydrous glucose standard solution in 6 test tubes, then adding 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mL of distilled water in order, and adding 0.5 mL of 6% phenol solution and 2.5 mL of concentrated sulfuric acid successively. After mixing, standing for 20 min at room temperature, and measuring the absorbance at a wavelength of 490 nm. The absorbance is plotted against the concentration of glucose to obtain a standard curve. The absorbance of 1 mL of each tube of the sample solution is measured under the same condition as that of the standard curve to calculate the total polysaccharide content in each tube of the sample solution.

(17) 4.5 dialysis: collecting the eluted fractions separately, transferring the concentrated fractions to a dialysis bag (3000 Da) and dialyzing at 4° C. for 48 hours, concentrating them under reduced pressure, and lyophilizing to obtain the myrtle fruit polysaccharide P1.

Example 2

(18) The myrtle fruit polysaccharide P1 obtained in Example 1 was subjected to UV analysis. 1 mg of the polysaccharide sample was weighed to prepare a 1 mg/ml polysaccharide solution, and the ultraviolet spectrum was scanned in the range of 200-500 nm.

(19) FIG. 2 is an ultraviolet spectrum of P1. It shows that the product has no obvious absorption peak at 260 nm and at 280 nm, indicating that it does not contain protein and nucleic acid substances.

Example 3

(20) The molecular weight of myrtle fruit polysaccharide P1 obtained in Example 1 was analyzed by the following method.

(21) The molecular weight was determined by gel permeation chromatography (GPC). 2 mg of polysaccharide sample was dissolved in a 0.02 mol/L phosphate buffer solution to prepare a 2.0 mg/mL solution. Then the solution was filtered with a 0.22 μm sterile filter membrane, and the filtered solution was stored for use. Chromatographic conditions were as follows: column temperature was 35° C., 0.02 mol/L phosphate buffer was used as mobile phase, flow rate was at 0.6 mL/min, injection volume was 20 μL, and Waters 2410 differential refractive index detector was used. A series of dextran solutions with different molecular weight (700, 400, 200, 100, 50, 30, 10, 5 kD) were used as standard to draw a standard curve, and the molecular weight of the sample was calculated with the standard curve according to its corresponding elution volume.

(22) The molecular weight of the purified myrtle fruit polysaccharide was measured as shown in Table 1 below:

(23) TABLE-US-00001 TABLE 1 The molecular weight of the myrtle fruit polysaccharide component Mn (Da) Mw (Da) Mw/Mn P1 6.44 × 10.sup.3 2.04 × 10.sup.4 3.17

Example 4

(24) The monosaccharide composition of the myrtle fruit polysaccharide P1 obtained in Example 1 was analyzed by the following method.

(25) 5 mL of 4 mol/L trifluoroacetic acid was added to 10 mg of the polysaccharide sample, and the mixture was hydrolyzed at 100° C. for 2 hours. The hydrolyzate was dried by a nitrogen gas blower and washed three times with chromatographically pure methanol to obtain a polysaccharide hydrolyzate. 10 mg of hydroxylamine hydrochloride, 1 mg of internal standard inositol and 2 mL of pyridine were sequentially added to the polysaccharide hydrolyzate. After 90 min of water bath at 90° C., 2 mL of acetic anhydride was added. After 90 min of water bath at 90° C., 2 mL of distilled water was added to terminate the reaction. The resulted mixture was extracted twice with 2 mL of dichloromethane, and the dichloromethane phase was combined, dried over anhydrous sodium sulfate, and filtered by a 0.22 μm organic microporous membrane.

(26) Gas chromatographic conditions: analytical column was HP-5MS (30 m×0.25 mm×0.25 μm); injection volume was 1 μL; inlet temperature was 250° C.; constant pressure was 20 PSI; programmed temperature: column temperature was kept at 100° C. for 0.5 min, raised to 140° C. at a speed of 20° C./min for 5 min, increased to 160° C. at a speed of 3° C./min, and then increased to 250° C. at a speed of 10° C./min for 5 min; split ratio was set as 10:1, the mobile phase was helium and the flow rate was 1 mL/min.

(27) Monosaccharide standard test: ribose, rhamnose, arabinose, xylose, mannose, glucose, galactose standard were tested according to the same method as above as standard controls.

(28) The measured monosaccharide composition of the myrtle fruit polysaccharide is shown in Table 2 below:

(29) TABLE-US-00002 TABLE 2 The monosaccharide composition of the myrtle fruit polysaccharide Molar ratio (%) component Rib Rha Ara Xyl Man Glu Gal P1 6.74 1.73 60.06 3.54 5.64 13.16 9.13

Example 5

(30) Fourier infrared spectroscopy analysis of the myrtle fruit polysaccharide P1 obtained in Example 1 was carried out.

(31) 2 mg of the polysaccharide sample was mixed with dried KBr in a mortar. The mixture was pressed into a tablet by a tableting machine, and a Fourier transform infrared spectrometer was used to scan a spectrum with wave number at 400-4000 cm.sup.−1.

(32) Functional groups have characteristic absorption peaks in infrared spectrum, and therefore, infrared spectrum can better analyze the structure of the polysaccharide. FIG. 4 is an infrared spectrum of the purified myrtle fruit polysaccharide P1, the absorption peak at 3354 cm.sup.−1 is resulted from OH stretching vibration, and the absorption peak at 2928 cm.sup.−1 is resulted from CH stretching vibration, and the absorption peak at 1458 cm.sup.−1 is resulted from CO stretching vibration. These peaks are characteristic peaks of polysaccharide, and it can be deduced that P1 should be a polysaccharide.

(33) In the infrared spectrum of P1 (FIG. 4), there is a distinct absorption peak at 1649 cm.sup.−1, which is a characteristic absorption peak of bound water; the absorption peak at 1236 cm.sup.−1 is resulted from the stretching vibration of S═O, indicating sulfate group is present in P1; the absorption peak at 1050 cm.sup.−1 illustrates the presence of glucose units.

Example 6

(34) The in vitro cholate binding capacity of the myrtle fruit polysaccharide P1 obtained in Example 1 was tested, and cholestyramine was used as a positive control to preliminarily characterize the hypolipidemic ability of the polysacchride. The method is as follows.

(35) Preparation of standard curve of cholate: 0.3 mmol/L sodium taurocholate, sodium glycocholate, and sodium cholate solution were prepared with 0.1 mol/L and pH=6.3 phosphate buffer solution, respectively. 0, 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the above solutions were added respectively in a 10 mL stoppered test tube. 0.1 mol/L, pH=6.3 phosphate buffer solution was further added to the test tubes until the volume became 2.5 mL. Then 7.5 mL of 60 wt % sulfuric acid solution was added and the temperature was kept at 70° C. in a water bath for 20 min. After that the test tubes were taken out and placed in an ice bath for 5 min, and absorbance at 387 nm wavelength was detected respectively. The absorbance was plotted against the concentration of cholate to prepare a standard curve.

(36) Simulation of the human gastrointestinal environment: 1 mL of the sample solution and 1 mL of 0.01 mol/L hydrochloric acid solution were added to a 10 mL stoppered test tube, and the mixture was shaken and digested at 37° C. for 1 h. Then pH was adjusted with a 0.1 mol/L sodium hydroxide solution to 6.3, followed by the addition of 4 mL of 10 mg/mL trypsin (prepared with 0.1 mol/L phosphate buffer at pH 6.3). The mixture was further shaken and digested at 37° C. for 30 min.

(37) In vitro binding of cholate: 4 mL of 0.3 mmol/L cholate solutions (sodium taurocholate, sodium glycocholate, sodium cholate) were added to the simulated human gastrointestinal environment respectively. The solutions were shaken at 37° C. for 1 h, then transferred to a centrifuge tube and centrifuged at 4000 r/min for 20 min. The content of cholate in the obtained supernatants were analyzed: 2.5 ml of each of the supernatants and 7.5 ml of 60% sulfuric acid solution were added sequentially to a stoppered test tube. The test tubes were kept in a water bath at 70° C. for 20 min, and then in an ice bath for 5 min. The absorbance was measured at 387 nm. The concentration of cholate in the sample solution was determined from the standard curve, which may be subtracted from the concentration of the added cholate to calculate the concentration of bound cholate.

(38) FIG. 3 shows the ability of the purified myrtle fruit polysaccharide P1 to bind cholate in vitro. The result shows that cholestyramine (a hypolipidemic drug which achieves a hypolipidemic effect by combination with cholate) was used as a positive control. Taking the binding rate of cholestyramine to each cholate as 100%, the relative binding rate of the myrtle polysaccharide P1 to sodium taurocholate, sodium glycocholate and sodium cholate was 25.28%, 44.56%, and 50.10%, respectively.

(39) The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications thereof made without departing from the spirit and scope of the invention should all be equivalent replacements and be included in the scope of the present invention.