CN103265644B - Preparation method of white muscardine silkworm anticancer-activity polysaccharide BBPW-2 - Google Patents
Preparation method of white muscardine silkworm anticancer-activity polysaccharide BBPW-2 Download PDFInfo
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to a preparation method of white muscardine silkworm anticancer-activity polysaccharide BBPW-2, which comprises the following steps: degreasing white muscardine silkworm powder under reflux of acetone-petroleum ether and 80% ethanol to remove glycosides and alkaloids, and extracting with distilled water at 80-100 DEG C to obtain crude polysaccharide; and after removing proteins from the crude polysaccharide by a Sevag process, separating out the neutral polysaccharide component by DEAE (diethylaminoethanol) sepharose ion-exchange chromatography, carrying out propylene dextrangel S-300 chromatography, taking the second eluting peak, concentrating under reduced pressure, carrying out propylene dextrangel S-200 chromatography for further purification, and carrying out freeze-drying to obtain the white muscardine silkworm anticancer-activity polysaccharide BBPW-2. The product prepared by the method provided by the invention has uniform purity, and the molecular weight is 2.0*10<3>Da; and the product has an inhibiting action on growth of human cervical cancer cells Hela and human liver cancer cells HepG2, does not have any adverse effect on growth of normal human embryo kidney cells HEK293 and mouse macrophages RAW264.7, and can be used for developing anticancer products.
Description
Technical field
The present invention relates to the preparation method of a kind of Bombyx Batryticatus active anticancer Polysaccharide B BPW-2.
Background technology
From the natural polymer polysaccharide of high animal and plant cells film and microorganism wall, be one of 4 large base substances forming vital movement, play an important role in the processing and transhipment of the identification of cell, secretion and albumen etc.Find that granulose has anti-tumor activity the end of the fifties in last century, after this extract from antibacterial, fungus, algae, plant and animal and multiplely there is the polysaccharide of active anticancer and be applied to clinical, due to the effective antitumaous effect of natural polysaccharide and lower toxicity, be subject to people and pay attention to more and more widely.
Bombyx Batryticatus be silkworm (
bombyx mori) larva pathogenic infection muscardine (
beauveria bassiana) lethal afterwards dry bacterium polypide, be the Chinese medicine material that China is famous and precious, it is medicinal still very extensive, also throughout the year for export at present.Bombyx Batryticatus property is put down, salty in the mouth, pungent, and have the pharmacological actions such as expelling wind to relieve convulsion, vanishing sputum and dispelling knot, convulsion, anticoagulant, hypnosis, antibacterial, anticancer, blood sugar lowering, blood fat reducing, its pharmacological action widely contains uniqueness active component with it is closely related.But Bombyx Batryticatus medical mechanism particularly anticancer mechanism is not clear, this has encumbered the medicinal level of Bombyx Batryticatus and has improved and medicinal expanded range.Bombyx Batryticatus is rich in polysaccharide, and its active polysaccharide is common in the pathogenic course after cause of disease muscardine (fungus) invades silkworm larva body generation, and separation and purification also prepares Bombyx Batryticatus active anticancer polysaccharide, has larger value for the medicinal exploitation of propelling Bombyx Batryticatus.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of Bombyx Batryticatus active anticancer Polysaccharide B BPW-2, adopt reflux, extract, DEAE-Sepharose Fast Flow ion exchange column to be separated with Sephacryl S-300 chromatographic column, the method of Sephacryl S-200 column chromatography, preparation Bombyx Batryticatus active anticancer Polysaccharide B BPW-2.
In order to achieve the above object, the technical solution used in the present invention is as follows:
1) Bombyx Batryticatus vacuum drying 36 h at-50 DEG C, micronizing 5 min at-15 DEG C, cross 80 ~ 100 mesh sieves, obtain Bombyx Batryticatus powder;
2) Bombyx Batryticatus powder is got, be that 1:3 adds acetone-petroleum ether mixed liquor by W:V, acetone and petroleum ether volume ratio are 1:1, and reflux defat 1 h under 60 ° of C, repeat 2 times, sucking filtration, residue is that 80% ethanol refluxes 1 h under 95 ° of C by 5 times of volume mass concentration, and removing glycoside and alkaloid, repeat 3 times, sucking filtration, residue is dried in 40 ° of C;
3) solid residue is that 1:5 adds distilled water by W:V, after ultrasonic Treatment 40 ~ 50 min, extracts 1 ~ 1.5 h, from Bombyx Batryticatus, extract crude polysaccharides under 80 ~ 100 ° of C;
4) crude polysaccharides is that 1:10 adds distilled water dissolving by W:V, be the chloroform of 4:1 and n-butyl alcohol removing protein 6 times by volume ratio, iodine color reaction and ninhydrin reaction all can't detect protein, steam in 50 ° of C backspins to remove chloroform and n-butyl alcohol, adding mass concentration is 80% ethanol, make ethanol mass concentration reach 80%, fully stir rearmounted refrigerator overnight, precipitation obtains polysaccharide;
5) polysaccharide distilled water dissolves and is mixed with 20 mg/mL solution, and centrifugal 10 min of 8000 rpm, get supernatant, cross 0.45 μm of filter membrane, upper DEAE-Sepharose Fast Flow ion exchange column, with distilled water with 3.0 ~ 3.5 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, collect the eluent having chromogenic reaction, concentrating under reduced pressure, vacuum drying at-50 DEG C, obtain neutral polysaccharide component, be named BBPW;
6) BBPW distilled water dissolves and is mixed with 10 mg/mL solution, cross 0.45 μm of filter membrane, upper Sephacryl S-300 chromatographic column, with distilled water with 1.0 ~ 1.2 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, and collect the eluent of the 2nd eluting peak, concentrating under reduced pressure becomes concentration to be 10 mg/mL;
7) Sephacryl S-200 chromatographic column on concentrated solution, with distilled water with 1.0 ~ 1.2 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, and collects the eluent having chromogenic reaction, vacuum drying at-50 DEG C, obtain homogeneous polysaccharide component, be named BBPW-2.
The beneficial effect that the present invention has is:
Bombyx Batryticatus active anticancer Polysaccharide B BPW-2 prepared by the present invention, purity is homogeneous, and molecular weight is 2.0 × 10
3da, obvious inhibitory action is all shown to the growth of human cervical carcinoma cell Hela and human liver cancer cell HepG2, normal cell embryo nephrocyte HEK293 and mouse macrophage RAW264.7 growth is all had no adverse effects, can be used for the exploitation of antineoplastic product, this, for lifting Bombyx Batryticatus medical value, has positive Social benefit and economic benefit.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of BBPW-2 purity detecting.
Fig. 2 is the basic block diagram of BBPW-2
Fig. 3 is the suppression figure that BBPW-2 grows human cervical carcinoma cell Hela and human liver cancer cell HepG2.
Detailed description of the invention
Before enforcement, first by Bombyx Batryticatus vacuum drying 36 h at-50 DEG C, micronizing 5 min at-15 DEG C, crosses 80 ~ 100 mesh sieves, obtains Bombyx Batryticatus powder;
Get Bombyx Batryticatus powder, be that 1:3 adds acetone-petroleum ether mixed liquor by W:V, acetone and petroleum ether volume ratio are 1:1, and reflux defat 1 h under 60 ° of C, repeat 2 times, sucking filtration, residue is that 80% ethanol refluxes 1 h under 95 ° of C by 5 times of volume mass concentration, and removing glycoside and alkaloid, repeat 3 times, sucking filtration, residue is dried in 40 ° of C.
Embodiment 1:
Got the Bombyx Batryticatus powder of 80 mesh sieves, and defat as stated above, except after glycoside and alkaloid, added distilled water by 1:5 solid-liquid ratio, ultrasonic Treatment 40 min, extracts 1 h, extracts crude polysaccharides under 100 ° of C; Crude polysaccharides with after Sevag method (volume ratio chloroform: n-butyl alcohol=4:1) removing protein, upper DEAE-Sepharose Fast Flow ion exchange column, with distilled water with 3.0 mL/min flow velocity eluting, automatic collector collect, obtain neutral polysaccharide B component BPW; BBPW upper Sephacryl S-300 chromatographic column, with distilled water with 1.0 mL/min flow velocity eluting, automatic collector is collected, and collects the eluent of the 2nd eluting peak, concentrating under reduced pressure; Sephacryl S-200 chromatographic column on concentrated solution, with distilled water with 1.0 mL/min flow velocity eluting, automatic collector is collected, and obtains polysaccharide component BBPW-2.BBPW-1 purity homogeneous (as shown in Figure 1), molecular weight 2.0 × 10
3da, containing mannose, glucose and galactose 3 kinds of monosaccharide, mol ratio is mannose: glucose: galactose=1: 0.74: 0.59, with β-D-1, 2, 6-glucose and β-D-1, 2, 6-mannose is main chain, with α-D-1, 2-galactose and α-D-1, 3-mannose is side chain, the oligosaccharide (as shown in Figure 2) that α-D-MANNOSE and β-D-Glucose form for end group, suppression ratio human cervical carcinoma cell Hela and human liver cancer cell HepG2 grown when concentration 2.5 mg/mL be respectively 55.8% and 39.1%(as shown in Figure 3), normal cell embryo nephrocyte HEK293 and mouse macrophage RAW264.7 growth is had no adverse effects.
Embodiment 2:
Got the Bombyx Batryticatus powder of 100 mesh sieves, and defat as stated above, except after glycoside and alkaloid, added distilled water by 1:5 solid-liquid ratio, ultrasonic Treatment 50 min, extracts 1.5 h, extracts crude polysaccharides under 100 ° of C; Crude polysaccharides with after Sevag method (volume ratio chloroform: n-butyl alcohol=4:1) removing protein, upper DEAE-Sepharose Fast Flow ion exchange column, with distilled water with 3.5 mL/min flow velocity eluting, automatic collector collect, obtain neutral polysaccharide B component BPW; BBPW upper Sephacryl S-300 chromatographic column, with distilled water with 1.2 mL/min flow velocity eluting, automatic collector is collected, and collects the eluent of the 2nd eluting peak, concentrating under reduced pressure; Sephacryl S-200 chromatographic column on concentrated solution, with distilled water with 1.2 mL/min flow velocity eluting, automatic collector is collected, and obtains polysaccharide component BBPW-2.BBPW-1 purity homogeneous (similar to the Fig. 1 in embodiment 1), molecular weight 2.0 × 10
3da, containing mannose, glucose and galactose 3 kinds of monosaccharide, mol ratio is mannose: glucose: galactose=1: 0.74: 0.59, with β-D-1, 2, 6-glucose and β-D-1, 2, 6-mannose is main chain, with α-D-1, 2-galactose and α-D-1, 3-mannose is side chain, α-D-MANNOSE and the oligosaccharide that β-D-Glucose forms for end group (Fig. 2 in embodiment 1 is similar), the suppression ratio grown human cervical carcinoma cell Hela and human liver cancer cell HepG2 when concentration 2.5 mg/mL is respectively 56.3% similar to the Fig. 3 in embodiment 1 with 39.5%(), normal cell embryo nephrocyte HEK293 and mouse macrophage RAW264.7 growth is had no adverse effects.
Embodiment 3:
Got the Bombyx Batryticatus powder of 90 mesh sieves, and defat as stated above, except after glycoside and alkaloid, added distilled water by 1:5 solid-liquid ratio, ultrasonic Treatment 40 min, extracts 1.2 h, extracts crude polysaccharides under 100 ° of C; Crude polysaccharides with after Sevag method (volume ratio chloroform: n-butyl alcohol=4:1) removing protein, upper DEAE-Sepharose Fast Flow ion exchange column, with distilled water with 3.2 mL/min flow velocity eluting, automatic collector collect, obtain neutral polysaccharide B component BPW; BBPW upper Sephacryl S-300 chromatographic column, with distilled water with 1.1 mL/min flow velocity eluting, automatic collector is collected, and collects the eluent of the 2nd eluting peak, concentrating under reduced pressure; Sephacryl S-200 chromatographic column on concentrated solution, with distilled water with 1.1 mL/min flow velocity eluting, automatic collector is collected, and obtains polysaccharide component BBPW-2.BBPW-2 purity homogeneous (similar to the Fig. 1 in embodiment 1), molecular weight 2.0 × 10
3da, containing mannose, glucose and galactose 3 kinds of monosaccharide, mol ratio is mannose: glucose: galactose=1: 0.74: 0.59, with β-D-1, 2, 6-glucose and β-D-1, 2, 6-mannose is main chain, with α-D-1, 2-galactose and α-D-1, 3-mannose is side chain, α-D-MANNOSE and the oligosaccharide that β-D-Glucose forms for end group (Fig. 2 in embodiment 1 is similar), the suppression ratio grown human cervical carcinoma cell Hela and human liver cancer cell HepG2 when concentration 2.5 mg/mL is respectively 56.8% similar to the Fig. 3 in embodiment 1 with 39.9%(), normal cell embryo nephrocyte HEK293 and mouse macrophage RAW264.7 growth is had no adverse effects.
Claims (1)
1. a preparation method of Bombyx Batryticatus active anticancer Polysaccharide B BPW-2, is characterized in that:
1) Bombyx Batryticatus vacuum drying 36 h at-50 DEG C, micronizing 5 min at-15 DEG C, cross 80 ~ 100 mesh sieves, obtain Bombyx Batryticatus powder;
2) Bombyx Batryticatus powder is got, be that 1:3 adds acetone-petroleum ether mixed liquor by W:V, acetone and petroleum ether volume ratio are 1:1, and reflux defat 1 h at 60 DEG C, repeat 2 times, sucking filtration, residue is that 80% ethanol refluxes 1 h at 95 DEG C by 5 times of volume mass concentration, and removing glycoside and alkaloid, repeat 3 times, sucking filtration, residue is in 40 DEG C of oven dry;
3) solid residue is that 1:5 adds distilled water by W:V, after ultrasonic Treatment 40 ~ 50 min, extracts 1 ~ 1.5 h, from Bombyx Batryticatus, extract crude polysaccharides at 80 ~ 100 DEG C;
4) crude polysaccharides is that 1:10 adds distilled water dissolving by W:V, be the chloroform of 4:1 and n-butyl alcohol removing protein 6 times by volume ratio, iodine color reaction and ninhydrin reaction all can't detect protein, steam in 50 DEG C of backspins to remove chloroform and n-butyl alcohol, add ethanol, make ethanol mass concentration reach 80%, fully stir rearmounted refrigerator overnight, precipitation obtains polysaccharide;
5) polysaccharide distilled water dissolves and is mixed with 20 mg/mL solution, and centrifugal 10 min of 8000 rpm, get supernatant, cross 0.45 μm of filter membrane, upper DEAE-Sepharose Fast Flow ion exchange column, with distilled water with 3.0 ~ 3.5 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, collect the eluent having chromogenic reaction, concentrating under reduced pressure, vacuum drying at-50 DEG C, obtain neutral polysaccharide component, be named BBPW;
6) BBPW distilled water dissolves and is mixed with 10 mg/mL solution, cross 0.45 μm of filter membrane, upper Sephacryl S-300 chromatographic column, with distilled water with 1.0 ~ 1.2 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, and collect the eluent of the 2nd eluting peak, concentrating under reduced pressure becomes concentration to be 10 mg/mL;
7) Sephacryl S-200 chromatographic column on concentrated solution, with distilled water with 1.0 ~ 1.2 mL/min flow velocity eluting, automatic collector is collected, phend-sulphuric acid detects, and collects the eluent having chromogenic reaction, vacuum drying at-50 DEG C, obtain homogeneous polysaccharide component, be named BBPW-2;
8) adopt the 1525 type highly effective liquid phase chromatographic systems being furnished with TSK PWXL G4000 glycan analysis post, with differential and UV-detector Parallel testing BBPW-2, present single symmetrical peak, molecular weight calculated by reference standard opisometer is 2.0 × 10
3da, containing mannose, glucose and galactose 3 kinds of monosaccharide, mol ratio is mannose: glucose: galactose=1: 0.74: 0.59, with β-D-1,2,6-glucose and β-D-1,2,6-mannose be main chain, with α-D-1,2-galactose and α-D-1,3-mannose be side chain, the oligosaccharide that forms for end group of α-D-MANNOSE and β-D-Glucose; Mtt assay detects the growth of BBPW-2 to human cervical carcinoma cell Hela and human liver cancer cell HepG2 and all shows obvious inhibitory action, suppression ratio when concentration 2.5 mg/mL is respectively 56.3% and 39.5%, all has no adverse effects to normal cell embryo nephrocyte HEK293 and mouse macrophage RAW264.7 growth.
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| CN101676304A (en) * | 2008-09-16 | 2010-03-24 | 上海医药工业研究院 | Low-molecular-weight glucan, its preparation method and use |
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