CN1443772A - Narcissus seed glycopeptide and its application - Google Patents

Narcissus seed glycopeptide and its application Download PDF

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CN1443772A
CN1443772A CN 02110170 CN02110170A CN1443772A CN 1443772 A CN1443772 A CN 1443772A CN 02110170 CN02110170 CN 02110170 CN 02110170 A CN02110170 A CN 02110170A CN 1443772 A CN1443772 A CN 1443772A
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glycopeptide
group
seed
narcissus seed
narcissus
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CN1168739C (en
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王淦
宋淑玉
宋波
杜晓敏
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ZIBO SHUNDA ENTERPRISE CORP
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ZIBO SHUNDA ENTERPRISE CORP
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Abstract

The narvissus seed glycopeptide is the water soluble active matter extracted from narcissus seed, in which its glycoprotein content is 75-97%, said glycoprotein is a polysaccharide and peptide combined structure, its molecular weight is 2600-4200, in which polysaccharide content (by anhydrous glucose) is not less than 25.0% of total weight of the glycopeptide (by dried product weight), and the piptide content is not less than 50% of total weighto f the glycopeptide (by dried product weight). Said invented narcissus seed glycopeptide can be used for preparing antitumor medicine.

Description

Narcissus seed glycopeptide and uses thereof
Technical field
The active substance narcissus seed glycopeptide and the purposes that the present invention relates to from Semen Narcissi Chinensis, extract.
Background technology
Occurring in nature plant, microorganism, animal all contain polysaccharide, and outside existing divided by free state, the form that also can combine with protein exists.Sugar becomes glycoprotein with protein bound, is the structural material of some histoorgans.The at present common chondroitin sulfate that the achyranthes bidentata glycopeptide that extracts from the root of bidentate achyranthes, the heparin that extracts from the small intestine of pig is arranged, from the hog snout laryngeal cartilage, extract etc.Also put down in writing the Lycium chinense glycopeptide that from wolfberry fruit, extracts in the patent documentation.Because raw material sources difference, the biological activity of extract are also different.Be subjected to the different restriction of raw material sources simultaneously, none is unified the extraction process of standard.
Semen Narcissi Chinensis claims Chrysomyia megacephala (Fabricius), engineering fly maggot again, and extracting chitosan from Semen Narcissi Chinensis has prior art.But do not find from Semen Narcissi Chinensis, to extract the record of glycopeptide at present.
Summary of the invention
Technical problem to be solved by this invention provides a kind of narcissus seed glycopeptide that extracts from Semen Narcissi Chinensis, propose the purposes of narcissus seed glycopeptide simultaneously.
Narcissus seed glycopeptide of the present invention is characterized in that the water-soluble actives for extracting from Semen Narcissi Chinensis, wherein the content of glycoprotein is 75～97%.
The content of glycoprotein is preferably 75～92%, is polysaccharide and peptide integrated structure, and molecular weight is 2600～4200.Wherein polysaccharide content is not less than 25.0% of glycopeptide (by dry product) total amount in dextrose anhydrous, and peptide content is not less than 50% of glycopeptide (by dry product) total amount.Preferred polysaccharide content is 25～35%, and peptide content is 50～70%.
The purposes of narcissus seed glycopeptide is the preparation antitumor drug.Glycoprotein wherein is the effective active component.
The extracting method of Semen Narcissi Chinensis, a kind of method provided by the invention is:
With the protein coagulating in the Semen Narcissi Chinensis, make the boiled water fairy maiden, add water and pull an oar into slurries, carry out enzymolysis then and get enzymolysis solution, the deactivation enzymolysis solution, centrifugation obtains supernatant liquor, then supernatant liquor is handled.
Semen Narcissi Chinensis forms earlier the boiled water fairy maiden, makes protein coagulated method, can be according to the multiple mode of prior art, and the most common is method by boiling.Concrete technology can be preferred: the water of Semen Narcissi Chinensis and 1～2 times of weight boiled under 100 ℃ 5～10 minutes.
Boiled water fairy maiden water is pulled an oar into slurries, and the water of preferred boiled water fairy maiden and 1～3 times of weight is pulled an oar.Slurries are that enzymolysis obtained enzymolysis solution in 3～5 hours under 7～7.5,40～45 ℃, neutral protease at PH, and the deactivation enzymolysis solution makes the enzyme termination reaction then.The deactivation enzymolysis solution carries out centrifugation, obtains supernatant liquor and slag, isolates supernatant liquor.When enzymolysis, neutral protease can adopt suitable proteolytic enzyme, preferred 1398 neutral proteinase.
The supernatant liquor that more than obtains, its treating processes comprise with organic solvent and precipitating, and throw out are dissolved purification, purification liquid drying.Provide following a kind of concrete operation steps:
Supernatant liquor removes water through vacuum concentration and becomes concentrated solution, centrifugal slagging-off, and the clear liquid ethanol sedimentation is isolated precipitation, and precipitation is used water dissolution, the centrifugal again removal of impurities of the aqueous solution that obtains, clear liquid obtains narcissus seed glycopeptide 120～170 ℃ of following spraying dryings.
The concentration of described concentrated solution is 20～25%.Used concentration of ethanol is best 〉=and 95%, sedimentation time was at least 12 hours.Also can adopt other organic solvent, as other alcohols, acetone.Consumption of ethanol can be selected according to the clear liquid before the precipitation: ethanol=1: 4～5 (volume ratio), amount of alcohol is more more not to influence whole technology.
The above-mentioned narcissus seed glycopeptide that obtains is brown or pale brown look amorphous powder, and gas is little, and it is special to distinguish the flavor of, have draw moist.This product is easily molten in water, and is insoluble in ethanol, acetone, chloroform.
Below be relevant experimental result:
One, adopts the molecular weight and the molecular weight distribution of efficient gel chromatography determination narcissus seed glycopeptide
With the glucose of different molecular weight product in contrast, as sample determination, the result is with three batches of narcissus seed glycopeptides:
Lot number 1 number-average molecular weight (Mn) 3251 weight-average molecular weight (Mw) 3403 molecular weight distribution (Mw/Mn) 1.047 2 3267 3456 1.058 3 3161 3329 1.053
See Fig. 1 at the detected glycopeptide color atlas of RI-6A differential refraction detector.
Test-results shows that the molecular weight of narcissus seed glycopeptide (glycoprotein) is 2600～4200, and molecular weight distribution is 1.0～1.1.By the color atlas of Fig. 1, go out the place, peak at sample and have only an independent peak shape and do not have other peak shape appearance, can infer that narcissus seed glycopeptide (glycoprotein) is the one-piece construction that polysaccharide and peptide link together.
Two, the composition of narcissus seed glycopeptide.
1, measure the composition of peptide in the narcissus seed glycopeptide by uv detection method: total amino acid content is that 600～700 μ g/mg, free aminoacid content are 570～660 μ g/mg 39～51 μ g/mg, component peptide class in conjunction with aminoacids content.
2, amino acidanalyser is measured amino acid whose kind in the peptide: narcissus seed glycopeptide is made up of 18 seed amino acids, and the aminoacids content that constitutes peptide is 500～530 μ g/mg.The composition of 18 seed amino acids and ratio are:
Title g/100g title g/100g
Aspartic acid 6.43-6.96 Threonine 1.67-1.92
Serine 1.91-2.21 L-glutamic acid 10.00-10.65
Glycine 2.44-2.55 L-Ala 2.28-2.40
Gelucystine 0.19-0.26 Xie Ansuan 1.50-1.58
Methionine(Met) 0.60-0.66 Isoleucine 0.96-1.01
Leucine 1.43-1.52 tyrosine 1.55-1.65
Phenylalanine 1.73-1.85 Methionin 5.11-5.49
Histidine 1.63-1.74 arginine 3.05-3.18
Proline(Pro) 1.38-1.45 tryptophane 0.45-0.51
3, the composition of polysaccharide in the narcissus seed glycopeptide: the gas chromatographic analysis result, the polysaccharide fraction in the narcissus seed glycopeptide mainly is made up of glucose and Fucose, and the two accounts for 83.4% and 16.6% of total reducing sugar amount in the glycopeptide respectively.
4, measurement of the polysaccharide content in the narcissus seed glycopeptide: according to spectrophotometry, measure with six batch samples, polysaccharide content is between 25～35%.
Owing at present the glycopeptide class is not still had the ideal measuring method, therefore above experiment is used for having proved that narcissus seed glycopeptide belongs to the glycopeptide structure.From the experimental data of sample as can be seen, the molecular weight of narcissus seed glycopeptide (glycoprotein) is about 2600～4200, and wherein polysaccharide content is 25～35%, mainly is made up of glucose and Fucose, and the ratio of the two is 83.4: 16.6; Peptide is made up of 18 seed amino acids, and content is 500～700mg/g, and these known components constitute more than 75% of narcissus seed glycopeptide, are the main effect places of pharmacodynamics.In addition, also has not principal component of the parts such as substituting group that may exist in moisture, sodium ion and the saccharide residue.
Four: the main pharmacodynamics research of narcissus seed glycopeptide
Narcissus seed glycopeptide is the compound of the biologically active that extracts in the five cereals polypide, according to its chemical structure, compositional analysis and test of pesticide effectiveness screening, we from regulate immunity, press down knurl, three aspects such as attenuation have carried out preliminary pharmacodynamic study to it, existing test-results is reported as follows.
＜one 〉, test materials
1. medicine: the narcissus seed glycopeptide Shandong Zibo provides lot number 970323,991028 along reaching main office of group of enterprises;
FT-207 (FT207) Jinan pharmaceutical factory produces, lot number 951207,981003; Endoxan Shenyang
Pharmaceutical factory of pharmaceutical university produces, lot number 961120; Donga, complex prescription glue mucilage Shandong donkey-hide gelatin group produces, lot number
960913。2. animal: Kunming mouse, BALB/C mice, SD kind rat are provided by Shandong Medical University laboratory animal (conformity certification Shandong kinoplaszm 960102,970101,970103), the department of the Chinese Academy of Sciences of laboratory animal section of Beijing Medical University (the moving word of conformity certification number doctor 01-3046 number) respectively.3. knurl strain and target cell: S 180Knurl strain, H 22Knurl strain, the strain of ehrlich carcinoma knurl, by Chinese medical courses in general institute medicine introduced, generation passes; NK sensitive cells Yac-1 strain, basis institute of Shandong medical courses in general institute epi chamber provides; IL-2 relies on the strain ctll cell, and Shandong Quan Cheng biotechnology research provides.4. reagent: 3H-TdR (tritium-labeled deoxythymidine) Chinese Academy of Sciences Institute for Atomic Research (20ci/mM) 961019; RPMI1640 GI810 laboratoyies Cat:NO430-1800EB; FCS (foetal calf serum) institute of biological products, Dalian 96810; MTT (tetrazolium bromide) USA.Sigma Spo120-3; ConA (concanavalin A) USA.Sigma typeVI, activity＜64ug/ml, 11028-71-0; The Chinese Military Medical Science Institute of IL-2,911201 400Iu/Amp that tire; CD 4(L 3T 4Antibody), CD 8(Lyt-2 antibody) test kit, immunity teaching and research room of Beijing Medical University provides; 5. instrument: microplate reader day island proper Tianjin BIO-TEK Intrument Incomp MicroplateEL309; ZT-NB type bull cell harvestor Zhejiang Dong Pu medical apparatus and instruments factory; β-liquid scintillation counter, the EG2101 type, homemade; 96 holes, 24 holes trace Tissue Culture Plate Costar produce; Thermostat container, fluorescent microscope (Olimpas), Germany produces.＜two 〉, the influence of experimental technique and 1.1 pairs of tumor-bearing mice peritoneal macrophages of result's 1. immunity tests phagocytic function is got 20～24g healthy mice, reference literature＜new drug (Western medicine) preclinical study governing principle compilation〉method experimentizes.Calculate macrophage phagocytic percentage and phagocytic index, the results are shown in Table 1.(experimentation slightly)
The influence of table 1. pair tumor-bearing mice peritoneal macrophage phagocytic function (the group dosage number of animals phagocytic rate P value phagocytic index P value of X ± SD)
(g/kg) (only) (%) normal control group-10 44.60 ± 5.52＜0.001 0.80 ± 0.09＜0.001 lotus knurl control group-15 35.29 ± 3.38-0.49 ± 0.04-complex prescription glue mucilage 2 10 60.80 ± 3.91＜sub-glycopeptide group 4 10 41.40 ± 4.17 of 0.001 1.11 ± 0.09＜0.001 narcissus＜sub-glycopeptide group 2 10 58.22 ± 6.78 of 0.001 0.59 ± 0.09＜0.01 narcissus＜sub-glycopeptide group 1 10 49.11 ± 4.99 of 0.001 0.80 ± 0.10＜0.001 narcissus＜0.001 0.78 ± 0.11＜0.001
Phagocytic percentage=[engulfing the phagocytic cell number/200 scavenger cell number (gulp down and do not gulp down) of CRBC] * 100%
CRBC number/200 scavenger cell that phagocytic index=quilt is engulfed
By table 1 result as seen, the peritoneal macrophage phagocytic rate and the phagocytic index of lotus knurl control group all are starkly lower than the normal control group, the peritoneal macrophage phagocytic function that shows lotus knurl group mouse is low, and the These parameters of large, medium and small three the dosage groups of narcissus seed glycopeptide illustrates that all apparently higher than lotus knurl control group (P＜0.001) narcissus seed glycopeptide can obviously improve the peritoneal macrophage phagocytic function of tumor-bearing mice.
1.2 influence to the tardy parasexuality reaction of tumor-bearing mice
Get body weight 19～21g healthy mice, reference literature＜new drug (Western medicine) preclinical study governing principle compilation〉the 135th page of method experimentize, and the results are shown in Table 2.(experimentation slightly)
The influence that table 2. narcissus seed glycopeptide reacts tumor-bearing mice DTH (X ± SD)
Group number of animals dosage DTH P value
(only) be (mg) (g/kg)
Normal control group 10-2.70 ± 0.64＜0.001
Lotus knurl control group 9-1.22 ± 1.03-
The blank group 9-1.11 ± 0.99 of lotus knurl-
Complex prescription glue mucilage 92 3.44 ± 0.83＜0.001
Narcissus seed glycopeptide group 10 4 3.10 ± 1.51＜0.001
Narcissus seed glycopeptide group 10 2 3.56 ± 1.42＜0.001
Narcissus seed glycopeptide 10 1 3.33 ± 1.41＜0.001
Table 2 result shows that the DTH value of lotus knurl control group is starkly lower than the normal control group, and is basic identical with the blank group of unsensitized lotus knurl; The DTH value of three dosage groups of narcissus seed glycopeptide compares there was no significant difference then apparently higher than lotus knurl control group (P＜0.001) with the normal control group; Show that narcissus seed glycopeptide can improve the DTH reaction of tumor-bearing mice, makes its impaired immunologic function recover normal.
1.3 influence to the tumor-bearing mice immune organ weight
Get 18～22g Kunming mouse, aseptic condition extracts the S that went down to posterity 7 days down 180The oncocyte suspension (is equivalent to 1 * 10 approximately with the physiological saline dilution 7Individual oncocyte/ml), behind the right armpit skin of mouse iodine disinfection, subcutaneous injection oncocyte dilution suspension 0.2ml/ only; Be divided at random after 24 hours: three dosage groups of model control group, FT207 positive controls (0.125g/kg) and narcissus seed glycopeptide large, medium and small (4g, 2g, 1g/kg), except that model control group is irritated stomach physiological saline, all the other each treated animal gastric infusions every day once, continuous 9 days (irritate body of stomach long-pending be 0.25ml/10g) after the last administration 2 days, taken off cervical vertebra and put to death mouse, get thymus gland, spleen, weigh, calculate thymus index and spleen index, the results are shown in Table 3.
Table 3. narcissus seed glycopeptide is to the influence of tumor-bearing mice immune organ weight (X ± SD)
Group number of animals dosage thymus index spleen index
(only) (g/kg) (mg/10g) (mg/10g)
Lotus knurl control group 9-22.26 ± 11.13 81.83 ± 22.19
FT207 organizes 10 0.125 15.68 ± 7.89 72..22 ± 34.34
Narcissus seed glycopeptide group 10 4 30.62 ± 12.94 *92.02 ± 18.24
Narcissus seed glycopeptide group 92 21.93 ± 11.21 73.83 ± 11.22
Narcissus seed glycopeptide group 10 1 28.83 ± 8.58 *76.00 ± 13.66
Compare with FT207 *: P＜0.05 *: P＜0.01
Table 3 is the result show, chemotherapeutic FT207 can make the thymus index of tumor-bearing mice obviously reduce (relatively reducing by 29.56% with lotus knurl control group), narcissus seed glycopeptide then can obviously improve the thymus index of tumor-bearing mice, with chemotherapeutic FT207 group significant difference (P＜0.05) is arranged relatively.
1.4 to the active influence of tumor-bearing mice IL-2
The results are shown in Table 4.
Table 4. narcissus seed glycopeptide is to the influence of tumor-bearing mice IL-2 level (X ± SD)
Group dosage number of animals body weight OD value IL-2 activity
(g/kg) begin to finish to change (g)
Normal control group-10 10-1.57 0.0595 ± 0.04 2.281 ± 2.108
Lotus knurl control group-14 14-0.91 0.0535 ± 0.03 2.187 ± 1.656
FT207 organizes 0.125 14 12-3.59 0.0784 ± 0.05 2.655 ± 2.060
Narcissus seed glycopeptide 2 14 12-1.90 0.1942 ± 0.04 8.125 ± 1.749 *#
Narcissus seed glycopeptide 1 14 13-1.71 0.3414 ± 0.05 31.25 ± 2.061 *#
*: compare P＜0.001, # with lotus knurl group: compare P＜0.001 with the FT207 group
Table 4 is the result show, narcissus seed glycopeptide can make the IL-2 level of tumor-bearing mice obviously raise, and to regulating the tumor-bearing mice body's immunity, suppressing tumour has positive effect, relatively has significant difference (P＜0.001) with lotus knurl treated animal.
1.5 influence to tumor-bearing mice NK cytoactive
Table 5. narcissus seed glycopeptide is to the cytotoxic influence of tumor-bearing mice NK (X ± SD)
Group dosage number of animals NK cytotoxicity P value
(g/kg) begin to finish (%) * # ★
Normal control group-10 10 22.74 ± 6.00＜0.05＜0.051-
Lotus knurl control group-14 12 33.48 ± 11.00-＞0.05＜0.05
FT207 organizes 0.125 12 10 32.00 ± 10.60＞0.05-＜0.05
Narcissus seed glycopeptide 4 12 12 61.61 ± 14.00＜0.001＜0.001＜0.001
Semen Narcissi Chinensis sugar 2 12 12 51.46 ± 5.00＜0.001＜0.001＜0.001
Narcissus seed glycopeptide 1 12 12 45.67 ± 11.00＜0.001＜0.001＜0.001
*: compare # with lotus knurl group: compare ★ with the FT207 group: compare with normal group
Cytoactive and induce the active enhancing of IL-2 has been brought into play active effect to suppressing tumor growth.
1.6 influence to tumor-bearing mice T cell subsets
At the quiet pot-life of T lymphocyte, the different glycoprotein molecule of antigenicity that its surface of cell membrane occurs, its effect is also different.Because these molecules are at the expression quite stable of T cell surface, these have different antigenic molecules, and being called t cell surface antigen or surface marker again can be in order to differentiate T cell and functional status.Be present in that mouse is sophisticated and kill and wound (TC)/suppress the Lyt-2 antigen of (TS) T cell surface, be called CD 8Molecule, its energy enhancement antigen identification receptor (TCR) are to exotic antigen and the antigenic combination of self MHC I class, to strengthen identification; Be present in that mouse is sophisticated to be killed and wounded/L on suppressor T cell surface 3T 4Molecule is called CD 4Molecule, its energy enhancement antigen identification receptor (TCR) is to exotic antigen and the antigenic combination of self MHC II class.Detect the CD in the T cell subsets 4, CD 8The expression of surface molecular and proportionlity help to understand the anti-tumor activity of medicine and the relation between the immunologic function, and therefore, we have carried out narcissus seed glycopeptide to pure lines tumor-bearing mice (S 180) the test of T cell subsets influence, existing test-results is reported as follows.The results are shown in Table 6～8.Experimentation slightly.
Table 6. narcissus seed glycopeptide is to S 180Tumor-bearing mice T cell subsets CD 4Influence (X ± SD)
Group dosage number of animals CD 4The P value
(g/kg) begin to finish and lotus knurl group ratio and normal group ratio
Normal control group-10 10 29.40 ± 4.59＜0.001/
Lotus knurl control group-10 10 19.60 ± 5.18/＜0.001
FT207 organizes 0.15 10 8 18.29 ± 2.25＞0.05＜0.001
Narcissus seed glycopeptide 4 10 10 16.60 ± 5.39＞0.05＜0.001
Narcissus seed glycopeptide 2 10 10 18.50 ± 6.97＞0.05＜0.001
Narcissus seed glycopeptide 1 10 10 14.10 ± 3.48＜0.05＜0.001
Table 7. narcissus seed glycopeptide is to S 180Tumor-bearing mice T cell subsets CD 8Influence (X ± SD)
Group dosage number of animals CD 8The P value
(g/kg) begin to finish and lotus knurl group ratio and normal group ratio
Normal control group-10 10 23.20 ± 5.00＜0.001/
Lotus knurl control group-10 10 8.50 ± 2.94/＜0.001
FT207 organizes 0.15 10 8 12.43 ± 3.50＜0.05＜0.001
Narcissus seed glycopeptide 4 10 10 23.00 ± 9.66＜0.001＞0.05
Narcissus seed glycopeptide 2 10 10 29.00 ± 7.27＜0.001＞0.05
Narcissus seed glycopeptide 1 10 10 16.10 ± 6.58＜0.001＜0.05
Table 8. narcissus seed glycopeptide is to S 180Tumor-bearing mice T cell subsets CD 4/ CD 8Influence (X ± SD)
Group dosage number of animals CD 4/ CD 8The P value
(g/kg) begin to finish and lotus knurl group ratio and normal group ratio
Normal control group-10 10 1.32 ± 0.31＜0.001/
Lotus knurl control group-10 10 2.53 ± 0.91/＜0.001
FT207 organizes 0.15 10 8 1.57 ± 0.42＜0.05＞0.05
Narcissus seed glycopeptide 4 10 10 0.84 ± 0.41＜0.001＜0.01
Narcissus seed glycopeptide 2 10 10 0.65 ± 0.21＜0.001＜0.001
Narcissus seed glycopeptide 1 10 10 0.99 ± 0.40＜0.001＞0.05
Table 6～8 results shows, tumor-bearing mice except that low dose of narcissus seed glycopeptide group, the CD of all the other each administration groups 4Value compares there was no significant difference with lotus knurl control group, and each lotus knurl group mouse and normal group more all have significant difference; After inoculated tumour is described, to the helper T cell CD of animal 4Subgroup is influential, but narcissus seed glycopeptide is to CD 4Variable effect little; Except that low dose of narcissus seed glycopeptide group, each administration group mouse CD 8Value and normal group relatively there are no significant difference, and each dosage group of narcissus seed glycopeptide and lotus knurl control group comparison CD 8, significant difference (the P value is all＜0.001) is then arranged, the cytotoxic T cell CD of narcissus seed glycopeptide to tumor animal is described 8Subgroup has a significant effect; Owing to be subjected to the erosion of oncocyte in the tumor-bearing mice body, its normal T cell subsets ratio is subjected to the influence of many-sided factor and overbalance, so CD after the administration 4/ CD 8Variation also very obvious, each dosage group of narcissus seed glycopeptide and lotus knurl control group more all have significant difference (P＜0.001), except that low dose of narcissus seed glycopeptide group, each administration group mouse and normal group more also have significant difference.
Under morbid state, the T cell and the functional status thereof of local infiltration are related to the healing of the pathologic process and the disease of disease.
Above-mentioned test-results shows that narcissus seed glycopeptide has immunity of organism regulating effect preferably; By strengthening tumor-bearing mice peritoneal macrophage phagocytic function, regulate body's immunity, influence activity, the raising NK cytoactive of T cell subsets and inducing the active enhancing of IL-2, brought into play positive effect to suppressing tumor growth.
2. inhibition test
2.1 to S 180The restraining effect of knurl strain (experimentation slightly)
The results are shown in Table 9～11.
Table 9 narcissus seed glycopeptide is to tumor-bearing mice S 180The influence of knurl bulk-growth (one)
Group dosed administration number of animals knurl body weight (g) tumour inhibiting rate
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 24 21 1.25 ± 0.60-
FT207 group 0.125 * 9 P.0 10 9 0.49 ± 0.24 60.81 * *
Narcissus seed glycopeptide 4 * 9 P.0 10 9 0.70 ± 0.45 44.00 *
Narcissus seed glycopeptide 2 * 9 P.0 10 9 0.80 ± 0.24 36.00 *
Narcissus seed glycopeptide 1 * 9 P.0 10 9 1.44 ± 0.52-15.20
Compare with lotus knurl group * *P＜0.001 *P＜0.05
Table 10 narcissus seed glycopeptide is to tumor-bearing mice S 180The influence of knurl bulk-growth (two)
Group dosed administration number of animals knurl body weight tumour inhibiting rate
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 25 23 1.28 ± 0.88-
FT207 group 0.1259 P.0 10 9 0.40 ± 0.25 68.75 *
Narcissus seed glycopeptide 4 * 9 P.0 10 9 0.53 ± 0.34 58.59 *
Narcissus seed glycopeptide 2 * 9 P.0 10 9 0.66 ± 0.22 48.44 *
Narcissus seed glycopeptide 1 * 9 P.0 10 8 0.82 ± 0.50 35.94
Compare with lotus knurl group *P＜0.01 *P＜0.05
Table 11 narcissus seed glycopeptide is to tumor-bearing mice S 180The influence of knurl bulk-growth (three)
Group dosed administration number of animals knurl body weight tumour inhibiting rate
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 25 23 1.91 ± 0.79-
FT207 group 0.125 * 9 P.0 10 9 0.77 ± 0.33 59.69 * *
Narcissus seed glycopeptide 4 * 9 P.0 10 8 0.83 ± 0.32 56.54 * *
Narcissus seed glycopeptide 2 * 9 P.0 10 8 1.21 ± 0.57 36.65 *
Narcissus seed glycopeptide 1 * 9 P.0 10 9 1.48 ± 0.83 22.51
Compare with lotus knurl group * *P＜.0.001 *P＜0.05
Table 9～11 results show: by three times to tumor-bearing mice S 180The test of knurl bulk-growth influence, the average tumour inhibiting rate of large, medium and small three the dosage groups of narcissus glycopeptide is respectively: 53.04%, 40.36%, 14.42%; Compare with lotus knurl control group, the big or middle dosage group of narcissus seed glycopeptide is to S 180The knurl bulk-growth all has obvious suppression effect (P＜0.01-0.001); The result shows that also narcissus seed glycopeptide is to mouse S 180There is certain dose-effect relationship in the restraining effect of knurl bulk-growth.
2.2 to H 22The restraining effect of knurl strain
The results are shown in Table 12～14.
Table 12 narcissus seed glycopeptide is to tumor-bearing mice H 22The influence of knurl bulk-growth (one)
Group dosed administration number of animals knurl body weight (g) tumour inhibiting rate
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 25 22 1.87 ± 0.21-
FT207 group 0.125 * 9 P.0 10 9 0.41 ± 0.19 79.68 * *
Narcissus seed glycopeptide group 4 * 9 P.0 10 9 1.01 ± 0.28 45.99 * *
Narcissus seed glycopeptide group 2 * 9 P.0 10 9 1.48 ± 0.20 20.86 *
Narcissus seed glycopeptide group 1 * 9 P.0 10 9 1.82 ± 0.24 2.67
Compare with lotus knurl group * *P＜0.001 *P＜0.001
Table 13 narcissus seed glycopeptide is to tumor-bearing mice H 22The influence of knurl bulk-growth (two)
Heavy (g) tumour inhibiting rate of group dosed administration number of animals knurl
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 25 24 1.98 ± 0.37-
FT207 group 0.125 * 9 P.0 10 10 0.31 ± 0.11 84.38 * *
Narcissus seed glycopeptide group 4 * 9 P.0 10 10 0.96 ± 0.33 51.52 * *
Narcissus seed glycopeptide group 2 * 9 P.0 10 10 1.03 ± 0.53 47.98 * *
Narcissus seed glycopeptide group 1 * 9 P.0 10 9 1.27 ± 0.73 35.86 *
Compare with lotus knurl group * *P＜0.001 *P＜0.01
Table 14 narcissus seed glycopeptide is to tumor-bearing mice H 22The influence of knurl bulk-growth (three)
Heavy (g) tumour inhibiting rate of group dosed administration number of animals knurl
(g/kg) approach begins to finish (X ± SD) (%)
Model control group 25ml * 9 P.0 25 24 2.15 ± 0.43-
FT207 group 0.125 * 9 P.0 10 10 1.03 ± 0.45 52.09 * *
Narcissus seed glycopeptide group 4 * 9 P.0 10 9 1.39 ± 0.48 35.35 * *
Narcissus seed glycopeptide group 2 * 9 P.0 10 9 1.34 ± 0.56 37.67 * *
Narcissus seed glycopeptide group 1 * 9 P.0 10 9 1.44 ± 0.44 33.02 * *
Compare with lotus knurl group * *P＜0.001
Table 12～14 results show: by three times to tumor-bearing mice H 22The test of knurl bulk-growth influence, the average tumour inhibiting rate of large, medium and small three the dosage groups of narcissus seed glycopeptide is respectively: 44.29%, 35.50%, 23.85%, compare with lotus knurl control group, big or middle dosage group is to mouse H 22The growth of solid tumor all has obvious suppression effect (P＜0.01～0.001).Experimental result also shows: narcissus seed glycopeptide is to mouse H 22There is certain dose-effect relationship in the restraining effect of knurl growth.
2.3 influence to the ehrlich carcinoma survival time of mice
The results are shown in Table 15～17.
Table 15 narcissus seed glycopeptide is to the influence () of ehrlich carcinoma survival time of mice
Group dosage number of animals body weight survival time T/C P value
(g/kg) (only) changes (g) day (X ± SD)
Physiological saline group 25ml * 10 25 4.18 16.04 ± 2.86--
FT207 group 0.125 * 10 10 6.20 20.70 ± 5.33 1.29＜0.01
Narcissus seed glycopeptide group 4 * 10 10 11.20 20.10 ± 5.06 1.25＜0.01
Narcissus seed glycopeptide group 2 * 10 10 8.40 19.20 ± 5.39 1.19＜0.05
Narcissus seed glycopeptide group 1 * 10 10 10.09 17.60 ± 2.12 1.10＞0.05
Table 16 narcissus seed glycopeptide is to the influence (two) of ehrlich carcinoma survival time of mice
Group dosage number of animals body weight (g) survival time T/C P value
(g/kg) (only) changes day (X ± SD)
Physiological saline group 25ml * 10 26 3.81 14.46 ± 0.90--
FT207 group 0.125 * 10 10 4.10 15.40 ± 2.50 1.06＞0.05
Narcissus seed glycopeptide group 4 * 10 10 7.50 15.70 ± 0.67 1.09＜0.001
Narcissus seed glycopeptide group 2 * 10 10 5.90 15.50 ± 0.85 1.07＜0.01
Narcissus seed glycopeptide group 1 * 10 10 2.40 15.20 ± 1.14 1.05＜0.05
Table 17 narcissus seed glycopeptide is to the influence (three) of ehrlich carcinoma survival time of mice
Group dosage number of animals body weight survival time T/C P value
(g/kg) (only) changes (g) day (X ± SD)
Physiological saline group 25ml * 10 27 7.62 14.70 ± 0.72
FT207 group 0.125 * 10 8 4.10 16.88 ± 2.30 1.15＜0.001
Narcissus seed glycopeptide group 4 * 10 10 8.80 16.50 ± 1.08 1.12＜0.001
Narcissus seed glycopeptide group 2 * 10 10 3.20 15.50 ± 2.22 1.05＞0.05
Narcissus seed glycopeptide group 1 * 10 10 7.70 16.20 ± 4.66 1.10＞0.05
Table 15～17 results show: the heavy dose of group of narcissus seed glycopeptide has certain effect to prolonging the ehrlich carcinoma mouse storaging current, and survival time and lotus knurl group relatively have significant difference (P＜0.01～0.001); Middle dosage group also has certain prolongation effect lifetime, and survival time and lotus knurl group more also have significant difference (P＜0.01～0.001); The effect of small dose group is not obvious.Three times test-results is carried out analysis-by-synthesis, and the big or middle dosage group of narcissus seed glycopeptide and lotus knurl group relatively have the effect of lifetime of certain prolongation tumor-bearing mice.
2.4 use mouse H with cyclophosphamide combined 22The restraining effect of knurl
The results are shown in Table 18.
Table 18 narcissus seed glycopeptide and endoxan share H 22The influence of knurl strain growth (g, X ± SD)
The heavy tumour inhibiting rate P of the average knurl of group dosage number of animals value
(g/kg) begin to finish (%)
Physiological saline group 25ml * 10 20 18 1.29 ± 0.47--
Endoxan group 0.025 * 10 10 10 0.74 ± 0.43 42.6＜0.01
Narcissus seed glycopeptide group 2 * 10 10 9 0.75 ± 0.48 41.9＜0.01
Narcissus seed glycopeptide+2 * 10+
Endoxan group 0.025 * 10 10 10 0.64 ± 0.18 50.39＜0.001
Table 18 is the result show, independent medication group: the tumour inhibiting rate of endoxan (25ml/kg), narcissus seed glycopeptide (2g/kg) is respectively 42.6% and 41.9%, the tumour inhibiting rate of drug combination group (endoxan 25ml/kg+ narcissus seed glycopeptide 2g/kg) then is 50.39%, compare with independent medication group, tumour inhibiting rate all increases; From above-mentioned test-results as can be seen: narcissus seed glycopeptide share with low dose of chemotherapeutic, not only can alleviate the toxic side effect of chemotherapeutic, also can improve tumour inhibiting rate simultaneously, has shown the advantage of drug combination.
3. attenuation test
Assay sees Table 20～22.Experimentation slightly
Table 19 endoxan rat model liver, kidney biochemical indicator detected value (project animal dosage ALT (u/L) AST (u/L) CR (umol/L) BUN (mmol/L) of X ± S)
(number), (g./kg) Normal group 7/36.29 ± 4.96 72.57 ± 11.44 32.79 ± 5.62 7.60 ± 2.13 model control group 8/47.63 ± 13.73 79.50 ± 13.81 28.52 ± 19.75 8.63 ± 1.38 narcissus seed glycopeptides 74 34.00 ± 13.56 77.00 ± 12.58 26.36 ± 8.15 7.92 ± 0.73 narcissus seed glycopeptides 72 40.56 ± 11.98 67.33 ± 8.19 33.79 ± 20.14 7.53 ± 1.02 narcissus seed glycopeptide 71 43.86 ± 15.67 88.14 ± 17.57 27.11 ± 13.17 8.18 ± 0.99
Compare P＞0.05 with the normal control group
(X ± SD) group dosage number of animals bone marrow nucleated cell is counted the P value to table 20 narcissus seed glycopeptide to the influence of endoxan rat marrow karyocyte number
(g/kg) (only) (10 9/ L) compare normal control group-7 6.88 ± 2.16＜0.05 endoxan group-8 3.45 ± 1.23-narcissus seed glycopeptide, 47 5.94 ± 2.46＜0.05 narcissus seed glycopeptide, 27 5.81 ± 2.93＜0.05 narcissus seed glycopeptide 17 3.65 ± 0.76＞0.05 with the endoxan group
Table 20 is the result show, behind the rat injection endoxan, bone marrow nucleated cell quantity significantly descended; After one week of administration, endoxan model group detected value and normal control group relatively reduce by 49.85%: and the big or middle dosage group of narcissus seed glycopeptide detected value is apparently higher than model group, compare no significant difference with the normal control group, more then have significant difference (P＜0.05) with model group; The narcissus seed glycopeptide that big or middle dosage is described has provide protection to bone marrow nucleated cell, and the hemopoietic function of bone marrow that chemotherapeutic is caused suppresses to have certain attenuation effect.
(the group animal dosage detected value of X ± SD) of hematological indices detected value before the table 21 endoxan rat model test
(number) be WBC (10 (g/kg) 9/ L) Hb (g/L) RBC (10 12/ L) PLC (10 9/ L) normal control group 10/18.56 ± 3.58 137.60 ± 8.65 8.14 ± 0.55 568.60 ± 82.01 model control groups 12/21.91 ± sub-glycopeptide 10 4 25.02 of 7.37 128.20 ± 10.37 7.48 ± 0.95 466.90 ± 82.42 narcissuses ± sub-glycopeptide 10 2 20.83 of 8.55 131.10 ± 10.04 7.03 ± 1.16 503.70 ± 81.91 narcissuses ± sub-glycopeptide 10 1 23.96 of 7.70 129.36 ± 11.38 8.26 ± 1.65 458.91 ± 71.89 narcissuses ± 4.13 134.70 ± 10.04 7.68 ± 0.68 474.40 ± 135.83
Compare P＞0.05 with the normal control group
(the group animal dosage detected value of X ± SD) of hematological indices detected value behind the table 22 endoxan rat test
(number) be WBC (10 (g/kg) 9/ L) Hb (g/L) RBC (10 12/ L) PLC (10 9/ L) normal control group 7/27.29 ± 5.31 133.70 ± 5.40 8.40 ± 3.33 608.20 ± 47.53 model control group 8/4.23 ± 2.02 121.20 ± 8.02 7.00 ± 0.72 299.60 ± 52.14 narcissus seed glycopeptide 74 4.11 ± 2.32 121.80 ± 7.07 7.12 ± 0.78 619.60 ± 123.39 *Narcissus seed glycopeptide 72 4.33 ± 1.37 125.20 ± 6.68 7.10 ± 0.51 523.00 ± 98.72 *Narcissus seed glycopeptide 71 4.98 ± 2.61 119.50 ± 6.34 7.33 ± 0.55 527.80 ± 104.19 *
Compare with the endoxan model group *: P＜0.01
Table 21 is the result show, behind the rat injection endoxan white corpuscle, thrombocyte had a significant effect, and its numerical value is significantly descended; After one week of administration, each organizes the thrombocyte observed value and the normal control group compares, and the drop-out value of endoxan group and narcissus seed glycopeptide small dose group is the most obvious; And the thrombocyte observed value of the big or middle dosage group of narcissus seed glycopeptide reduction amplitude is less, and its detected value relatively has significant difference (P＜0.05) apparently higher than model control group with model group; The thrombopenia that the big or middle dosage of prompting narcissus seed glycopeptide causes endoxan has antagonistic action preferably, can alleviate the toxic side effect of chemotherapeutic to a certain extent.
＜three 〉, brief summary
Above-mentioned every experimental result shows, from giving up the biologically active substance-narcissus seed glycopeptide that extracts in the ripe larva body of fly, have tangible adjusting body's immunity, suppress tumor growth, share the synergy that alleviates its toxic side effect and strengthen tumor killing effect with chemotherapeutic:
1. narcissus seed glycopeptide can improve body's immunological function: obviously improve tumor-bearing mice peritoneal macrophage phagocytic function and thymus index, recover the low DTH reaction of tumor animal, by influencing the activity of T cell quantity, enhancing NK cytotoxicity and IL-2, reach and regulate body's immunity and the dual function that presses down knurl.
2. narcissus seed glycopeptide can suppress S 180Knurl strain and H 22The growth of knurl strain, the tumour inhibiting rate of three dosage groups is respectively 53.04%, 40.36%, 14.42%% and 44.29%, 35.50%, 23.85%, to stable prolongation effect is arranged lifetime of ehrlich carcinoma mouse; Share with the low dose of endoxan that is lower than therapeutic dose simultaneously, can produce the synergy that presses down knurl, improve tumour inhibiting rate.
3. narcissus seed glycopeptide has provide protection to rat platelet minimizing and the minimizing of bone marrow nucleated cell quantity that endoxan causes, and prompting narcissus seed glycopeptide and chemotherapeutic combined utilization have certain attenuation.
In sum, by regulate immunity, press down knurl, the pharmacodynamics test of three aspects of attenuation studies show that: narcissus seed glycopeptide have enhance immunity function preferably, antitumor, alleviate the chemotherapeutic toxic side effect and with the collaborative tumor-inhibiting action of chemotherapeutic combined utilization.
Description of drawings
Fig. 1 is a narcissus seed glycopeptide efficient gel color atlas.
Embodiment
Embodiment one:
The water of 10kg Semen Narcissi Chinensis and 1.5 times of weight boiled under 100 ℃ 5 minutes, obtain the boiled water fairy maiden, mix with the water of 2 times of weight and pull an oar into slurries, slurries are 7 at PH, temperature is that enzymolysis obtained enzymolysis solution in 3 hours under 45 ℃, 1398 neutral proteinase, and 90 ℃ of following deactivations obtain the deactivation enzymolysis solution.The deactivation enzymolysis solution carries out centrifugation, removes slag, obtains supernatant liquor.Supernatant liquor becomes concentrated solution at 60 ℃ of following vacuum concentration, and concentration is 20～25%, centrifugal slagging-off, and clear liquid is with 95% ethanol sedimentation 12 hours, clear liquid: ethanol=1: 5 (volume ratio).Quiescent settling is isolated precipitation, and precipitation is used water dissolution, the centrifugal again removal of impurities of the aqueous solution that obtains, and clear liquid obtains narcissus seed glycopeptide 250 grams 120 ℃ of following spraying dryings.
Narcissus seed glycopeptide is brown or pale brown look amorphous powder, and gas is little, and it is special to distinguish the flavor of, have draw moist.Easily molten in water, insoluble in ethanol, acetone, chloroform.
Fastness to alkali: PH5.5～7.5
Weight loss on drying:＜6.0%
Ignition residue:＜13%
Heavy metal:＜20ppm
Arsenic salt:＜2ppm
Glycoprotein content is 90%, and wherein polysaccharide is 28%, peptide 62%.
Embodiment two:
The water of 10kg Semen Narcissi Chinensis and 1.5 times of weight boiled under 100 ℃ 5 minutes, obtain the boiled water fairy maiden, mix with the water of 2 times of weight and pull an oar into slurries, slurries are 7.5 at PH, temperature is that enzymolysis obtained enzymolysis solution in 4 hours under 40 ℃, 1398 neutral proteinase, and 90 ℃ of following deactivations obtain the deactivation enzymolysis solution.The deactivation enzymolysis solution carries out centrifugation, removes slag, obtains supernatant liquor.Supernatant liquor becomes concentrated solution at 60 ℃ of following vacuum concentration, and concentration is 20～25%, centrifugal slagging-off, and clear liquid is with 95% ethanol sedimentation 14 hours, clear liquid: ethanol=1: 4 (volume ratio).Quiescent settling is isolated precipitation, and precipitation is used water dissolution, the centrifugal again removal of impurities of the aqueous solution that obtains, and clear liquid obtains narcissus seed glycopeptide 245 grams 120 ℃ of following spraying dryings.
Narcissus seed glycopeptide is brown or pale brown look amorphous powder, and gas is little, and it is special to distinguish the flavor of, have draw moist.Easily molten in water, insoluble in ethanol, acetone, chloroform.
Fastness to alkali: PH5.5～7.5
Weight loss on drying:＜6.0%
Ignition residue:＜13%
Heavy metal:＜20ppm
Arsenic salt:＜2ppm
Glycoprotein content is 80%, and wherein polysaccharide is 26%, peptide 54%.

Claims (6)

1, narcissus seed glycopeptide is characterized in that the water-soluble actives for extracting from Semen Narcissi Chinensis, wherein the content of glycoprotein is 75～97%.
2, narcissus seed glycopeptide according to claim 1, the content that it is characterized in that glycoprotein is 75～92%.
3, narcissus seed glycopeptide according to claim 1 and 2 is characterized in that described glycoprotein is polysaccharide and peptide integrated structure, and molecular weight is 2600～4200.
4, narcissus seed glycopeptide according to claim 3 is characterized in that described polysaccharide content is not less than 25.0% of glycopeptide (by dry product) total amount in dextrose anhydrous, and peptide content is not less than 50% of glycopeptide (by dry product) total amount.
5, narcissus seed glycopeptide according to claim 4 is characterized in that described polysaccharide content is 25～35%, and peptide content is 50～70%.
6, the purposes of narcissus seed glycopeptide is the preparation antitumor drug.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974095A (en) * 2010-10-14 2011-02-16 南京林业大学 Method for extracting and separating Chinese narcissus polysaccharides
CN103271947A (en) * 2013-06-06 2013-09-04 四川科伦新光生物科技开发有限公司 Novel antitumor application of maggot

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974095A (en) * 2010-10-14 2011-02-16 南京林业大学 Method for extracting and separating Chinese narcissus polysaccharides
CN101974095B (en) * 2010-10-14 2012-09-05 南京林业大学 Method for extracting and separating Chinese narcissus polysaccharides
CN103271947A (en) * 2013-06-06 2013-09-04 四川科伦新光生物科技开发有限公司 Novel antitumor application of maggot

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