CN107602717B - Preparation method and application of lonicera confusa polysaccharide - Google Patents
Preparation method and application of lonicera confusa polysaccharide Download PDFInfo
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- CN107602717B CN107602717B CN201710910545.XA CN201710910545A CN107602717B CN 107602717 B CN107602717 B CN 107602717B CN 201710910545 A CN201710910545 A CN 201710910545A CN 107602717 B CN107602717 B CN 107602717B
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Abstract
The invention relates to a preparation method of lonicera confusa polysaccharide, which comprises the steps of degreasing lonicera confusa with ethanol, drying in the air, extracting with boiling water, heating and concentrating an extracting solution, dialyzing, heating and concentrating an obtained dialyzed internal solution, adding 15 v% trichloroacetic acid under an ice bath condition, uniformly stirring, neutralizing with sodium hydroxide, centrifuging, removing precipitate, taking a supernatant for dialysis, adding 80% ethanol into the obtained dialyzed internal solution, standing overnight, centrifuging, collecting precipitate, drying to obtain crude lonicera confusa polysaccharide, and purifying with a DEAE cellulose anion column and a Sephacryl S-200HR column. The preparation method disclosed by the invention is simple in process and convenient to operate, and the prepared lonicera confusa polysaccharide can obviously inhibit the formation of HMEC-1 cell lumens, particularly has a strong inhibiting effect on the formation of HMEC-1 cell lumens when the polysaccharide concentration is 13.5 mu M, and can be developed into an anti-angiogenesis medicine.
Description
Technical Field
The invention relates to a preparation method of lonicera confusa polysaccharide, and belongs to the technical field of medicines.
Background
Angiogenesis is the process by which new blood vessels grow in the existing capillary network, with new angiogenesis in wound healing, rheumatoid arthritis, tumor growth and a variety of skin diseases. The important role of angiogenesis in the development of various diseases suggests that our inhibition of angiogenesis may be of positive significance in the treatment of these diseases.
From the 70 s in the 20 th century, Folkman proposed that angiogenesis plays an important role in the development and metastasis of tumors, and proposed the idea of treating tumors by inhibiting angiogenesis, thereby limiting the tumors from acquiring oxygen supply and nutrients and starving the tumors.
The polysaccharide is macromolecular substance, widely distributed in various organisms, and has the effects of resisting tumor, virus and aging, regulating immunity, lowering blood pressure, reducing blood lipid, reducing cholesterol, reducing blood sugar and the like. The action mechanism has the characteristics of multiple effects, multiple targets, multiple layers, multiple ways and the like. The polysaccharide has the advantages of low toxic and side effects, wide sources, high safety and the like, and has attracted extensive attention in research.
In the prior art, many polysaccharides, oligosaccharides or derivatives thereof have anti-angiogenic activity, most glycosaminoglycans (such as heparin) and fucoidans occupy the majority, and other polysaccharides have less anti-angiogenic activity. The research on the lonicera confusa polysaccharide is fresh at present. The prior research reports that lonicera confusa polysaccharide (lonicera macranthoides) has antioxidant activity, but the preparation method and the function of lonicera macranthoides in the aspect of lumen formation resistance are not reported.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a preparation method of the lonicera confusa polysaccharide, which has simple process and convenient operation, and the prepared lonicera confusa polysaccharide can inhibit the formation of HMEC-1 cell lumens and can be developed into anti-angiogenesis medicines.
Technical scheme
A preparation method of lonicera confusa polysaccharide comprises the following steps:
(1) polysaccharide extraction: degreasing flos Lonicerae with ethanol, air drying, extracting with boiling water for 2-6 times, each time for 3-5h, mixing extractive solutions, heating and concentrating to obtain concentrated solution, dialyzing, heating and concentrating the obtained dialyzed internal solution, adding trichloroacetic acid under ice bath condition, stirring well to make the concentration of trichloroacetic acid in the dialyzed internal solution 15g/100ml, neutralizing with sodium hydroxide, centrifuging, removing precipitate, collecting supernatant, dialyzing for two days, adding 80% ethanol into the obtained dialyzed internal solution, standing overnight, centrifuging, collecting precipitate, washing with anhydrous ethanol, and drying in a vacuum drying oven to obtain crude polysaccharide of flos Lonicerae with water extraction;
(2) polysaccharide purification: purifying the crude polysaccharide with DEAE cellulose anion column, sequentially purifying with H2Eluting with O, 0.1M NaCl and 0.2M NaCl, and collecting 0.2M NaCl eluateDialyzing, lyophilizing the dialyzed solution to obtain intermediate (primarily purified polysaccharide LF-02), purifying the intermediate with Sephacryl S-200HR column, eluting with 0.2M NaCl, collecting eluate, dialyzing, and lyophilizing the dialyzed solution to obtain flos Lonicerae polysaccharide (LF-02-2).
Further, in the step (1), the ethanol degreasing method comprises the following steps: soaking flos Lonicerae in 95% ethanol for 7 days.
Further, in the step (1), the concentrated solution is filled into a dialysis bag and dialyzed with flowing water for two days. The method of the two times of dialysis in the step (1) is the same.
Further, in the step (1), the rotation speed of the centrifugal operation is 8000-10000rpm, and the time is 10-25 min. The rotating speed and the time of the two times of centrifugation in the step (1) are the same.
Further, in the step (1), the volume ratio of the dialyzed solution to 80% ethanol is 1: 4.
Further, in the step (1), the temperature of the vacuum drying is 45-55 ℃.
Further, in the step (2), a dialysis bag with MW of 3500Da is used for the dialysis, and the dialysis is performed with purified water. The method of the two times of dialysis in the step (2) is the same.
Further, in the step (2), the freeze-drying is performed in a vacuum freeze-dryer.
The polysaccharide prepared by the preparation method has strong inhibition effect on the formation of HMEC-1 cell lumen when the polysaccharide concentration is 13.5 μ M, and can be used for preparing anti-angiogenesis medicines.
Has the advantages that: the high performance gel filtration chromatography (HPGPC) determination shows that the flos Lonicerae polysaccharide has weight average molecular weight of 74.1kDa, and contains rhamnose, galactose, galacturonic acid, and arabinose at a ratio of 10.4:14.9:6.7: 68.0. The reaction is carried out by methyl iodide until sugar is completely methylated, and then the reaction is completely hydrolyzed by acid, reduced, acetylated, extracted and concentrated, and then GC analysis is carried out. Combining methylation results, nuclear magnetism, literature confirms that the sugar is formed by connecting 1,2-Rhap and 1,4-GalpA alternately to form a main chain, and part of rhamnose is branched and substituted at the C4 position. Pharmacological experiments show that the lonicera confusa polysaccharide can obviously inhibit the formation of HMEC-1 cell lumens, particularly has a strong inhibiting effect on the formation of the HMEC-1 cell lumens when the concentration of the polysaccharide is 13.5 mu M, and can be developed into anti-angiogenesis drugs.
Drawings
FIG. 1 shows the polysaccharide of lonicera confusa prepared in example 113C NMR spectrum;
FIG. 2 shows the degradation products of the polysaccharide of lonicera confusa prepared in example 113C NMR spectrum;
FIG. 3 is an electron micrograph of the polysaccharide of lonicera confusa prepared in example 1 inhibiting the formation of HMEC-1 cell lumen.
Detailed Description
The invention is further illustrated by the following figures and examples, without restricting the content of the invention. It should be noted that: in the following examples, the raw material of Lonicera confusa was purchased from Guizhou Zunyi. The membrane used for dialysis is a dialysis membrane for a common-grade experiment. DEAE-cellulose packing was purchased from Whatman and Sephacryl S-200HR packing was purchased from GE Healthcare.
Example 1
(1) Polysaccharide extraction: soaking flos Lonicerae in 95% ethanol for 7 days, removing ethanol, and air drying in ventilated place. Extracting with boiling water for 4 hr each time, and detecting sugar content of the extractive solution by phenol-sulfuric acid method until sugar reaction is not obvious, and extracting for 6 times in total. Combining the extracting solutions, heating and concentrating to obtain a concentrated solution, filling the concentrated solution into a dialysis bag, dialyzing flowing water for 2 days, heating and concentrating the obtained dialyzed internal solution, adding trichloroacetic acid under an ice bath condition, uniformly stirring to ensure that the concentration of the trichloroacetic acid in the dialyzed internal solution is 15g/100ml, neutralizing with sodium hydroxide, centrifuging (the rotating speed is 9000rpm and the time is 20min), removing precipitates, taking supernatant for dialysis for two days, adding 80% ethanol into the obtained dialyzed internal solution, standing, centrifuging (the rotating speed is 9000rpm and the time is 20min), collecting precipitates, washing with absolute ethanol, and finally drying in a vacuum drying box at 45 ℃ to obtain the crude lonicera confusa water extract.
(2) Polysaccharide purification: subjecting the crude polysaccharide to step-by-step extraction with DEAE cellulose anion columnPurification by sequential use of H2Eluting with 0.1M NaCl and 0.2M NaCl, collecting 0.2M NaCl eluate, dialyzing with dialysis bag (MW 3500Da) and pure water, lyophilizing (in vacuum freeze-dryer) the dialyzed solution to obtain polysaccharide LF-02, purifying LF-02 with Sephacryl S-200HR column, eluting with 0.2M NaCl, collecting eluate, dialyzing, and lyophilizing the dialyzed solution to obtain flos Lonicerae polysaccharide (LF-02-2).
The weight average molecular weight of flos Lonicerae polysaccharide (LF-02-2) is 74.1kDa by High Performance Gel Permeation Chromatography (HPGPC). Monosaccharide composition analysis of the product shows that the product contains rhamnose, galactose, galacturonic acid and arabinose in a ratio of 10.4:14.9:6.7: 68.0.
And (3) carrying out methylation and nuclear magnetic analysis on the prepared lonicera confusa polysaccharide:
the results of methylation analysis show that: the lonicera confusa polysaccharide LF-02-2 consists of T-Araf (19.6%), 1,5-Araf (20.3%), T-Galp (13.7%), 1,4,6-Galp (23.7%), 1,2-Rhap (9.9%), 1,2,4-Rhap (12.8%). After the polysaccharide was reduced to LF-02-2-R, it consisted of T-Araf (19.7%), 1,5-Araf (16.8%), T-Galp (8.2%), 1,4,6-Galp (21.3%), 1,2-Rhap (7.2%), 1,2,4-Rhap (10.7%), 1,4-Galp (15.3%). Comparing the connection mode before and after reduction to obtain the connection mode of galacturonic acid which is 1, 4-GalpA. Because arabinose is easily degraded during methylation, the content of arabinose is reduced compared with that of raw sugar.
LF-02-2-1 consisted of T-Galp (40.2%), 1,2-Rhap (12.1%), 1,2,4-Rhap (47.8%). LF-02-2-1 was reduced to consist of T-Galp (25.8%), 1,4,6-Galp (3.2%), 1,2-Rhap (22.3%), 1,2,4-Rhap (32.5%), 1,4-Galp (16.2%). Comparing the connection mode before and after reduction, and verifying the connection mode of the galacturonic acid to be 1,4-GalpA again.
FIG. 1 shows the preparation of polysaccharide (LF-02-2) from flos Lonicerae prepared in example 113C NMR spectrum, as can be seen from FIG. 1, within the anomeric carbon region, signals of δ 108.67, δ 108.41, δ 104.64, δ 105.62, δ 98.77, δ 99.02, δ 99.91 are assigned to T- α -Araf, 1,5- α -Araf, T- β -Galp, 1,4,6- β -Galp, 1,4- α -GalpA, 1,2- α -Rhap,1,2,4- α -Rhap, δ 175.42 are assigned to 1,4- α -GalpA carboxyl carbonPeak(s).
FIG. 2 shows the degradation product (LF-02-2-1) of the polysaccharide from lonicera confusa prepared in example 113C NMR spectrum, it can be seen that, in anomeric carbon region, signals of delta 99.06, delta 99.67, delta 104.67 and delta 98.74 are respectively assigned to 1,2- α -Rhap,1,2,4- α -Rhap, T- β -Galp, 1,4- α -GalpA and delta 105.59 signal is probably 1,4,6- β -Galp C1 and delta 175.92 signal is assigned to signal peak of 1,4- α -GalpA carboxyl carbon.
The influence of the lonicera confusa polysaccharide LF-02-2 prepared in example 1 on the formation of HMEC-1 cell lumens is tested by the following method:
HMEC-1 cell culture
HMEC-1 cells were cultured in MCDB131 medium supplemented with 15% FBS (V/V), 2mM L-glutamine, 10ng/mL EGF and 100U/mL penicillin and 100. mu.g/mL streptomycin. HMEC-1 cells at 5% CO2The culture was carried out at a constant temperature of 37 ℃.
HMEC-1 cell lumen formation assay
Matrigel (50 μ L) was thawed at 4 ℃ before being added to a 4 ℃ pre-cooled 96-well plate and allowed to solidify at 37 ℃ for 30 min. Different concentrations (0. mu.M, 0.5. mu.M, 1.5. mu.M, 4.5. mu.M, 13.5. mu.M) of LF-02-2 polysaccharide and HMEC-1 cells (4X 10. mu.M) were added4One) of MCDB131 broth (100 μ L) was added to the matrigel. Cells were in 5% CO2The cells were incubated overnight at 37 ℃ in an incubator. The recordings were taken with an inverted microscope at 4X magnification.
FIG. 3 is an electron micrograph of the polysaccharide of lonicera confusa prepared in example 1 inhibiting the formation of HMEC-1 cell lumen, and it can be seen from FIG. 3 that the angiogenesis inhibiting effect is more obvious with the increase of the polysaccharide concentration. When the concentration of the polysaccharide is 13.5 mu M, the polysaccharide has obvious inhibition effect on angiogenesis.
Claims (7)
1. The application of the lonicera confusa polysaccharide in preparing the anti-angiogenesis medicine comprises the following steps:
(1) polysaccharide extraction: degreasing flos lonicerae with ethanol, drying, extracting with boiling water for 2-6 times for 3-5 hours each time, combining extracting solutions, heating and concentrating to obtain a concentrated solution, dialyzing, heating and concentrating the obtained dialyzed internal solution, adding trichloroacetic acid under an ice bath condition, uniformly stirring to ensure that the concentration of the trichloroacetic acid in the dialyzed internal solution is 15g/100mL, neutralizing with sodium hydroxide, centrifuging, removing precipitate, collecting supernatant, dialyzing for two days, adding 80% ethanol into the obtained dialyzed internal solution, standing overnight, centrifuging, collecting precipitate, washing with absolute ethanol, and finally drying in a vacuum drying box to obtain crude flos lonicerae polysaccharide extracted with water;
(2) polysaccharide purification: purifying the crude polysaccharide with DEAE cellulose anion column, sequentially purifying with H2Eluting with 0.1M NaCl and 0.2M NaCl, collecting the 0.2M NaCl eluate, dialyzing, lyophilizing the dialyzed solution to obtain intermediate product, purifying the intermediate product with Sephacryl S-200HR column, eluting with 0.2M NaCl, collecting the eluate, dialyzing, and freeze-drying the dialyzed solution to obtain flos Lonicerae polysaccharide.
2. The use of claim 1, wherein in step (1), the ethanol degreasing method comprises: soaking flos Lonicerae in 95% ethanol for 7 days.
3. The use of claim 1, wherein in step (1), the dialysis is carried out by filling the concentrate into a dialysis bag and dialyzing the concentrate with running water for two days.
4. The use as claimed in claim 1, wherein in step (1), the rotation speed of the centrifugation is 8000-10000rpm for 10-25 min.
5. The use of claim 1, wherein in step (1), the volume ratio of the dialysate to 80% ethanol is 1: 4.
6. The use according to claim 1, wherein in step (1), the temperature of the vacuum drying is 45-55 ℃.
7. The use of any one of claims 1 to 6, wherein in step (2), the dialysis is performed using a dialysis bag of MW 3500Da, and the dialysis is performed with purified water.
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| CN101406509A (en) * | 2008-11-28 | 2009-04-15 | 江苏省中国科学院植物研究所 | Lonicera confusa extract and preparation method and application thereof |
| CN105399848A (en) * | 2015-11-20 | 2016-03-16 | 中国科学院上海药物研究所 | Fucosan sulfate as well as preparation method and application of fucosan sulfate |
| CN105796627A (en) * | 2016-03-09 | 2016-07-27 | 贵州省生物研究所 | Extraction method for flos lonicerae active materials |
| CN106243241A (en) * | 2015-06-03 | 2016-12-21 | 上海家化联合股份有限公司 | The preparation of a kind of Rhizoma Polygonati Pectic polysaccharides and application |
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| CN101406509A (en) * | 2008-11-28 | 2009-04-15 | 江苏省中国科学院植物研究所 | Lonicera confusa extract and preparation method and application thereof |
| CN106243241A (en) * | 2015-06-03 | 2016-12-21 | 上海家化联合股份有限公司 | The preparation of a kind of Rhizoma Polygonati Pectic polysaccharides and application |
| CN105399848A (en) * | 2015-11-20 | 2016-03-16 | 中国科学院上海药物研究所 | Fucosan sulfate as well as preparation method and application of fucosan sulfate |
| CN105796627A (en) * | 2016-03-09 | 2016-07-27 | 贵州省生物研究所 | Extraction method for flos lonicerae active materials |
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