CN118027237A - Morinda officinalis uniform polysaccharide and preparation method and application thereof - Google Patents
Morinda officinalis uniform polysaccharide and preparation method and application thereof Download PDFInfo
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- CN118027237A CN118027237A CN202410220790.8A CN202410220790A CN118027237A CN 118027237 A CN118027237 A CN 118027237A CN 202410220790 A CN202410220790 A CN 202410220790A CN 118027237 A CN118027237 A CN 118027237A
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- polysaccharide
- morinda
- uniform
- morinda officinalis
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Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicine extraction, and provides morinda officinalis uniform polysaccharide, and a preparation method and application thereof. The morinda officinalis uniform polysaccharide has a structure shown in the following formula (A is alpha-L-Araf, B is alpha-D-Galp, C is beta-D-Glcp, D is alpha-L-Rhap), and is a polysaccharide consisting of rhamnose, arabinose, galactose and glucose; taking morinda officinalis preparation as a raw material, and obtaining morinda officinalis uniform polysaccharide HLS-D2N1 through extraction, impurity removal, ion exchange purification and gel filtration chromatography purification, and the morinda officinalis uniform polysaccharide HLS-D2N1 has the characteristics of high extraction rate, high purity and the like; the morinda officinalis uniform polysaccharide HLS-D2N1 has good OB protection effect and remarkable immune activation characteristic, and has wide application in preparing products for treating and/or preventing osteoporosis, promoting fracture healing or regulating immunity.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and relates to morinda officinalis uniform polysaccharide, and a preparation method and application thereof.
Background
The morinda officinalis (Morinda officinalis How) belongs to vine plants of Rubiaceae, is a traditional name Guinan medicine in China, has various pharmacological effects of resisting osteoporosis, resisting oxidation, resisting aging, resisting fatigue, regulating immunity, resisting tumor, resisting depression, resisting inflammation, relieving pain, improving reproductive function and the like, and can be used for treating impotence, spermatorrhea, infertility due to cold womb, irregular menstruation, cold pain in lower abdomen, rheumatalgia and flaccidity of bones and muscles. The chemical components separated and identified from the morinda plants mainly comprise anthraquinone, iridoid, oligosaccharide, polysaccharide and the like, wherein the polysaccharide substance content is higher than 13.45-24.37%; pharmacological experiments show that the polysaccharide is taken as one of main active ingredients of morinda officinalis, has obvious biological activity in various aspects such as antioxidation, antifatigue, immunoregulation, anti-osteoporosis and the like, and has good development prospect.
But at the same time there are the following problems: the extraction rate of the morinda officinalis polysaccharide is low, and the morinda officinalis polysaccharide has defects in separation and purification; the structural research of morinda officinalis polysaccharide is focused on monosaccharide composition, side chain position and the like, and the research on higher structure, structure-activity relationship and the like is less; the polysaccharide composition and molecular weight distribution obtained by different extraction methods have great difference, and the chemical structure of the polysaccharide needs to be further clarified to explore the action mechanism of the structure and the function of the morinda officinalis polysaccharide, so that the extraction rate of the morinda officinalis polysaccharide needs to be improved, the research of the separation, purification and structure-activity relationship of the morinda officinalis polysaccharide is enhanced, and theoretical guidance is provided for the product development of the morinda officinalis polysaccharide in functional foods and medicines.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a morinda officinalis purified polysaccharide, and a preparation method and application thereof. Taking morinda officinalis preparation as a raw material, and obtaining morinda officinalis uniform polysaccharide HLS-D2N1 through extraction, impurity removal, ion exchange purification and gel filtration chromatography purification, and the morinda officinalis uniform polysaccharide HLS-D2N1 has the characteristics of high extraction rate, high purity and the like; the structure of the morinda officinalis uniform polysaccharide HLS-D2N1 is analyzed and determined, and the morinda officinalis uniform polysaccharide HLS-D2N1 has good OB protection effect and remarkable immune activation characteristic and can be widely applied to the preparation of products for treating and/or preventing osteoporosis.
The technical scheme of the invention is as follows:
a morinda citrifolia uniform polysaccharide having the structure shown below:
Wherein A is alpha-L-Araf, B is alpha-D-Galp, C is beta-D-Glcp, and D is alpha-L-Rhap.
Further, the morinda citrifolia uniform polysaccharide is a polysaccharide consisting of rhamnose, arabinose, galactose and glucose.
Still further, the molar ratio of rhamnose, arabinose, galactose and glucose in the morinda citrifolia uniform polysaccharide was 16.46:37.85:28.83:4.30.
Further, the weight average molecular weight of the morinda officinalis uniform polysaccharide is 90-100kDa; specifically 97.902kDa.
Further, the morinda officinalis uniform polysaccharide is morinda officinalis uniform polysaccharide HLS-D2 obtained by ion exchange purification of morinda officinalis crude polysaccharide by using 0.1M NaCl solution as eluent.
Preferably, the morinda citrifolia uniform polysaccharide is morinda citrifolia uniform polysaccharide HLS-D2N1 obtained by gel purification of morinda citrifolia uniform polysaccharide HLS-D2 with pure water as eluent.
The invention also provides a preparation method of the morinda officinalis uniform polysaccharide.
The preparation method comprises the following steps:
(1) Extracting the morinda officinalis crude polysaccharide: adding processed radix Morindae officinalis product into water, reflux extracting, and precipitating with ethanol to obtain radix Morindae officinalis crude polysaccharide;
(2) Removing impurities from the crude morinda officinalis polysaccharide;
(3) Ion exchange purification: dissolving the crude morinda officinalis polysaccharide subjected to impurity removal in water to obtain crude morinda officinalis polysaccharide mother solution; passing through ion exchange column, collecting eluate, drawing ion purification elution curve, mixing eluents corresponding to the same elution peak, concentrating, desalting, and drying to obtain corresponding Morinda officinalis polysaccharide;
(4) Purifying by gel filtration chromatography: dissolving each morinda officinalis polysaccharide obtained in the step (3) in water to obtain morinda officinalis polysaccharide mother liquor, passing through a gel chromatographic column, collecting eluent, drawing a gel filtration chromatographic purification elution curve, combining eluents corresponding to the same elution peak, concentrating and drying to obtain the morinda officinalis uniform polysaccharide.
Further, in the step (1), the mass ratio of the morinda officinalis preparation to the water is 1:8-10; the reflux extraction times are 2-3 times, and each extraction time is 1-2 hours.
Further, the morinda officinalis crude polysaccharide impurity removal comprises the following steps:
S1: dissolving radix Morindae officinalis crude polysaccharide in water, and adding papain for enzymolysis to obtain enzymolysis solution;
S2: sequentially adding a chloroform/n-butanol mixed solvent and petroleum ether into the enzymolysis liquid for extraction, and collecting a water phase;
s3: adding macroporous adsorbent resin AB-8 into the water phase, fully mixing, and adsorbing overnight;
s4: and collecting liquid, dialyzing and drying to obtain a crude polysaccharide sample after impurity removal.
Further, the ion exchange purification sequentially adopts pure water, 0.1M, 0.2M and 0.3M NaCl solution and the like for gradient elution.
Further, the gel purification is performed using water.
The invention also provides application of the morinda officinalis uniform polysaccharide prepared by any one of the above or any one of the above preparation methods in preparation of products for treating and/or preventing secondary osteoporosis, promoting fracture healing or regulating immunity.
Compared with the prior art, the invention has the following beneficial effects:
(1) The morinda officinalis processed product is used as a raw material, and the extraction and purification efficiency is high through the processes of extraction, impurity removal, ion exchange purification and gel filtration chromatography purification, so that the morinda officinalis uniform polysaccharide has high purity and the purity is 95.8%;
(2) The invention provides a novel morinda officinalis uniform polysaccharide HLS-D2N1 and identifies the structure thereof, wherein the morinda officinalis uniform polysaccharide HLS-D2N1 is prepared from rhamnose, arabinose, galactose and glucose according to a molar ratio of 16.46:37.85:28.83:4.30, a polysaccharide having a weight average molecular weight Mw of 97.902kDa;
(3) The morinda officinalis uniform polysaccharide has good OB protective effect and remarkable immune activation characteristic, and has wide application in the fields of preparing products for treating and/or preventing secondary osteoporosis, promoting fracture healing or regulating immunity, such as foods, medicines, health products and the like;
(4) The invention provides a theoretical basis for the application of the morinda officinalis polysaccharide in functional foods, health-care products, medicines and other products through the identification of the morinda officinalis polysaccharide.
Drawings
FIG. 1 shows the purification elution profile of morinda citrifolia uniform polysaccharide ions;
FIG. 2 shows the HLS-D2 gel filtration chromatography purification elution profile of morinda officinalis uniform polysaccharide;
FIG. 3 is a graph of absolute molecular weight analysis of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 4 is a graph showing molecular configuration analysis of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 5 is a mixed standard ion chromatogram;
FIG. 6 shows a HLS-D2N1 ion chromatogram of Morinda citrifolia uniform polysaccharide;
FIG. 7 is a GC-MS total ion flow diagram of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 8 is a nuclear magnetic one-dimensional 1 H spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 9 is a nuclear magnetic one-dimensional 13 C spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 10 is a nuclear magnetic two-dimensional COSY spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 11 is a nuclear magnetic two-dimensional NOESY spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 12 is a nuclear magnetic two-dimensional HSQC spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 13 is a nuclear magnetic two-dimensional HMBC spectrum of morinda officinalis uniform polysaccharide HLS-D2N 1;
FIG. 14 shows the effect of morinda officinalis uniform polysaccharide HLS-D2N1 on MC3T3-E1 cells (### compared to Ctrl group, P < 0.001);
FIG. 15 is a graph showing the effect of morinda officinalis uniform polysaccharide HLS-D2N1 on Dex-induced MC3T3-E1 cell viability (### vs. Ctrl group, P < 0.001; *** vs. Dex group, P < 0.001);
FIG. 16 shows the effect of morinda officinalis uniform polysaccharide HLS-D2N1 on RAW264.7 cell proliferation (** vs. blank, P < 0.01; *** vs. blank, P < 0.001);
FIG. 17 shows the effect of morinda officinalis uniform polysaccharide HLS-D2N1 on phagocytic capacity of RAW264.7 cells (### vs. Con. Group, P < 0.001; *** vs. LPS group, P < 0.001);
FIG. 18 shows the effect of the Morinda citrifolia uniform polysaccharide HLS-D2N1 (A) and LPS+Morinda citrifolia uniform polysaccharide HLS-D2N1 (B) on the secretion of NO by RAW264.7 cells (### vs. Con. Group, P < 0.001; ** vs. LPS group, P < 0.01, *** vs. LPS group, P < 0.001);
FIG. 19 shows the effect of the morinda officinalis homogeneous polysaccharide HLS-D2N1 (A) and LPS+morinda officinalis homogeneous polysaccharide HLS-D2N1 (B) on TNF- α secretion by RAW264.7 cells (### P < 0.001 compared to the blank; ** P < 0.01 compared to the LPS group; *** P < 0.001 compared to the LPS group);
FIG. 20 shows the effect of the morinda officinalis uniform polysaccharide HLS-D2N1 (A) and LPS+morinda officinalis uniform polysaccharide HLS-D2N1 (B) on the secretion of IL-6 by RAW264.7 cells (### P < 0.001 compared to the blank control; *** P < 0.001 compared to the LPS group);
FIG. 21 shows the effect of the morinda officinalis homogeneous polysaccharide HLS-D2N1 (A) and LPS+morinda officinalis homogeneous polysaccharide HLS-D2N1 (B) on the secretion of IL-10 by RAW264.7 cells (### vs. blank control group P < 0.001; * vs. LPS group P <0.05, ** vs. LPS group P <0.01, *** vs. LPS group P < 0.001).
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. The following is merely exemplary of the scope of the invention as claimed and many variations and modifications of the invention will be apparent to those skilled in the art in light of the disclosure, which are intended to be within the scope of the invention as claimed.
The embodiment of the invention uses the morinda officinalis preparation product as the salt-roasted morinda officinalis, and the specific preparation method comprises the following steps: weighing salt according to the proportion of 2% of the medicinal materials, dissolving in 5 times of water, adding the radix morindae officinalis, stirring uniformly, moistening until the salt water is absorbed, placing in a medicine steaming box, steaming at 100 ℃ for 2.5-3.5 hours until the radix morinda officinalis is completely steamed, taking out the wood core while hot, cutting into sections of 10-15mm, drying at 70 ℃ for 2-2.5 hours until the water content is less than 15%, taking out, cooling and standing for later use.
Example 1: preparation of morinda officinalis homogeneous polysaccharide HLS-D2N1
1. Extracting the morinda officinalis crude polysaccharide:
Taking 2kg of morinda officinalis preparation products according to a feed liquid ratio of 1:8 adding the extract into water, and extracting under reflux for 2 times each for 2 hours; mixing the extractive solutions, concentrating to 4L, adding ethanol to reach ethanol content of 80%, standing overnight to precipitate, removing supernatant, washing the precipitate with 8L of 95% ethanol, volatilizing to remove ethanol smell, and drying to obtain radix Morindae officinalis crude polysaccharide.
2. Removing impurities from the morinda officinalis crude polysaccharide:
(1) Taking 30g of morinda officinalis crude polysaccharide, adding the morinda officinalis crude polysaccharide into 3L of pure water, fully dissolving the crude polysaccharide, adding 1.5wt% of papain, and carrying out overnight enzymolysis to obtain an enzymolysis solution;
(2) Adding 750mL of chloroform and n-butanol into the enzymolysis solution (water phase), fully mixing, and collecting upper water phase;
(3) Continuously adding 1.5L of petroleum ether into the water phase, fully and uniformly mixing, and collecting a lower water phase;
(4) Adding macroporous adsorbent resin AB-8400g into the water phase, fully mixing, and adsorbing overnight;
(5) Collecting the liquid, dialyzing with dialysis bag (3000 Da) for 48 hr, removing small molecular components, and lyophilizing the polysaccharide liquid to obtain 16g of purified radix Morindae officinalis crude polysaccharide with purity of 80.0%.
3. Ion exchange purification:
(1) Dissolving a proper amount of purified morinda officinalis crude polysaccharide sample in pure water to prepare morinda officinalis crude polysaccharide mother solution with the concentration of 20 mg/mL;
(2) Centrifuging the crude polysaccharide mother solution of radix Morindae officinalis at 10000r/min for 10min, collecting supernatant, purifying with ion exchange column Cellulose DEAE-52, and flowing at 4mL/min; sequentially adopting pure water, 0.1M, 0.2M and 0.3M NaCl solution to perform gradient elution, collecting one tube every 15mL, and collecting all eluents;
(3) Accurately measuring an appropriate amount of eluent in each collecting pipe, adding sulfuric acid-phenol reagent, performing light-proof reaction for 10min, measuring the light absorption value at 490nm, and measuring the total sugar content by adopting a sulfuric acid-phenol method; drawing ion purification elution curves of the samples by taking the number of the eluent tubes as an abscissa and taking the total sugar content and the NaCl concentration in the eluent as an ordinate respectively (see figure 1);
As can be seen from fig. 1, the present ion purification mainly yields four eluting peak components, ion eluting peak 1: tube 7-16, record HLS-D1; ion elution peak 2: tubes 28-37, designated HLS-D2; ion elution peak 3: tube 52-55, designated HLS-D3; ion elution peak 4: tubes 73-79, designated HLS-D4;
(4) Combining the eluent of each collecting pipe corresponding to the same eluting peak;
(5) Concentrating the eluent in each collecting pipe by rotary evaporation to 1/5 of the original volume;
(6) Dialyzing with dialysis bag with molecular weight of 3000Da for 48 hr to remove salt, and lyophilizing the desalted solution.
4. Purifying by gel filtration chromatography:
(1) Taking 21.0g of morinda officinalis uniform polysaccharide HLS-D with highest peak concentration after ion purification, and adding pure water to prepare 2mg/mL morinda officinalis uniform polysaccharide HLS-D2 mother solution;
(2) Centrifuging 10000g of Morinda officinalis uniform polysaccharide HLS-D2 mother liquor at 10000r/min for 10min, separating and purifying supernatant with gel chromatography column Superdex20010/300GL at flow rate of 1mL/min, eluting with pure water for 1.5 times of column volume, collecting one tube every 12mL, and collecting all eluents;
(3) Determining the total sugar content of the eluent in each collecting pipe by a sulfuric acid-phenol method, and drawing a gel purification elution curve of the sample by taking the number of the eluent pipes as an abscissa and the total sugar content as an ordinate (see figure 2);
as can be seen from fig. 2, the present gel filtration chromatography purification mainly provides an elution peak component, gel elution peak 1: tubes 27-37, designated HLS-D2N1;
(4) Combining the eluent of each collecting pipe corresponding to the eluting peak, and concentrating by rotary evaporation to 1/5 of the original volume;
(5) Freeze drying, and identifying the polysaccharide, content and purity after gel purification by adopting a sulfuric acid-phenol method to obtain the morinda officinalis homogeneous polysaccharide HLS-D2N10.61g, wherein the comprehensive yield is 61.0%, and the polysaccharide purity is 95.8%.
Example 2: structural identification of morinda officinalis homogeneous polysaccharide HLS-D2N1
(1) Determination of morinda officinalis uniform polysaccharide HLS-D2N1 molecular weight by high performance gel chromatography:
(a) Chromatographic conditions: gel chromatography-differential-multi-angle laser light scattering system was used: the liquid phase system is U3000 (Thermo, USA), the differential detector is Optilab T-rex (Wyatt technology, CA, USA), and the laser light scattering detector is DAWN HELEOS II (Wyatt technology, CA, USA); the gel exclusion chromatographic column Ohpak SB-805HQ (300X 8 mm) and Ohpak SB-803HQ (300X 8 mm) were connected in series; column temperature: 45 ℃; sample injection amount: 100. Mu.L; mobile phase: 0.02wt% NaN 3,0.1M NaNO3; flow rate: 0.6mL/min; isocratic elution: 75min.
(B) Sample preparation: the sample was dissolved in 0.1M NaNO 3 aqueous solution (0.02% NaN 3, w/w) to a final concentration of 1mg/mL and filtered through a filter with a pore size of 0.45 μm and then run on a machine.
(C) Measurement results: the absolute molecular weight analysis chart is shown in figure 3, and the molecular configuration analysis chart is shown in figure 4; the weight average molecular weight Mw= 97.902kDa of the morinda citrifolia homogeneous polysaccharide HLS-D2N1 was calculated according to Mark-Houwink Equation equation.
(2) Analysis of morinda officinalis homogeneous polysaccharide HLS-D2N1 composition using ion chromatograph:
(a) Chromatographic conditions:
Chromatographic system: a Thermo ICS 5000+ ion chromatography system (ICS 5000+, thermo FISHER SCIENTIFIC, USA) for analysis and detection of monosaccharide components using an electrochemical detector; liquid chromatography column Dionex TMCarboPacTM PA20 (150 x 3.0mm,10 μm); sample injection amount: 5. Mu.L; mobile phase: mobile phase a is H 2 O, mobile phase B is 0.1M NaOH, mobile phase C is 0.1M NaOH (containing 0.2M NaAc); flow rate: 0.5mL/min; column temperature: 30 ℃; elution gradient: 0min: phase A/B/phase C (95:5:0, V/V); 26min: phase A/B/phase C (85:5:10, V/V); 42min: phase A/B/phase C (85:5:10, V/V); 42.1min: phase A/B/phase C (60:0:40, V/V); 52min: phase A/B/phase C (60:40:0, V/V); 52.1min: phase A/B/phase C (95:5:0, V/V); 60min: phase A/B/phase C (95:5:0, V/V).
(B) The preparation and calculation method of the standard solution comprises the following steps:
Taking 13 monosaccharide standard substances (fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA) and guluronic acid (Gul-UA)), and preparing into a standard substance mother solution with the concentration of about 10 mg/mL; mixing a proper amount of standard substance mother liquor single standard to prepare a standard substance mixed standard with the highest index concentration of 60 mug/mL, 50 mug/mL or 40 mug/mL, and preparing a series of standard substances required by a machine according to the concentration gradient of the table 1;
TABLE 1 monosaccharide mixing series concentration table
(C) Acid hydrolysis: 5.04mg of morinda officinalis homogeneous polysaccharide HLS-D2N1 sample is weighed, 1mL of 2M TFA acid solution is added, and the mixture is heated at 121 ℃ for 2 hours; introducing nitrogen and drying; adding 99.99% methanol for cleaning, drying, and repeating methanol cleaning for 3 times. Adding double distilled water for dissolution, and performing ion chromatography detection.
(D) Measurement results:
the results of the HLS-D2N1 ion chromatogram of the standard substance and the morinda officinalis uniform polysaccharide are shown in fig. 5 and 6.
The content of each component in the sample (μg/mg) =c x V x F/M,
Wherein: c is concentration in μg/mL; v is the volume of the sample extracting solution, and the unit is mL; f is a dilution factor; m is the total amount of the sample weighed in mg.
Through calculation, compared with a monosaccharide standard substance, the morinda officinalis uniform polysaccharide HLS-D2N1 is obtained and is a polysaccharide consisting of rhamnose, arabinose, galactose and glucose; the molar ratio of each monosaccharide in HLS-D2N1 is rhamnose: arabinose: galactose: glucose 16.46:37.85:28.83:4.30.
(3) The bonding mode of the glycosidic bond of morinda officinalis uniform polysaccharide HLS-D2N1 was analyzed by methylation and gas chromatography-mass spectrometry (GC-MS):
(a) Analysis conditions: 7890A-5977B gas chromatograph-mass spectrometer (Agilent Technologies Inc. CA, UAS) of Agilent company, the model of the autosampler is G4567A; chromatographic column: BPX70 (30 m×0.25mm×0.25 μm, SGE, australia); sample injection amount: 1 μl; split ratio: 10:1; carrier gas: high purity helium gas; the initial temperature of the column incubator is kept at 140 ℃ for 2.0min, and the temperature is programmed to be increased to 230 ℃ at 3 ℃/min and kept for 3min; mass spectrometry conditions: electron bombardment ion source (EI) and MassHunter station, analyte detection in full SCAN (SCAN) mode, mass SCAN range (m/z): 50-350.
(B) Sample treatment: taking 2mg of morinda officinalis uniform polysaccharide HLS-D2N1 sample, adding 500 mu L of DMSO for dissolution, adding 1mg of NaOH, and incubating for 30min; adding 50 mu L of methyl iodide solution for reaction for 1h, adding 1mL of water and 2mL of dichloromethane, mixing uniformly by vortex, centrifuging, and discarding the water phase; sucking the dichloromethane phase of the lower layer and drying with nitrogen; adding 100 mu L of 2M TFA, reacting at 121 ℃ for 90min, and evaporating at 30 ℃; adding 50 mu L of 2M ammonia water, 50 mu L of 1M NaBD 4, uniformly mixing, reacting for 2.5 hours at room temperature, adding 20 mu L of acetic acid to terminate the reaction, drying by nitrogen, washing twice by 250 mu L of methanol, and drying by nitrogen; adding 250 mu L of acetic anhydride, mixing by vortex, reacting for 2.5h at 100 ℃, adding 1mL of water, standing for 10min, adding 500 mu L of dichloromethane, mixing by vortex, centrifuging, discarding the water phase, taking the dichloromethane phase at the lower layer, and detecting by GC-MS.
(C) Test results:
the GC-MS total ion flow results of morinda officinalis uniform polysaccharide HLS-D2N1 are shown in FIG. 7 and Table 2;
TABLE 2 results of bond Structure analysis of Morinda citrifolia homogeneous polysaccharide HLS-D2N1 polysaccharide samples
(4) Nuclear magnetic signature assignment of sugar residues by nuclear magnetic resonance spectroscopy:
(a) Instrument conditions: nuclear magnetic resonance spectrometer: bruk (germany) 500MHz; the scanning temperature is 25 ℃; QXI 1H/31P/13C/15N 5mm four resonance reverse detection probe (Z-gradient, ATM Acc), technical parameters: signal to noise ratio (1H): 888, resolution (Hz): 0.32 (rotation); BBFO 1H-19F,31P-15 N,1H coupling/observe multicore forward detection probe (Z-gradien, ATM), technical parameters: signal to noise ratio (1 H) 798, resolution (Hz) 0.26 (rotation); signal to noise ratio (13 C) 32, resolution (Hz) 0.1.
(B) Sample treatment: taking a proper amount of morinda officinalis uniform polysaccharide HLS-D2N1, fully dissolving into D 2 O, and preparing into polysaccharide solution with the concentration of 40 mg/mL; the dissolved solution was transferred to a nuclear magnetic tube with an addition of 0.5mL.
(C) Test results: the nuclear magnetic tube is put into a nuclear magnetic resonance spectrometer to scan a one-dimensional 1 H spectrum, a 13 C spectrum and a two-dimensional COSY, NOESY, HSQC, HMBC spectrum, and the results are shown in figures 8-13.
Sugar residue a: the anomeric signal δ=5.18/109.21 ppm (H1/C1) indicates that residue a is an arabinose residue of the α -configuration, the H1 chemical shift δ=5.18 ppm for sugar residue a determined from 1 H NMR, and the H2 chemical shift δ=4.16 ppm for residue a determined from COSY pattern cross peak δ=5.18/4.16 ppm. In the same way, the H3-H5 signal, i.e. the H3-H5 chemical shift of sugar residue a, can be determined sequentially by COSY maps, belonging to δ=4.25 ppm, δ=4.1 ppm and δ=3.66 (3.85) ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shift of C2-C5 on the sugar ring can be attributed to the HSQC signal, which is delta=81.51 ppm, 84.91ppm, 86.98ppm and 61.04ppm respectively. Wherein the chemical shift of C1 is shifted to a low field, which indicates that the residue is substituted at the O-1 position of the sugar ring, and the sugar residue A is determined to be alpha-L-Araf- (1- & gt) by combining with a methylation analysis result;
sugar residue B: the anomeric signal delta=5.02/107.44 ppm (H1/C1) indicates that residue B is an arabinose residue of the alpha-configuration. The H1 chemical shift δ=5.02 ppm for sugar residue B as determined by 1 H NMR, the H2-H5 signal can be determined sequentially by COSY pattern cross peaks (the method is the same as for sugar residue a, the following is the same), and the sugar residues B H-H5 chemical shifts are assigned δ=4.07 ppm, 3.67ppm, 3.24ppm and 3.74ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shift of C2-C5 on the sugar ring can be attributed by HSQC signal, delta= 81.12ppm, 80.17ppm, 82.06ppm and 66.43ppm in this order. Wherein the chemical shifts of C1 and C5 shift toward the low field, indicating that the residue was substituted at the O-1 and O-5 positions of the sugar ring, and determining that sugar residue B is → 5) - α -L-Araf- (1 → in combination with methylation analysis results.
Sugar residue C: the anomeric signal delta=5.08/106.86 ppm (H1/C1) indicates that residue C is a galactose residue of the alpha-configuration. The H1 chemical shift δ=5.08 ppm for sugar residue C, determined by 1 H NMR, can be determined in turn by COSY pattern cross peaks as H2-H6 signals, with H2-H6 chemical shifts for sugar residue C belonging to δ=4.08 ppm, 3.79ppm, 3.66ppm, 3.43ppm and 3.84 (4.03) ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shift of C2-C6 on the sugar ring can be attributed by HSQC signal, delta=72.8 ppm, 81.02ppm, 73ppm, 71.18ppm and 68.89ppm in this order. Wherein the chemical shifts of C1, C3 and C6 shift to the lower field, indicating that the residue is substituted at the O-1, O-3 and O-6 positions of the sugar ring, and the result of methylation analysis is combined to infer that the sugar residue C is → 3, 6) -alpha-D-Galp- (1 →.
Sugar residue D: the anomeric signal delta=5.17/106.39 ppm (H1/C1) indicated that residue D was a galactose residue of the alpha-configuration. The H1 chemical shift δ=5.17 ppm for sugar residue D, determined by 1 H NMR, the H2-H6 signal can be determined sequentially by COSY pattern cross peaks, with H2-H6 chemical shifts for sugar residue D belonging to δ=3.88 ppm, 3.76ppm, 4.35ppm, 4.05ppm and 3.76 (3.57) ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shift of C2-C6 on the sugar ring can be attributed by HSQC signal, delta= 73.39ppm, 72.3ppm, 77ppm, 69.94ppm and 61.09ppm in this order. Wherein the chemical shifts of C1 and C4 shift toward the low field, indicating that the residue is substituted at the O-1 and O-4 positions of the sugar ring, and, in combination with the methylation analysis result, deducing that the sugar residue D is → 4) - α -D-Galp- (1 → is provided.
Sugar residue E: the anomeric signal delta=4.4/103.12 ppm (H1/C1) indicates that residue E is a glucose residue in the β -configuration. The H1 chemical shift δ=4.4 ppm for sugar residue E, determined by 1 H NMR, can be determined in turn by COSY pattern cross peaks as H2-H6 signals, with H2-H6 chemical shifts for sugar residue E belonging to δ=3.28 ppm, 3.63ppm, 4.06ppm, 3.6ppm and 3.41ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shifts of C2 to C6 on the sugar ring can be attributed by HSQC signals, which are δ=72.72 ppm, 74.9ppm, 76.26ppm, 72.5ppm and 59.97ppm in order. Wherein the chemical shifts of C1 and C4 shift toward the lower field, indicating that the residue is substituted at the O-1 and O-4 positions of the sugar ring, and in combination with the methylation analysis result, deducing that the sugar residue E is → 4) - β -D-Glcp- (1 → is disclosed.
Sugar residue F: the anomeric signal δ=5.2/98.4 ppm (H1/C1) indicates that residue F is a rhamnose residue in the α -configuration. The H1 chemical shift δ=5.2 ppm for sugar residue F, determined by 1 H NMR, can be determined in turn by COSY pattern cross peaks as H2-H6 signals, with H2-H6 chemical shifts for sugar residue F belonging to δ=4.05 ppm, 3.9ppm, 3.97ppm, 4.04ppm and 1.23ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shifts of C2 to C6 on the sugar ring can be attributed through HSQC signals, namely delta= 83.97ppm, 70.2ppm, 76.58ppm, 69.94ppm and 16.56ppm in sequence. Wherein the chemical shifts of C1, C2 and C4 shift to the low field, indicating that the residue is substituted at the O-1, O-2 and O-4 positions of the sugar ring, and the result of methylation analysis is combined to infer that the sugar residue F is → 2, 4) - α -L-Rhap- (1 →.
Sugar residue G: the anomeric signal delta=4.95/97.47 ppm (H1/C1) indicates that residue G is a rhamnose residue in the alpha-configuration. The H1 chemical shift δ=4.95 ppm for sugar residue G, determined by 1 H NMR, can be determined by COSY pattern cross peaks, which in turn determine the H2-H6 signal, with H2-H6 chemical shifts for sugar residue G belonging to δ=3.84 ppm, 3.47ppm, 4.61ppm, 3.59ppm and 1.18ppm, respectively. After the chemical shift of hydrogen on the sugar ring is completed, the chemical shift of C2-C6 on the sugar ring can be attributed by HSQC signal, delta=76.5 ppm, 75.09ppm, 71.43ppm, 69.98ppm and 16.86ppm in this order. Wherein the chemical shifts of C1 and C2 shift toward the low field, indicating that the residue is substituted at the O-1 and O-2 positions of the sugar ring, and, in combination with the methylation analysis result, deducing that the sugar residue G is → 2) - α -L-Rhap- (1 → is provided.
The signal of the sugar residue of the sample was attributed to the combination of one-dimensional and two-dimensional NMR spectra, as shown in Table 3.
TABLE 3 chemical shifts of sugar residues 1 H and 13 C
In conclusion, the presence of structure and linkage in the polysaccharide was analyzed in combination with HMBC and NOESY spectra based on the chemical shift of each sugar residue 13 C and 1 H in the sample. Judging and predicting the connection sequence of each residue in the polysaccharide by combining an HMBC spectrogram: the sugar residue C-H1 had a cross peak delta=5.08/77 ppm with residue D-C4 and a cross peak delta 5.08/76.26ppm with residue E-C4. Judging and presuming the connection sequence of each residue in the polysaccharide by combining NOESY spectrogram: sugar residue A-H1 exhibited a cross-peak delta=5.18/3.84 ppm with residue C-H6 and a cross-peak delta=5.18/3.97 ppm with residue F-H4. The sugar residue B-H1 had a cross peak delta=5.02/3.79 ppm with residue C-H3. The sugar residue C-H1 had a cross peak delta=5.08/4.06 ppm with residue E-H4. The sugar residue D-H1 had a cross peak delta=5.17/3.74 ppm with residue B-H5. Sugar residue E-H1 had a cross peak delta=4.4/4.05 ppm with residue F-H2. The sugar residue F-H1 had a cross peak delta=5.2/4.35 ppm with residue D-H4.
By combining one-dimensional nuclear magnetism, two-dimensional nuclear magnetism information and methylation result analysis, it is deduced that the polysaccharide is mainly composed of → 5) -alpha-L-Galp- (1 → 3, 6) -alpha-D-Galp- (1 → 4) -alpha-D-Galp- (1 → and a small amount of → 4) -beta-D-GlcP- (1 → and the like which are connected to form a main chain, and the branched chain is mainly composed of alpha-L-Galp- (1 → connected to the O-6 position of sugar residue → 3, 6) -alpha-D-Galp- (1 → and the O-4 position of → 2, 4) -alpha-L-Rhap- (1 → and the like, so that the structure of the polysaccharide chain is as follows:
effect example 1: effect of morinda officinalis Uniform polysaccharide HLS-D2N1 on MC3T3-E1 cell viability
Fracture is a common sudden injury, often accompanied by fracture nonunion, delayed union and other complications, which not only can not enable a patient to recover functions as early as possible, but also directly increases the medical cost of the patient. When fracture occurs, the body activates a large number of osteoblasts, which promote the bone formation process. Therefore, a sufficient number and abundant activity of osteoblasts is of great importance for bone regeneration. Osteoblasts are bone forming cells, bone progenitor cells or preosteoblasts (osteoprogenitor cell) derived from the mesenchymal stem cells (BMSCs). Therefore, the invention selects the preosteoblast MC3T3-E1 of the mice as an experimental cell, and researches the influence of the morinda officinalis uniform polysaccharide HLS-D2N1 with different concentrations on the activity of the MC3T3-E1 cell.
(1) MC3T3-E1 cell culture:
MC3T3-E1 cells were cultured in DMEM medium (containing 10% FBS,100U/mL penicillin and 100. Mu.g/mL streptomycin) at 37℃in a 5% CO 2 incubator with cell culture medium changed every 3 days.
(2) Proliferation Activity assay of morinda officinalis homogeneous polysaccharide HLS-D2N1 on MC3T3-E1 cells:
The effect of morinda officinalis uniform polysaccharide HLS-D2N1 on MC3T3-E1 cell proliferation activity was examined by CCK-8 method. Selecting MC3T3-E1 cells with good growth state, discarding the culture medium, adding trypsin (0.25%) for incubation for 2min, adding the culture medium (containing 10% FBS) for stopping digestion, collecting digestive juice for centrifugation (800 rpm,5 min), discarding supernatant, resuspending cell sediment, inoculating to a 96-well plate (5.0X10 3/well), adding morinda officinalis uniform polysaccharide HLS-D2N1 DMEM solution with different concentrations (2.5, 5, 10, 20 and 40 mu g/mL) into the administration group after the cells are attached, adding an equal volume of DMEM culture medium into the CTRL group, and incubating for 24h. The effect of the different concentrations of morinda officinalis uniform polysaccharide components on MC3T3-E1 cell proliferation activity was compared as shown in FIG. 14 by adding 10. Mu.L of CCK-8 solution to each group, incubating for 4 hours in the absence of light, detecting absorbance at a wavelength of 450nm, and calculating cell survival.
(3) Experimental results:
As can be seen from fig. 14, the proliferation effect of the MC3T3-E1 cells and the concentration of the morinda officinalis uniform polysaccharide HLS-D2N1 are in positive correlation, and the difference has a statistical significance, which indicates that the morinda officinalis uniform polysaccharide has a good effect of promoting the activity of the MC3T3-E1 cells, thus having potential application in the aspect of treating fracture healing.
Effect example 2: effect of morinda officinalis uniform polysaccharide HLS-D2N1 on Dex-induced MC3T3-E1 cell viability
Secondary osteoporosis (osteoporosis, OP) is a common type of OP in orthodontic clinics, and long-term or high-dose administration of glucocorticoids is one of the important pathogenesis of current secondary OPs. Dexamethasone (Dexamethasone, dex) is a glucocorticoid commonly used in clinic, which can lead to the occurrence of OP by inhibiting proliferation of OB and inducing apoptosis. According to the invention, dex is used for acting on an osteoblast precursor cell MC3T3-E1, an in-vitro OP cell model is constructed, and the protection effect of morinda officinalis uniform polysaccharide HLS-D2N1 sugar with different concentrations on the MC3T3-E1 cell is explored.
(1) MC3T3-E1 cell culture:
MC3T3-E1 cells were cultured in DMEM medium (containing 10% FBS,100U/mL penicillin and 100. Mu.g/mL streptomycin) at 37℃in a 5% CO 2 incubator with cell culture medium changed every 3 days.
(2) Effect of morinda officinalis uniform polysaccharide HLS-D2N1 on Dex-induced MC3T3-E1 cell viability
The effect of morinda officinalis uniform polysaccharide HLS-D2N1 on MC3T3-E1 cell proliferation activity was examined by CCK-8 method. Selecting MC3T3-E1 cells with good growth state, discarding the culture medium, adding trypsin (0.25%) for incubation for 2min, adding the culture medium (containing 10% FBS) for stopping digestion, collecting digestive juice for centrifugation (800 rpm,5 min), discarding supernatant, resuspending cell sediment, inoculating to a 96-well plate (5.0X10 3/well), after the cells are attached to the wall, adding morinda officinalis uniform polysaccharide HLS-D2N1 DMEM solutions with different concentrations (2.5, 5, 10, 20 and 40 mu g/mL) into the administration group, adding equal volumes of DMEM culture medium into the CTRL group and the Dex group, incubating for 2h, and adding Dex (4 mu M) DMEM solution for incubation for 24h. The effect of the uniform polysaccharide fractions of morinda officinalis on MC3T3-E1 cell proliferation activity at different concentrations was compared by adding 10. Mu.L of CCK-8 solution to each group, incubating for 4 hours in the absence of light, detecting absorbance at a wavelength of 450nm, and calculating the cell viability, and the results are shown in FIG. 15.
(3) Experimental results:
As can be seen from fig. 15, dex significantly reduced the viability of MC3T3-E1 cells (P < 0.001), while different concentrations of morinda citrifolia purified polysaccharide (P < 0.001) significantly reversed the Dex-induced reduction in viability of MC3T3-E1 cells, indicating that morinda citrifolia uniform polysaccharide promoted proliferation and OB protection of preosteoblasts, thus having potential application in the treatment and/or prevention of secondary osteoporosis.
Effect example 3: effect of Morinda citrifolia uniform polysaccharide HLS-D2N1 on immune cell modulation
(1) RAW264.7 cell culture:
RAW264.7 cells were cultured in a DMEM medium (containing 10% FBS,100U/mL penicillin and 100. Mu.g/mL streptomycin) at 37℃in a 5% CO 2 incubator with cell culture medium replacement every 3 days.
(2) Toxicity detection of morinda officinalis homogeneous polysaccharide HLS-D2N1 and LPS on RAW264.7 cells:
Cells in the logarithmic growth phase were selected, inoculated in 96-well plates at 5X 10 4/mL and 100. Mu.L/well, incubated overnight in a CO 2 incubator, and 100. Mu.L of morinda officinalis-homogeneous polysaccharide DMEM solutions of different concentrations (6.25, 12.5, 25, 50, 100. Mu.g/mL) were added. LPS solutions (0.1, 0.5, 1,5 and 10 mug/mL) with different concentration gradients are simultaneously prepared, and a blank control group is not added with medicine and added with an equal volume of medicine dispensing solvent. A background control well was additionally prepared, 6 duplicate wells were prepared for each group, incubated at 37℃for 24h, 10. Mu.L of CCK-8 solution was added to each well 1h before the end, and absorbance was measured at 450 nm. The drug to cell survival rate was calculated according to the following formula:
cell viability = (OD experimental well-OD background well)/(OD blank well-OD background well) ×100%.
As shown in FIG. 16, the morinda officinalis uniform polysaccharide HLS-D2N1 with the concentration of 6.25-100 mug/mL has a remarkable proliferation promoting effect on RAW264.7 cells, and the cell viability is maximum at the concentration of 6.25 and 100 mug/mL. The subsequent experiments were performed with sample concentrations of 6.25-100 μg/mL based on the non-toxic effect of the processed morinda citrifolia uniform polysaccharide HLS-D2N1 on RAW264.7 cells at each concentration.
(3) Determination of phagocytic capacity of morinda officinalis uniform polysaccharide HLS-D2N1 on RAW264.7 cells
Phagocytic capacity is an important index for evaluating the immunological activity of phagocytes, neutral red is a small molecule, weakly basic pH indicator, which is often used to evaluate phagocytic capacity of phagocytes.
Cells in the logarithmic growth phase were selected, inoculated in a 96-well plate at 2.5X10 4/mL and 100. Mu.L/well in a CO 2 incubator overnight, and then cultured in a DMEM solution of morinda officinalis homogeneous polysaccharide HLS-D2N1 of different concentrations (6.25, 12.5, 25, 50, 100. Mu.g/mL) for 24 hours with the DMED medium as a blank control group and LPS (1. Mu.g/mL) as a positive control group. After the culture was completed, the supernatant was discarded, and after washing with pre-chilled PBS for 2 times, 1% neutral red dye was added to incubate for 1 hour. The neutral red was then aspirated, washed 2 times with PBS, and then extracted with cell lysate (50% ethanol, 1% acetic acid), and absorbance was measured at 540nm, as shown in FIG. 17.
Similar to the pro-proliferative effect exhibited by morinda citrifolia uniform polysaccharide HLS-D2N1 in FIG. 16, the activation of the phagocytic capacity of RAW264.7 cells by morinda citrifolia uniform polysaccharide HLS-D2N1 increased with increasing dose over the concentration range of 6.25-100 μg/mL. Compared with the blank control group, the phagocytic capacity of RAW264.7 cells to neutral red (P < 0.001) can be greatly promoted by the concentration of 6.25 and 100 mug/mL.
(4) Effects of morinda officinalis uniform polysaccharide HLS-D2N1 on secretion of inflammatory factors by RAW264.7 cells:
The activation of macrophage immune function by plant polysaccharide is mainly realized by the following two ways, namely, the direct activation of macrophage proliferation differentiation and phagocytic capacity; secondly, the cell membrane is identified by a pattern recognition receptor on the cell membrane, so that a relevant signal path is further excited, and secretion of cell molecules (NO and the like), tumor necrosis factor-alpha (Tumor necrosis factor-alpha), interleukin-6 (Interleukin-6, IL-6) and the like is promoted, so that an indirect activation path of immune regulation is realized. Interleukin 10 (IL-10) is an anti-inflammatory and immunosuppressive factor, and the secretion of IL-10 in RAW264.7 cell supernatant is significantly reduced under the treatment of LPS and morinda officinalis uniform polysaccharide, and the release of IL-10 is reduced with the increase of morinda officinalis uniform polysaccharide concentration, which shows a dose-dependent relationship.
① Establishment of RAW264.7 cell model:
A blank group, an LPS group, a morinda officinalis uniform polysaccharide group, and a morinda officinalis uniform polysaccharide+lps group were set, each at 5 duplicate wells). Selecting cells in logarithmic growth phase, inoculating 5×10 4 cells/mL cells/hole into 96-well plate, placing into CO 2 incubator, culturing overnight, sucking out culture solution, changing into serum-free DMEM solution, starving for 2 hr to make cells at the same level, adding the same concentration of Morinda officinalis polysaccharide solution into Morinda officinalis polysaccharide group and Morinda officinalis polysaccharide+LPS group, and adding same amount of DMEM medium into blank group and LPS group. After 24 hours of culture, LPS with a final concentration of 1. Mu.g/mL was added to the LPS group and the morinda officinalis homogeneous polysaccharide+LPS group for 24 hours, and the release amounts of NO, IL-6, TNF-. Alpha.and IL-10 in the culture medium were examined according to the kit instructions, and the results are shown in FIGS. 18 to 21.
② Experimental results:
As can be seen from fig. 18A, the release amount of NO from RAW264.7 cells treated with morinda officinalis uniform polysaccharide HLS-D2N1 at the experimental concentration was significantly improved (P < 0.001) compared to the blank group, indicating that morinda officinalis purified polysaccharide has an accelerating effect on the secretion of NO from RAW264.7 cells, and shows a dose-dependent relationship, and the release amount of NO from RAW264.7 cells at the concentration of 100 μg/mL is about 1.5 times that of LPS (1 μg/mL) group. The results from fig. 18B show that: morinda citrifolia uniform polysaccharide HLS-D2N1 still promotes LPS-induced NO release. The morinda citrifolia purified polysaccharide has certain immune activation characteristics.
As can be seen from fig. 19A and 20A, TNF- α and IL-6 secreted by the blank group were both at normal levels, while the secretion of TNF- α and IL-6 was significantly increased in the supernatant of RAW264.7 cells treated with morinda officinalis uniform polysaccharide HLS-D2N1 (P < 0.001). The release amount of TNF-alpha is basically consistent with that of a positive control group (LPS, 1 mug/mL) at the concentration of 6.25-25 mug/mL, and the release amount of TNF-alpha of RAW264.7 cells is increased along with the increase of the concentration of morinda officinalis purified polysaccharide, and the release amount of TNF-alpha is higher than that of the positive control group at the concentration of 50 and 100 mug/mL. The morinda officinalis uniform polysaccharide HLS-D2N1 can promote RAW264.7 cells to release inflammatory mediator IL-6 compared with a blank control group, and has a dose-dependent relationship, but has weaker effect than the positive control group. As can be seen from fig. 19B and 20B, the release amounts of TNF- α and IL-6 in the lps+morinda officinalis uniform polysaccharide group further confirm that morinda officinalis uniform polysaccharide HLS-D2N1 has an immune activating activity, and can promote the release of inflammatory factors.
As can be seen from fig. 21, compared with the blank control group, the secretion amount of IL-10 in the supernatant of RAW264.7 cells treated with morinda officinalis uniform polysaccharide HLS-D2N1 at the experimental concentration was significantly reduced (P < 0.001), and at the concentration of 6.25-25 μg/mL, the secretion amount of IL-10 was substantially consistent with that of the positive control group (LPS, 1 μg/mL), and as the concentration of morinda officinalis purified polysaccharide was increased, a dose-effect dependency was exhibited to some extent, and at the concentration of 50, 100 μg/mL, the secretion amount of IL-10 was lower than that of the positive control group, indicating that morinda officinalis uniform polysaccharide HLS-D2N1 could effectively inhibit the secretion of anti-inflammatory factor IL-10. In FIG. 21B, the release amount of LPS+Morinda citrifolia uniform polysaccharide group IL-10 further demonstrates the immune activating activity of Morinda citrifolia uniform polysaccharide HLS-D2N 1.
In conclusion, in the experimental concentration range (6.25-100 mug/mL), the morinda officinalis purified polysaccharide can obviously promote proliferation and differentiation of RAW264.7 cells, enhance phagocytic capacity of RAW264.7 cells, promote release of NO molecules and secretion of inflammatory factors +TNF-alpha and IL-6, inhibit secretion of anti-inflammatory factor IL-10, and show dose-effect dependency to a certain extent, so that the morinda officinalis purified polysaccharide has obvious immune activation characteristics.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A morinda citrifolia uniform polysaccharide, wherein the morinda citrifolia uniform polysaccharide has the structure shown below:
Wherein A is alpha-L-Araf, B is alpha-D-Galp, C is beta-D-Glcp, and D is alpha-L-Rhap.
2. The morinda citrifolia uniform polysaccharide according to claim 1, wherein said morinda citrifolia uniform polysaccharide is a polysaccharide consisting of rhamnose, arabinose, galactose and glucose.
3. The morinda citrifolia uniform polysaccharide according to claim 1, wherein the molar ratio of rhamnose, arabinose, galactose and glucose in the morinda citrifolia uniform polysaccharide is 16.46:37.85:28.83:4.30.
4. The morinda citrifolia uniform polysaccharide according to claim 1, wherein the morinda citrifolia uniform polysaccharide has a weight average molecular weight of 90-100kDa.
5. The morinda officinalis uniform polysaccharide according to claim 1, wherein said morinda officinalis uniform polysaccharide is morinda officinalis uniform polysaccharide HLS-D2 obtained by ion exchange purification of morinda officinalis crude polysaccharide with 0.1M NaCl solution as eluent;
Preferably, the morinda citrifolia uniform polysaccharide is morinda citrifolia uniform polysaccharide HLS-D2N1 obtained by gel purification of morinda citrifolia uniform polysaccharide HLS-D2 with pure water as eluent.
6. The method for preparing the morinda citrifolia uniform polysaccharide according to any one of claims 1 to 5, comprising the following steps:
(1) Extracting the morinda officinalis crude polysaccharide: adding processed radix Morindae officinalis product into water, reflux extracting, and precipitating with ethanol to obtain radix Morindae officinalis crude polysaccharide;
(2) Removing impurities from the crude morinda officinalis polysaccharide;
(3) Ion exchange purification: dissolving the crude morinda officinalis polysaccharide subjected to impurity removal in water to obtain crude morinda officinalis polysaccharide mother solution; passing through ion exchange column, collecting eluate, drawing ion purification elution curve, mixing eluents corresponding to the same elution peak, concentrating, desalting, and drying to obtain corresponding Morinda officinalis polysaccharide;
(4) Purifying by gel filtration chromatography: dissolving each morinda polysaccharide obtained in the step (3) in water to obtain morinda polysaccharide mother liquor, passing through a gel chromatographic column, collecting eluent, drawing a gel filtration chromatographic purification elution curve, combining eluents corresponding to the same elution peak, concentrating and drying to obtain the morinda uniform polysaccharide.
7. The method of claim 6, wherein in step (1), the mass ratio of morinda officinalis preparation to water is 1:8-10; the reflux extraction times are 2-3 times, and each extraction time is 1-2 hours.
8. The method of claim 6, wherein the removing of the crude morinda polysaccharide comprises the steps of:
S1: dissolving radix Morindae officinalis crude polysaccharide in water, and adding papain for enzymolysis to obtain enzymolysis solution;
S2: sequentially adding a chloroform/n-butanol mixed solvent and petroleum ether into the enzymolysis liquid for extraction, and collecting a water phase;
s3: adding macroporous adsorbent resin AB-8 into the water phase, fully mixing, and adsorbing overnight;
s4: and collecting liquid, dialyzing and drying to obtain a crude polysaccharide sample after impurity removal.
9. The method according to claim 6, wherein the ion exchange purification is performed sequentially by pure water, 0.1M, 0.2M, and 0.3M NaCl solution in an isocratic elution; the gel purification is performed using water.
10. Use of a morinda officinalis homogeneous polysaccharide according to any one of claims 1-5 or prepared by a method according to any one of claims 6-9 for the preparation of a product for the treatment and/or prevention of secondary osteoporosis, for promoting fracture healing or for immunomodulation.
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