阅读说明：本技术 一种黑果枸杞多糖lrp3-s1、其制备方法及其用途 (Lycium ruthenicum polysaccharide LRP3-S1, and preparation method and application thereof ) 是由 丁侃 陈霞 张世海 何菲 于 2019-04-04 设计创作，主要内容包括：本发明提供了从黑果枸杞果实当中获得一种黑果枸杞多糖LRP3-S1的制备方法和该多糖抑制胰腺癌细胞增殖和侵袭的用途、在制备预防和/或治疗胰腺癌的药物或保健品中的用途。采用水提醇沉的方法及层析柱分离纯化得到均一多糖,通过化学和物理方法鉴定了该多糖的RG-I结构。经体外实验证明,该多糖能抑制胰腺癌细胞的增殖并减弱其侵袭能力。因此,所述多糖具有潜在的治疗胰腺癌的作用,可能成为一种抗胰腺癌的糖类药物。(The invention provides a preparation method of lycium ruthenicum polysaccharide LRP3-S1 obtained from lycium ruthenicum fruits, application of the polysaccharide in inhibiting proliferation and invasion of pancreatic cancer cells, and application in preparing a medicament or a health-care product for preventing and/or treating pancreatic cancer. Homogeneous polysaccharide is obtained by adopting a water extraction and alcohol precipitation method and chromatographic column separation and purification, and the RG-I structure of the polysaccharide is identified by chemical and physical methods. In vitro experiments prove that the polysaccharide can inhibit the proliferation of pancreatic cancer cells and weaken the invasive ability of the pancreatic cancer cells. Therefore, the polysaccharide has potential effect of treating pancreatic cancer, and can be used as a carbohydrate drug for resisting pancreatic cancer.)

1. The lycium ruthenicum polysaccharide LRP3-S1 is characterized in that the monosaccharide composition in mole percentage is as follows: 14.4% of rhamnose, 17.7% of galacturonic acid, 26.6% of galactose, 16.4% of xylose and 24.9% of arabinose.

2. The lycium ruthenicum polysaccharide LRP3-S1 of claim 1, having the structure:

3. the Lycium ruthenicum polysaccharide LRP3-S1 of claim 1 or 2, wherein: the molecular weight is 100-120 kDa, and the preferred molecular weight is 114.8 kDa.

4. The preparation method of Lycium ruthenicum polysaccharide LRP3-S1 of any one of claims 1 to 3, comprising the following steps:

(1) and (3) extracting polysaccharide: extracting Lycium ruthenicum Murr with boiling water to obtain an extract, concentrating the extract, adding ethanol into the concentrated solution for precipitation, centrifuging, and collecting the precipitate to obtain crude polysaccharide;

(2) and (3) purifying polysaccharide: subjecting crude polysaccharide to DEAE Sepharose^TMSeparating by a Fast Flow anion exchange column, and collecting an elution component of 0.2MNaCl eluent; and further purifying the eluted component by a Sephacryl S-300HR gel column to obtain the Lycium ruthenicum Murr polysaccharide LRP 3-S1.

5. The method of claim 4, wherein: and (3) performing gradient elution by sequentially using 0.05M, 0.1M and 0.2M NaCl solutions during the separation of the anion exchange column in the step (2), and collecting elution components of 0.2M NaCl eluate.

6. The method of claim 4, wherein: and (3) when the Sephacryl S-300HR gel column in the step (2) is further purified, eluting by using 0.2M NaCl eluent, and collecting an elution component which is Lycium ruthenicum Murr polysaccharide LRP 3-S1.

7. A pharmaceutical composition characterized by: contains the Lycium ruthenicum Murr polysaccharide LRP3-S1 of any one of claims 1 to 3, and pharmaceutically acceptable adjuvants.

8. Use of the Lycium ruthenicum polysaccharide LRP3-S1 of any one of claims 1-3 in the preparation of a medicament or health product for preventing and/or treating pancreatic cancer.

9. Use of the pharmaceutical composition of claim 7 for the preparation of a medicament or health product for the prevention and/or treatment of pancreatic cancer.

Technical Field

The invention relates to an application of polysaccharide, in particular to RG-I type pectin extracted from Lycium ruthenicum Murr, and also provides a preparation method of the RG-I type pectin, an application of the RG-I type pectin in a pancreatic cancer treatment process, and an application of the RG-I type pectin in preparation of a medicine or a health-care product for preventing and/or treating pancreatic cancer.

Background

Pancreatic cancer (Pancreatic cancer) is one of the most malignant, worst prognosis, and lowest five-year survival solid tumors. Meanwhile, pancreatic cancer has high aggressiveness, usually metastasizes in the early stage of cancer, and invades stroma, blood vessels and peripheral tissues thereof, so that the death rate is greatly improved. Therefore, it is one of the serious diseases that people are currently concerned with.

Lycium ruthenicum Murr is a unique medicinal and edible plant in northwest China, has the effects of tonifying kidney and replenishing vital essence, nourishing liver and improving eyesight, enriching blood and soothing nerves, clearing heat and removing dampness and the like, is called as 'soft gold' in folk, and is mainly distributed in Ningxia, Gansu, Qinghai, Xinjiang and other provinces. Lycium ruthenicum Murr is mainly used for treating heart disease, hypertension, menoxenia, climacteric disorder, lithangiuria, gingival hemorrhage, etc. Polysaccharides, as a main component in lycium ruthenicum, have been reported to have anti-fatigue, immunoregulatory, anti-inflammatory, hypoglycemic, antioxidant effects, while few reports have been made on anti-tumor aspects.

Disclosure of Invention

The invention uses a simple and effective process and method for extracting and purifying plant polysaccharide, obtains a pectic polysaccharide by taking Lycium ruthenicum Murr of Qinghai producing area as a raw material, and proves that the polysaccharide can inhibit the growth of pancreatic cancer cells in vitro in a dose-dependent manner and weaken the invasive ability of the pancreatic cancer cells through in vitro experiments. The polysaccharide intervenes in the proliferation and invasion of pancreatic cancer cells by blocking FAK/AKT/GSK-3 beta and p38 signal pathways, thereby achieving the effect of resisting pancreatic cancer. Therefore, the polysaccharide has potential effect of treating pancreatic cancer, and is expected to be developed into a carbohydrate medicament for treating pancreatic cancer diseases.

In order to solve the aim, the invention provides lycium ruthenicum polysaccharide LRP3-S1, which comprises the following monosaccharides in percentage by mole: 14.4% of rhamnose, 17.7% of galacturonic acid, 26.6% of galactose, 16.4% of xylose and 24.9% of arabinose.

Preferably, the lycium ruthenicum polysaccharide LRP3-S1 has the following structure:

wherein the molecular weight of the Lycium ruthenicum Murr polysaccharide LRP3-S1 is 100-120 kDa, preferably 114.8 kDa; wherein the proportion of the three parts of a, b and c is fixed.

The invention also provides a preparation method of the lycium ruthenicum polysaccharide LRP3-S1, which comprises the following steps:

(1) and (3) extracting polysaccharide: extracting Lycium ruthenicum Murr with boiling water to obtain an extract, concentrating the extract, adding ethanol into the concentrated solution for precipitation, centrifuging, and collecting the precipitate to obtain crude polysaccharide;

(2) and (3) purifying polysaccharide: subjecting crude polysaccharide to DEAE Sepharose^TMSeparating by a Fast Flow anion exchange column, and collecting an elution component of 0.2M NaCl eluent; and further purifying the eluted component by a Sephacryl S-300HR gel column to obtain the Lycium ruthenicum Murr polysaccharide LRP 3-S1.

Preferably, in the step (1), before the extraction with boiling water, the lycium ruthenicum murr is soaked in ethanol for 3-10 days (for example, 7 days), and then is dried in the air;

preferably, in the step (1), ethanol used for precipitation is ethanol with a volume fraction of 95%; the volume of ethanol used for precipitation is 3-6 times (for example, 5 times) of that of the concentrated solution;

preferably, in the step (1), before ethanol precipitation, the concentrated solution is centrifuged, and then the centrifuged supernatant is dialyzed, wherein the dialysis time is preferably 24-48 h;

preferably, the crude polysaccharide obtained in step (1) is washed before the purification of the polysaccharide in step (2), with alternating washes of ethanol and acetone, each wash 2-4 times;

preferably, when the anion exchange column in the step (2) is used for separation, gradient elution is sequentially carried out by using 0.05M, 0.1M and 0.2M NaCl solutions, and elution components of 0.2M NaCl eluent are collected;

preferably, when the Sephacryl S-300HR gel column in the step (2) is further purified, 0.2M NaCl eluent is used for elution, and the collected elution component is Lycium ruthenicum Murr polysaccharide LRP 3-S1.

The identification of the obtained Lycium ruthenicum Murr polysaccharide LRP3-S1 comprises the steps of determining purity and molecular weight, and analyzing the structure by adopting methods such as partial acid hydrolysis, sugar composition determination, methylation, infrared and nuclear magnetic resonance and the like.

The invention also provides a pharmaceutical composition which contains the lycium ruthenicum polysaccharide LRP3-S1 and pharmaceutically acceptable auxiliary materials.

The invention also provides application of the lycium ruthenicum polysaccharide LRP3-S1 or the pharmaceutical composition in preparation of medicines or health products for preventing and/or treating pancreatic cancer.

Drawings

FIG. 1 is a graph of purity of Lycium ruthenicum polysaccharide LRP3-S1 high performance liquid chromatography prepared in preparation example 1.

FIG. 2 is an infrared spectrum of LRP3-S1, a Lycium ruthenicum polysaccharide prepared in preparation example 1.

FIG. 3 is a 1H NMR and 13C NMR spectra of Lycium ruthenicum polysaccharide LRP3-S1 prepared in preparation example 1.

FIG. 4 is a bar graph of the effect of Lycium ruthenicum polysaccharide LRP3-S1 prepared in preparation example 1 on pancreatic cancer cell and normal cell survival.

FIG. 5 is a diagram of a cell transmembrane experiment in which the Lycium ruthenicum polysaccharide LRP3-S1 prepared in preparation example 1 affects the invasion of BxPC-3 pancreatic cancer cells.

FIG. 6 is an immunoblot of the expression of protein levels in BxPC-3 cells by Lycium ruthenicum polysaccharide LRP3-S1 prepared in preparation example 1.

Detailed Description

Preparation example 1 extraction, separation, purification and structural characterization of Lycium ruthenicum polysaccharide LRP3-S1

(1) Extraction and separation of polysaccharides

Soaking Lycium ruthenicum Murr fruit in industrial alcohol for one week, air drying the soaked Lycium ruthenicum Murr fruit, extracting with boiling water, and detecting sugar content of the extractive solution with sulfuric acid-phenol method until sugar reaction is not obvious. Mixing extractive solutions, concentrating to small volume, and centrifuging to remove precipitate. The supernatant was dialyzed against convective water for two days. The internal liquid for dialysis is prepared from 95% ethanol by volume ratio of extract to ethanol of 1: 5(v/v) and then left to stand overnight. The excess ethanol in the upper layer is discarded, the precipitate is centrifuged, washed with ethanol and acetone alternately for three times, and dried to obtain crude polysaccharide (93.3g) extracted with water.

(2) Purification of polysaccharides

Dissolving 6g of crude polysaccharide in 80mL of deionized water, stirring overnight, centrifuging, collecting supernatant, and loading on DEAE Sepharose^TMThe Fast Flow anion exchange column was subjected to gradient elution with deionized water and NaCl solutions (0.05M, 0.1M, 0.2M) of different concentrations, the Flow rate was controlled at 13mL/15min, and the eluate was collected by an automatic collector. mu.L of each tube was developed by the sulfuric acid-phenol method and the absorbance thereof was measured at 490nm with a microplate reader, and the elution curve was plotted using the absorbance and the elution volume. Collecting separated polysaccharide according to elution curve, concentrating under reduced pressure, dialyzing, and freeze drying to obtain 0.2M NaCl eluate (secondary crude polysaccharide LRP 3).

200mg of LRP3, a secondary crude polysaccharide, was dissolved in 3mL of deionized water each time, and the supernatant was applied by centrifugation (4,000r/min) to a Sephacryl S-300HR gel column, eluted with 0.2M NaCl eluent at a flow rate of 5mL/15min, and collected by an automatic collector. Carrying out color development by a sulfuric acid phenol method, detecting absorbance by an enzyme-labeling instrument, drawing an elution curve, collecting the required separated components, carrying out concentration dialysis, and finally carrying out freeze drying to obtain the uniform lycium ruthenicum polysaccharide LRP3-S1(0.5 g).

(3) Structural identification of polysaccharides

Polysaccharide LRP3-S1 in tandem Ultrahydrogel^TM802 and Ultrahydrogel^TM804 the characteristic spectrum of the gel column is shown in figure 1, and the chromatographic conditions are as follows: mobile phase: 0.1M NaNO₃(ii) a Flow rate: 0.6 mL/min; column temperature: 40 ℃; agilent 1260 liquid chromatograph; a detector: a differential detector and an ultraviolet detector.

Taking an LRP3-S1 sample of Lycium ruthenicum Murr of about 2mg, and determining infrared spectrum of polysaccharide by potassium bromide tabletting method to obtain the result of 3429.52cm shown in figure 2^-1Is the absorption peak of the stretching vibration of O-H. 2931.54cm^-1The C-H stretching vibration absorption peak is shown. 1420.32cm^-1And 1050.29cm^-1Respectively the C-O stretching vibration outside and inside the ring. 1735.01cm^-1C ═ O elongation from carboxyl groupThe vibration indicates that the polysaccharide is an acidic polysaccharide.

Collecting Lycium ruthenicum Murr polysaccharide LRP3-S135mg, and adding D₂O0.5 mL, added 2.5. mu.L acetone as internal standard: (_H＝2.29ppm，_C31.5ppm), one-dimensional nmr spectra were determined on a BrukerAVANCE III 500M nmr spectrometer at 25 ℃ and are shown in figure 3. FIG. 3A shows a polysaccharide¹³C NMR spectra, chemical shifts 110.38ppm,108.71ppm and 108.34ppm assigned to T-linked α -L-Araf,1,5-linked α -L-Araf and 1,3,5-linked α -L-Araf, chemical shifts 105.44ppm and 104.86ppm assigned to 1,3,6-linked β -D-Galp and 1,3-linked β -D-Galp.104.69ppm and 104.60ppm assigned to T-linked β -D-Galp and 1,6-linked β -D-Galp.chemical shifts assigned to T-linked xylp.99.86ppm assigned to 1,2-linked α -L-Rhap and 1,2,4-linked α -L-Rhap, 98.76ppm assigned to 1,4-linked α -L-Rhap, respectively, and FIG. 3B-linked α -polysaccharide B-Galap assigned to 103.87ppm¹H NMR spectra with chemical shifts of 5.31ppm H-1.5.18 ppm and 5.15ppm H-1.5.07 ppm and 4.68ppm H-1.4.70 ppm which are assigned to 1,4-linked α -D-GalA and T-linked α -D-Galp respectively and 1,5-linked α -L-Araf respectively and which are assigned to 1,4-linked α -D-GalA and T-linked α -D-Galp respectively and H-1.4.70 ppm which are assigned to 1,3,6-linked β -D-Galp and 1,3-linked β -D-Galp respectively and H-1 ppm of galactose-1, 3-linked β -D-Galp and H-1 ppm and 4.58ppm H-1 which are assigned to 1,6-linked β -D-Gal and 1,6-linked β -D-Gal respectively and the chemical shifts of 4.7 ppm H-11% xylose-L-Galp and the chemical shifts of galactose-1, 17% xylose-17% L-xylose-galactose and 17% xylose-galactose-1-17% L-Galp, respectively, and the results with monosaccharide concentrations of xylose, and L-14% xylose, 7% L-17% xylose:

the relative molecular mass of Lycium ruthenicum polysaccharide LRP3-S1 determined by High Performance Gel Permeation Chromatography (HPGPC) is 114.8kDa

Example 1 Lycium ruthenicum polysaccharide LRP3-S1 was tested for anti-pancreatic cancer activity

(1) MTT experiment detects influence of Lycium ruthenicum Murr polysaccharide LRP3-S1 on proliferation of pancreatic cancer cells AspC-1, BxPC-3 and PANC-1

Pancreatic cancer cells or normal cells with good growth status were collected (cell density 5 × 10)⁴One) were seeded into 96-well plates. Add 100. mu.L per well, set 3 replicate wells, and set a blank (equal volume of medium) and a control (cells incubated with equal volume of medium) for overnight incubation. Adding LRP3-S1 solutions of Lycium ruthenicum Murr polysaccharide with different concentrations into 96-well plate, respectively, and mixing with blank group and control group at 37 deg.C and 5% CO₂The cell survival rate was calculated according to the following formula (OD value of experimental group-blank OD value)/(OD value of control group-blank OD value) × 100%. cell survival rate was ×% and as shown in fig. 4, when the administration concentration was 8.71 μ M, the inhibition rates of LRP3-S1 against AsPC-1, PANC-1 and BxPC-3 cell proliferation reached 30.1%, 29.0% and 67.1% (fig. 4A), but under the same conditions, the inhibition rates of polysaccharide LRP3-S1 against HPDE6-C7 and LO2 were only 10% (fig. 4B), and the inhibition rates were low, indicating that the polysaccharide had no significant toxic side effects.

(2) Transwell chamber transmembrane experiments examined the effect of LRP3-S1 on the invasive potential of BxPC-3 pancreatic cancer cells.

Experiments were carried out using a transwell chamber with a pore size of 8 μ M as the inner chamber and a 24-well plate as the outer chamber, Matrigel was solubilized at 4 ℃, diluted to 5mg/mL with serum-free pre-cooled medium, 80 μ L of the diluted gel was added to the transwell chamber, incubated at 37 ℃ for 5h, the gel was washed with serum-free medium to hydrate the basement membrane, LRP3-S1(0, 4.36, 8.71 μ M) and BxPC-3 cells (1.5 × 10) were added to the inner chamber⁵) 100. mu.L of the blood-free medium (BxPC-3), 600. mu.L of a medium containing 15% FBS was added to the outer chamber, the medium was discarded after further culturing for 24 hours, the cells were fixed with 90% by volume of ethanol for 30 minutes, stained with 0.1% crystal violet for 30 minutes, and the upper non-migrated cells were wiped off with a cotton swab and recorded by taking a photograph with an inverted microscope at a magnification of 200 ×. from FIG. 5, it can be observed that a large number of BxPC-3 cells invaded the lower layer of the Transwell chamber, and after 48 hours of administration treatment, the lower layer was removedThe layer cells decreased dramatically with increasing concentration. At 8.71. mu.M, the cell area of the lower layer cells was 43% lower than that at 0. mu.M, indicating that LRP3-S1 significantly inhibited the invasion of BxPC-3 cells.

(3) The immunoblotting experiment detects the expression of LRP3-S1 to FAK, AKT, GSK-3 beta and p38 and its phosphorylated protein.

Taking BxPC-3 cells in logarithmic growth phase, and culturing at 5 × 10⁵The cells are treated by LRP3-S1 of 4.63 mu M and 8.71 mu M lycium ruthenicum polysaccharide (LRP 3-S1) after 24h of culture, the supernatant is discarded, the cells are rinsed by precooled PBS, cell lysate RIPA (protease inhibitor cocktail, purchased from Biyunnan company) is added for 30min of ice lysis before use, the supernatant is collected by centrifugation, the proteins are denatured in a boiling sample device for 15-30min after 5 × of sample buffer solution is added, the proteins are stored at-80 ℃ after cooling, and the protein expression condition of FAK, AKT, GSK-3 β and p38 phosphorylation is detected by immunoblotting.

The results of the experiment are shown in fig. 6, with increasing concentrations (0, 4.63 μ M, 8.71 μ M) administered, the phosphorylation levels of AKT (fig. 6A), GSK-3 β (fig. 6B), FAK (fig. 6C) and P38 (fig. 6C) were all gradually reduced, while the protein expression levels of FAK, AKT and P38 were not significantly changed. The above experiments demonstrate that the FAK/AKT/GSK-3 beta and p38 signaling pathways are involved in the inhibition of proliferation and the reduction of invasive potential of LRP3-S1 regulated human pancreatic cancer cells.

In conclusion, the examples show that Lycium ruthenicum Murr polysaccharide LRP3-S1 can inhibit the proliferation of pancreatic cancer cells and weaken the invasion capacity of pancreatic cancer cells in a dose-dependent manner, has low toxic and side effects, and has no killing effect on normal human cells, and further molecular mechanism research shows that LRP3-S1 can obviously inhibit the expression of FAK, AKT, GSK-3 beta and p38 phosphorylation, so that the activity of pancreatic cancer cells is inhibited. The polysaccharide LRP3-S1 can be a potential carbohydrate drug for treating pancreatic cancer.