CN101486755A - House lizard, preparation and medical use - Google Patents
House lizard, preparation and medical use Download PDFInfo
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Abstract
The invention relates to a glycopeptide sauropus, a preparation method and the application in medicine thereof. The weight-average molecular weight of the glycopeptide sauropus is larger than 2,000,000 Da; the composition sugar contains rhamnose, fucose, mannose, glucose, semi-galactose, glucosamine, and uronic acid; the combination type protein contains 16 types of amino acids with the mass percentage content of 16.33%; the mass percentage content of sulfate base is 14.7%; the invention further studies and obtains scientific validation on the basis of the application of sauropus polysaccharide; compared with the sauropus polysaccharide, the glycopeptide sauropus has higher efficacy on malignant tumors, has the function of inhibiting the hepatoma cells from secretion of IL-8, which the sauropus polysaccharide does not have, and can be used for the treatment of malignant tumors separately and also used as an adjuvant therapy for tumor surgery, radiotherapy, chemotherapy and interventional treatment, as well as the prevention of virus infectious diseases such as AIDS and the like; and the invention can also be used as a new immunomodulator for the treatment of diseases such as tumors, chronic infection, and the like.
Description
Technical field
The present invention relates to have in a kind of animal Chinese medicinal materials activeconstituents and preparation method and its medical applications of very high pharmaceutical use, particularly a kind of house lizard glycopeptide and preparation method and its medical applications.
Background technology
The traditional Chinese medical science is the quintessence of Chinese culture of China, and Chinese medicinal materials is the material treasure-house of traditional Chinese medical theory, and Chinese medicinal materials is divided into botanical herbs material, animal Chinese medicinal materials and mineral Chinese medicine material; Theory, clinical and the modern study of the tradition traditional Chinese medical science shows that all it is a big characteristic of the traditional Chinese medical science that animal drug is treated malignant tumour, as in Chinese blister beetle, extract Cantharidin, cantharidate is applied to treat malignant tumour and obtains certain effect, and has certain antiviral effect.In general, most animals Chinese medicine all has certain toxicity to human body, has limited the dosage of this class medicine when treatment, makes result of treatment be subjected to bigger influence.How to reduce its detrimentally affect to human body when keeping medical active, be the focus that pharmacy men are studied.Simultaneously, the activeconstituents of clear and definite medicine with and the mechanism of action be the top priority of crude drug exploitation.In being arranged, the animal Chinese medicine of antitumous effect extracts that the active substance of antitumous effect is arranged then is the pharmacy man of China and even countries in the world, the focus of work of research and development institution.
House lizard is traditional Chinese medicinal materials, for the Gekkonidae animal does not have web gecko Gekko swinhonis G ū enther or other several geckoes [many warts gecko Gekko japonicus (Dum é ril ﹠amp; Bibron); No wart gecko Gekko subpalmatus G ü enther; Webbed toe gecko Gekko chinensis Gray] all, be the traditional salty-cold soft hard medicine of the traditional Chinese medical science.The traditional Chinese medical science is used house lizard treatment scrofula cancerous swelling (" blue or green capsule is assorted usurps ", " must join book on Chinese herbal medicine ", " Sichuan Chinese medicinal herbal ") for a long time, and the tame house lizard treatment malignant tumour of using of domestic more doctor has also obtained better curative effect.In vitro study finds that the house lizard aqueous solution can suppress the breathing of human liver cancer cell, and clear and definite house lizard contains the toxic substance similar to the hornet poison and organizes amine substance, fatty oil, multiple amino acids, trace element and VITAMIN, but mentioned component all is not enough to explain the antitumor action of house lizard, and we have antitumor, antiviral and immunoregulatory activeconstituents by the gecko polysaccharide of discovering previously; Referring to the applicant at first to file: ZL 2,005 1 0065309.X, about the patent of " gecko polysaccharide and preparation method and its medical applications ".
Polysaccharide or glycopeptide are class natural macromolecular materials, are one of base substances that constitutes life.Studies show that in a large number polysaccharide or glycopeptide have antiviral, antitumor and immunoregulation effect.Generally speaking, polysaccharide or glycopeptide are good immunomodulators.Polysaccharide or glycopeptide can activated macrophages, promote lymphocyte mitotic division, strengthen NK cell (natural killer cell) and LAK cell (killer cell of lymphokineactivation) activity, strengthen T
DCell (delayed hypersensitivity T cell) function induces interleukin-(especially IL-1, IL-2), IFN (Interferon, rabbit), TNF cytokines such as (tumour necrosis factors), and complement activation strengthens erythrocyte immune, regulates nerve-endocrine-immunological network.Studies show that recently: the polysaccharide of sulfur-bearing acid group or glycopeptide have the effect of anti-HIV, thereby sulfated polysaccharide or glycopeptide have become the focus of domestic and international research.
The IL-8 metastases has very confidential relation, and tumour cell can be secreted IL-8, promote its invasion and attack and shift, and the recurrence of tumour is the major cause for the treatment of malignant tumor failure with shifting, and also is to cause the final dead major reason of tumour patient.Therefore, suppressing tumor cell secretion promotes invasion and attack and the cytokine such as the IL-8 that shift to have important scientific meaning and wide application prospect.
Therefore, a kind of diseases such as human malignant tumor and viral infection of more effectively treating are provided, strengthening house lizard glycopeptide and preparation method and its medical applications of body immunity, bring benefit to the mankind, will be that this technical field scientific research personnel is badly in need of one of new problem of furtheing investigate exploitation.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, house lizard glycopeptide and preparation method and its medical applications of providing a kind of effect highly significant promptly to be derived from the house lizard of animal Chinese medicine.This house lizard glycopeptide has very strong restraining effect to malignant tumour, and simultaneously because this house lizard glycopeptide contains sulfate radical virus diseases such as HIV are had tangible antivirus action, the house lizard glycopeptide also is one to have the biopharmaceuticals of extensive immunoregulation effect; Another goal of the invention of the present invention provides the application at field of medicaments of the preparation method of this house lizard glycopeptide and house lizard glycopeptide.
Implementation of the present invention is as follows for achieving the above object:
The house lizard glycopeptide is characterized in that: weight-average molecular weight is greater than 2,000,000Da; Specific rotatory power is-60 °; The quality percentage composition of carbon is 23.39%, and the quality percentage composition of protium is 4.925%, and the quality percentage composition of nitrogen element is 6.51%; The sugar moieties of its composition contains rhamnosyl, Fucose, seminose, glucose, semi-lactosi, glucosamine, mol ratio is: rhamnosyl: Fucose: seminose: glucose: semi-lactosi: glucosamine=1.334:3.363:3.375:6.983:10.480:0.019; The mode of connection of monose is: rhamnosyl is with 1,3-, 1,3,4-and 1,2, the form that 3-connects exists, and Fucose is with 1-, 1,4-and 1, the form that 2-connects exists, and seminose is with 1, the form that 4-connects exists, and glucose is with 1-, and 1,3, the form that 4-connects exists, and semi-lactosi is with 1,4-, 1,4, the form that 6-connects exists, and glucosamine is with 1, and the form that 4-connects exists; The quality percentage composition of uronic acid is 3.08%; Its mating type protein portion contains 16 seed amino acids, and the quality percentage composition is 16.33%; The type of cardohydrata-peptide linkage contains O-and connects; The quality percentage composition of sulfate is 14.7%.
The preparation method of house lizard glycopeptide, it is characterized in that: with the house lizard drying, broken back goes out lipid-soluble substance with the organic solvent extracting, with the dregs of a decoction after the degreasing with by 1-10 times of hot water of house lizard quality under 30-100 ℃ of condition lixiviate 2-12 hour, the water extract filters back gradation adding ethanol to 15%-80% concentration (V/V) and precipitates, with resolution of precipitate in water, 1000-10000r/min centrifuging and taking supernatant, conventional sevage method is removed foreigh protein removing, dialysis, be dried to powder, be dissolved in again powder quality 30-180 doubly contain 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH6.5, add the papoid of powder quality 0.2%-2% behind the papoid and the 0.5-2 toluene doubly that add powder quality 0.5%-4%, 50 ℃ of-80 ℃ of water-bath 4h-48h, continue water-bath 4h-24h; Hydrolyzed solution concentrates the back with ethanol sedimentation, and collecting precipitation is used dehydrated alcohol successively, and washing with acetone is water-soluble again, filters, and conventional sevage method is removed foreigh protein removing, concentrate, and dialysis, drying gets the house lizard glycopeptide.
Above-mentioned house lizard glycopeptide is made the injection type that meets the pharmaceutics requirement.
Above-mentioned house lizard glycopeptide is made the oral dosage form that meets the pharmaceutics requirement.
The application of above-mentioned house lizard glycopeptide in the medicine of preparation treatment malignant tumour.
The application of above-mentioned house lizard glycopeptide in the medicine of preparation treatment liver cancer.
The application of above-mentioned house lizard glycopeptide in the medicine of preparation treatment disease of viral infection.
The application of above-mentioned house lizard glycopeptide in the medicine that preparation treatment HIV infects.
The application of above-mentioned house lizard glycopeptide in the preparation immunomodulator.
The invention has the beneficial effects as follows: the present invention furthers investigate and finds that gecko polysaccharide and peptide mortise are together, further the mixture (abbreviation gecko polysaccharide) of natural gecko polysaccharide and peptide is used protease hydrolysis, obtain a new house lizard glycopeptide (being called for short the house lizard glycopeptide), have than the better biological activity of natural gecko polysaccharide.All cell proliferation has obvious restraining effect to people's liver cancer SMMC-7721 with low dose group (10 μ g/ml) for house lizard glycopeptide high dosage (200 μ g/ml), middle dosage (100 μ g/ml).And gecko polysaccharide only high dosage (200 μ g/ml), middle dosage group (100 μ g/ml) cell proliferation has obvious restraining effect to people's liver cancer SMMC-7721, cell proliferation does not have restraining effect to low dose group (10 μ g/ml) to people's liver cancer SMMC-7721.As seen the house lizard glycopeptide can suppress people's liver cancer SMMC-7721 cell proliferation in 10 μ g/ml concentration, and gecko polysaccharide suppresses people's liver cancer SMMC-7721 cell proliferation in 100 μ g/ml concentration.In addition, the IL-8 metastases has very confidential relation, and the recurrence of tumour is the major cause for the treatment of malignant tumor failure with shifting, and also is to cause the final dead major reason of tumour patient.House lizard glycopeptide high dosage (200 μ g/ml), middle dosage group (100 μ g/ml) all have obvious restraining effect to people's liver cancer SMMC-7721 emiocytosis IL-8.And gecko polysaccharide does not have restraining effect to people's liver cancer SMMC-7721 emiocytosis IL-8.Above result shows that the house lizard glycopeptide has than the better biological activity of gecko polysaccharide.
The present invention is further research on the basis of using gecko polysaccharide, and obtain scientific validation; House lizard glycopeptide quite gecko polysaccharide has higher curative effect to malignant tumour, shown high tumor-suppression activity, and can suppress the cytokine IL-8 that tumor cell secretion promotes metastases, can be used for the treatment of malignant tumour separately, also can be used for the assisting therapy of malignant tumor operation, radiotherapy, chemotherapy and interventional therapy; The house lizard glycopeptide contains sulfate radical, has extremely bright application prospect in the control of diseases of viral infection such as acquired immune deficiency syndrome (AIDS); Glycopeptide has good immunoregulation effect usually, uses house lizard treatment immunocompromised that good result of treatment is also arranged, and the house lizard glycopeptide can be used as a kind of new immunomodulator, is used for treatment of diseases such as malignant tumour, chronic infection; House lizard has limited its clinical heavy dose of application to the toxicity of human body, so the general 1.5～4.5g only of the daily dosage portion of house lizard, the house lizard glycopeptide has played good synergism and attenuation.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of gecko polysaccharide.
Fig. 2 is the high-efficient liquid phase chromatogram of house lizard glycopeptide.
Fig. 3 is the infrared spectrogram of house lizard glycopeptide.
Fig. 4 is the gas chromatogram of standard sugary sugar nitrile acetic ester derivative;
Among the figure: 1 rhamnosyl, 2 pectinoses, 3 Fucoses, 4 seminoses, 5 glucose, 6 semi-lactosis, 7 inositols.
Fig. 5 is the gas chromatogram of the sugared nitrile acetic ester derivative of house lizard glycopeptide;
Among the figure: 1 rhamnosyl, 2 Fucoses, 3 seminoses, 4 glucose, 5 semi-lactosis, 6 inositols.
Fig. 6 is the gas chromatogram of standard sugary alditol acetate derivative;
Among the figure: 1 inositol, 2 galns, 3 glucosamines.
Fig. 7 is the gas chromatogram of the alditol acetate derivative of house lizard glycopeptide;
Among the figure: 1 inositol, 2 glucosamines.
Fig. 8 is the total ion current figure of house lizard glycopeptide.
Fig. 9 is that gecko polysaccharide is analyzed synoptic diagram to the influence curve of people's liver cancer SMMC-7721 cell proliferation;
Among the figure: 1 blank group, 2 gecko polysaccharide high dose group (200 μ g/ml), dosage group in 3 gecko polysaccharides (100 μ g/ml), 4 gecko polysaccharide low dose group (10 μ g/ml).
Figure 10 is that the house lizard glycopeptide is analyzed synoptic diagram to the influence curve of people's liver cancer SMMC-7721 cell proliferation;
Among the figure: 1 blank group, 2 house lizard glycopeptide high dose group (200 μ g/ml), dosage group in the 3 house lizard glycopeptides (100 μ g/ml), 4 house lizard glycopeptide low dose group (10 μ g/ml).
Embodiment
Below in conjunction with accompanying drawing and preferred embodiment, to details are as follows according to embodiment provided by the invention:
The house lizard glycopeptide is characterized in that: weight-average molecular weight is greater than 2,000,000Da; Specific rotatory power is-60 °; The quality percentage composition of carbon is 23.39%, and the quality percentage composition of protium is 4.925%, and the quality percentage composition of nitrogen element is 6.51%; The sugar moieties of its composition contains rhamnosyl, Fucose, seminose, glucose, semi-lactosi, glucosamine, mol ratio is: rhamnosyl: Fucose: seminose: glucose: semi-lactosi: glucosamine=1.334:3.363:3.375:6.983:10.480:0.019; The mode of connection of monose is: rhamnosyl is with 1,3-, 1,3,4-and 1,2, the form that 3-connects exists, and Fucose is with 1-, 1,4-and 1, the form that 2-connects exists, and seminose is with 1, the form that 4-connects exists, and glucose is with 1-, and 1,3, the form that 4-connects exists, and semi-lactosi is with 1,4-, 1,4, the form that 6-connects exists, and glucosamine is with 1, and the form that 4-connects exists; The quality percentage composition of uronic acid is 3.08%; Its mating type protein portion contains 16 seed amino acids, and the quality percentage composition is 16.33%; The type of cardohydrata-peptide linkage contains O-and connects; The quality percentage composition of sulfate is 14.7%.
The preparation of house lizard glycopeptide: get all 1000g of house lizard, be cut into the long fragment of 0.3cm, use 4000ml respectively, the methyl alcohol of 2000ml soaks 3h, filter, the dregs of a decoction dry in room temperature underlying ventilation, use 10000ml, 8000ml then, the hot water of 6000ml extracts three times for 100 ℃, extraction time is respectively 6h, 4h, 3h.United extraction liquid filters, and is concentrated into 800ml, the ethanol sedimentation that adds final concentration 80% (V/V), centrifugal (2000r/min, 20min), collecting precipitation, will precipitate multiple water-soluble, centrifugal (2000r/min, 20min), get supernatant, conventional sevage method is removed floating preteins 10 times, concentrates, dialysis, drying.Getting dried powder 24g is dissolved in 3000ml and contains 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH6.5), add 240mg papoid and 30ml toluene, 65 ℃ of water-bath 12h, after add the papoid of 120mg, continue 65 ℃ of water-bath 12h.Enzymolysis solution is filtered, be evaporated to 500ml, 80% (V/V) ethanol sedimentation leaves standstill 1h.Collecting precipitation is used dehydrated alcohol successively, washing with acetone, and 65-70 ℃ is dried to pressed powder.Water-soluble again powder, filter, collect filtrate, conventional sevage method is removed foreigh protein removing 6 times, concentrating under reduced pressure, dialysis, drying gets the house lizard glycopeptide.
The preparation of house lizard glycopeptide: get all 1000g of house lizard, be cut into the long fragment of 0.2cm, use 6000ml respectively, the methyl alcohol of 3000ml soaks 2h, filter, the dregs of a decoction dry in room temperature underlying ventilation, use 8000ml, 6000ml then, the hot water of 4000ml extracts three times for 100 ℃, extraction time is respectively 6h, 4h, 3h.United extraction liquid filters, and is concentrated into 800ml, the ethanol sedimentation that adds final concentration 80% (V/V), centrifugal (3200r/min, 10min), collecting precipitation, will precipitate multiple water-soluble, centrifugal (3200r/min, 10min), get supernatant, conventional sevage method is removed floating preteins 10 times, concentrate, dialysis concentrates drying.Getting dried powder 24g is dissolved in 1200ml and contains 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH6.5), add 120mg papoid and 10ml toluene, 60 ℃ of water-bath 48h, after add the papoid of 60mg, continue 60 ℃ of water-bath 24h.Enzymolysis solution is filtered, be evaporated to 500ml, 80% (V/V) ethanol sedimentation leaves standstill 12h.Collecting precipitation is used dehydrated alcohol successively, washing with acetone, and 65-70 ℃ is dried to pressed powder.Water-soluble again powder, filter, collect filtrate, conventional sevage method is removed foreigh protein removing 6 times, concentrating under reduced pressure, dialysis, drying gets the house lizard glycopeptide.Other is with reference to embodiment 1.
The preparation of house lizard glycopeptide: get all 1000g of house lizard, pulverize and be thick end, cross sieve No. 1, soak 12h with the methyl alcohol of 3000ml, filter, the dregs of a decoction dry in room temperature underlying ventilation, and the hot water with 10000ml extracts three times for 60 ℃ then, and extraction time is 6h.United extraction liquid filters, and is concentrated into 1000ml, adds the ethanol sedimentation of final concentration 70% (V/V), and 4 ℃ leave standstill 24h, centrifugal (4000r/min, 6min), collecting precipitation will precipitate multiple water-solublely, and it is centrifugal that (4000r/min 6min), gets supernatant, dialyses drying.Getting dried powder 24g is dissolved in 4200ml and contains 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH6.5), add 480mg papoid and 40ml toluene, 70 ℃ of water-bath 8h, after add the papoid of 240mg, continue 70 ℃ of water-bath 4h.Enzymolysis solution is filtered, be evaporated to 500ml, 80% (V/V) ethanol sedimentation leaves standstill 24h.Collecting precipitation is used dehydrated alcohol successively, washing with acetone, and vacuum-drying becomes pressed powder.Water-soluble again powder, filter, collect filtrate, conventional sevage method is removed foreigh protein removing 4 times, concentrating under reduced pressure, dialysis, drying gets the house lizard glycopeptide.Other is with reference to embodiment 1.
The preparation of house lizard glycopeptide: get all 1000g of house lizard, be cut into the long fragment of 0.3cm, use 4000ml respectively, the soaked in absolute ethyl alcohol 4h of 2000ml filters, and the dregs of a decoction dry in room temperature underlying ventilation, hot water with 10000ml extracts three times for 30 ℃ then, and extraction time is 12h.United extraction liquid filters, and is concentrated into 800ml, add the ethanol sedimentation of final concentration 30% (V/V), 4 ℃ leave standstill 24h, centrifugal (10000r/min, 5min), collecting precipitation will precipitate multiple water-soluble, it is centrifugal that (10000r/min 5min), gets supernatant, conventional sevage method is removed floating preteins 4 times, concentrates dialysis, concentrate drying.Getting dried powder 24g is dissolved in 3000ml and contains 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH6.5), add 240mg papoid and 30ml toluene, 60 ℃ of water-bath 12h, after add the papoid of 120mg, continue 60 ℃ of water-bath 24h.Enzymolysis solution is filtered, be evaporated to 500ml, 40% (V/V) ethanol sedimentation leaves standstill 12h.Collecting precipitation is used dehydrated alcohol successively, and washing with acetone is lyophilized into pressed powder.Water-soluble again powder, filter, collect filtrate, conventional sevage method is removed foreigh protein removing 8 times, concentrating under reduced pressure, dialysis, drying gets the house lizard glycopeptide.Other is with reference to embodiment 1.
The structural analysis of house lizard glycopeptide
(1) gecko polysaccharide purity check
1, materials and methods:
Adopt high performance liquid chromatography (HPLC) to detect gecko polysaccharide purity.Liquid-phase chromatographic column is ShodexKS-805, and moving phase is bi-distilled water, and flow velocity is 1ml/min, 35 ℃ of column temperatures.The aqueous solution of preparation sample, sample introduction.
2, result:
Referring to Fig. 1, single symmetrical peak appearred at 5.883 minutes, and the prompting gecko polysaccharide is the homogeneous component.
(2) house lizard glycopeptide purity check and relative molecular mass are measured
1, materials and methods:
Adopt high performance liquid chromatography (HPLC) to detect house lizard glycopeptide purity and relative molecular mass.Employing standard dextran Dextran T series production standard curve according to the retention time of house lizard glycopeptide sample under identical chromatographic conditions, calculates the relative molecular mass of this sample with typical curve.Liquid-phase chromatographic column is ShodexKS-805, and moving phase is bi-distilled water, and flow velocity is 1mL/min, and column temperature is 35 ℃.Preparation glucose, each standard T10, T40, T70, T11, T500,2000,2% (W/V) solution of blue dextran obtains corresponding liquid phase collection of illustrative plates behind each solution sample introduction, draw out the typical curve of retention time and molecular weight relation, draws regression equation.According to the retention time of house lizard glycopeptide, try to achieve molecular weight from regression equation.
2, result:
2.1 purity:
Referring to Fig. 2, single symmetrical peak appearred at 7.980 minutes, and prompting house lizard glycopeptide is the homogeneous component.The appearance time of gecko polysaccharide is 5.883 minutes (see figure 1)s, and prompting house lizard glycopeptide is little than the molecular weight of gecko polysaccharide, illustrates that the house lizard glycopeptide is the new compound that gecko polysaccharide obtains through protease hydrolysis.
2.2 molecular weight:
Try to achieve house lizard glycopeptide weight-average molecular weight (Mw) greater than 2,000 according to regression equation, 000Da.
(3) mensuration of house lizard glycopeptide specific rotatory power
1, materials and methods:
Get house lizard glycopeptide 4mg, be dissolved in the 2ml distilled water, measure specific rotation, with formula [α]=α/(1 * C) calculates specific rotatory power with automatic polarimeter.[a] is specific rotatory power, and a is a rotation angle, and 1 for (dm) grown in the pond, c is concentration (g/ml).
2, result such as following table:
| Rotation angle | Concentration (g/ml) | Specific rotatory power | |
| The house lizard glycopeptide | -0.03 | 2 | -60° |
(4) ultimate analysis of house lizard glycopeptide such as following table:
(5) house lizard glycopeptide infrared spectra
1, materials and methods:
Get 1-2mg house lizard glycopeptide, with carrying out Infrared spectroscopy behind the pressing potassium bromide troche.
2, result:
Referring to Fig. 3, at 3421.4cm
-1The broad peak that the place occurs is that the stretching vibration of O-H causes 2929.5cm
-1The more weak absorption peak that occurs about the place, for the C-H stretching vibration causes, 1409.8cm
-1The absorption peak that the place occurs causes these 3 groups of characteristic peaks that absorption peak is a glucide for the vibration of C-H angle.1601.8cm
-1The strong absorption peak that the place occurs causes that for the vibration of N-H angle prompting contains glucosamine and protein, illustrates that this sample is polysaccharide and proteinic mixture.859.2cm
-1Place's absorption peak is to be caused by the C-O-S stretching vibration, and prompting contains sulfate.
(6) composition of house lizard glycopeptide monose and mode of connection
1, the composition of monose and ratio
1.1, thin-layer chromatography (TLC)
1.1.1, materials and methods:
Be the neutral sugar of detection house lizard glycopeptide and the composition of aminosugar, sample is used trifluoroacetic acid and hydrochloric acid hydrolysis respectively.The hydrolysising condition of trifluoroacetic acid is: take by weighing house lizard glycopeptide 6mg, add 4mol/L trifluoroacetic acid 6ml, tube sealing, 105 ℃ of hydrolysis 4h; The hydrolysising condition of hydrochloric acid is: take by weighing house lizard glycopeptide 10mg, add 6mol/L hydrochloric acid 10ml, tube sealing, 105 ℃ of hydrolysis 8h.Add 45 ℃ of evaporated under reduced pressure of methyl alcohol then respectively repeatedly, to remove trifluoroacetic acid and hydrochloric acid fully.In sample, add a spot of water more respectively and make sample dissolution, take a morsel and on silica-gel plate, carry out thin-layer chromatography, adopt the contrast of standard monose to form with preliminary definite sample monose.The standard monose contrast of neutral sugar is: pectinose, glucose, seminose, semi-lactosi, Fucose, rhamnosyl; The standard monose contrast of aminosugar is: glucuronic acid, galacturonic acid, N-ethanoyl glucose, galn, glucosamine.Ethyl acetate: methyl alcohol: acetate: water (12ml:3ml:3ml:2ml) launches, the aniline-phthalic acid colour developing.The kind of relatively coming to determine to form in the sample monose according to the Rf value (Rf value) of standard monose and sample.
1.1.2, the result:
1.1.2.1, the table composed as follows of house lizard glycopeptide neutral sugar:
Sample has two points after launching colour developing, its Rf value is identical with glucose and Fucose, contains glucose and Fucose in the prompting sample.
1.1.2.2, the table composed as follows of house lizard glycopeptide aminosugar:
Sample has two points after launching colour developing, the Rf value of one of them point is identical with glucosamine, contains glucosamine in the prompting sample.The Rf value of another one point may contain uronic acid near glucuronic acid in the prompting sample.
1.2, gas phase
1.2.1, the detection of neutral sugar
1.2.1.1, materials and methods:
Sample: precision takes by weighing house lizard glycopeptide 8.4mg, adds the 4mol/L trifluoroacetic acid, tube sealing, and 105 ℃ of hydrolysis 4h add methyl alcohol repeatedly and are evaporated to dried in 45 ℃.Add the 1mg inositol, 16mg oxammonium hydrochloride, 1ml pyridine, 90 ℃ of reacting by heating 30 minutes.Taking-up is cooled to room temperature; add the 1ml diacetyl oxide; 90 ℃ were continued reacting by heating 30 minutes; add methyl alcohol repeatedly and be evaporated to driedly in 45 ℃, then the sample after the acetylize is dissolved in the chloroform, add isopyknic distilled water wash chloroform layer again 3 times; eliminate water layer; the derivative for preparing sugared nitrile acetic ester, reaction product is dissolved with the 0.2ml chloroform, sample introduction 1 μ l.Mix mark: precision takes by weighing rhamnosyl, pectinose, and seminose, Fucose, glucose, each 1mg of semi-lactosi, inositol 1mg adds oxammonium hydrochloride 14mg, pyridine 1ml, the same operation, reaction product is dissolved with the 0.2ml chloroform, sample introduction 1 μ l.The kind of relatively coming to determine to form in the sample monose according to the retention time of standard monose and sample.The mol ratio of coming to form in the calculation sample monose according to the peak area of standard monose and sample and sample size.
1.2.1.2, result such as following table:
| Monose | Retention time (min) | Mol ratio |
| Rhamnosyl | 9.748 | 1.334 |
| Fucose | 10.477 | 3.636 |
| Seminose | 16.844 | 3.375 |
| Glucose | 17.171 | 6.983 |
| Semi-lactosi | 18.085 | 10.480 |
Sample contains rhamnosyl, Fucose, seminose, glucose, semi-lactosi.
Mol ratio is: rhamnosyl: Fucose: seminose: glucose: semi-lactosi=1.334:3.363:3.375:6.983:10.480, and referring to Fig. 4, Fig. 5.
1.2.2, the detection of aminosugar
1.2.2.1, materials and methods:
Sample: precision takes by weighing house lizard glycopeptide 8mg, adds 6mol/L hydrochloric acid, tube sealing, and 105 ℃ of hydrolysis 8h add 45 ℃ of evaporated under reduced pressure of methyl alcohol, repeatedly to remove hydrochloric acid fully.Add the 1mg inositol again, tetrahydro boron sodium 20mg, distilled water 2ml, room temperature reduction 3h uses the excessive tetrahydro boron sodium of 25% acetate (V/V) neutralization again, adds methyl alcohol repeatedly and is evaporated to driedly in 45 ℃, puts P then
20
5Moisture eliminator in spend the night, next day 80 ℃ the heating 15min remove moisture.Add the 3ml diacetyl oxide; tube sealing; i00 ℃ of reacting by heating 1h adds 45 ℃ of evaporated under reduced pressure of toluene repeatedly, removes diacetyl oxide; then the sample after the acetylize is dissolved in the chloroform; add isopyknic distilled water wash chloroform layer again 3 times, eliminate water layer, the derivative of preparation alditol acetate; reaction product is dissolved with the 0.2ml chloroform, sample introduction 1 μ l.Mix mark: precision takes by weighing glucosamine, galn, and each 1mg of inositol, tetrahydro boron sodium 20mg, the same operation, reaction product is dissolved with the 0.1ml chloroform, sample introduction 1 μ l.The kind of relatively coming to determine to form in the sample monose according to the retention time of standard monose and sample.The mol ratio of coming to form in the calculation sample monose according to the peak area of standard monose and sample and sample size.
1.2.2.2, the result:
Sample contains glucosamine.Retention time 14.151min, mole number are 0.019, referring to Fig. 6, Fig. 7.
1.3, glucuronic acid content measures
1.3.1, materials and methods:
Hydroxyl biphenyl between 0.15% (W/V) with the configuration of 0.5% (W/V) sodium hydroxide, keeps in Dark Place in refrigerator, and is stable in January;
Sodium tetraborate-sulphuric acid soln: the sulphuric acid soln of 0.0125mol/L sodium tetraborate;
Galacturonic acid standardized solution: 60ug/ml;
Precision is measured standardized solution 0,0.05,0.1,0.15,0.2, in the 0.25ml value test tube, adds to 0.25ml with distilled water, adds sodium tetraborate-sulphuric acid soln of 1.5ml in ice bath after the precooling.Mixing, after boiling is heated 5min in the water-bath, is cooled to room temperature with ice bath, add 25ul between hydroxyl biphenyl reagent, mixing is in the mensuration photoabsorption of 520nm place.Draw sample solution (1mg/ml) 0.25ml, the same operation, for avoiding the interference of non-hexuronic acid composition and sodium tetraborate in the sample-sulphuric acid soln reaction, hydroxyl biphenyl reagent between replacing with 0.5% (W/V) sodium hydroxide of 25ul, the absorbance value that records is deducted from the absorption of sample value.Content and its corresponding absorbancy according to the standard substance uronic acid are drawn out typical curve, draw regression equation.According to the absorbancy of house lizard glycopeptide, try to achieve the content of uronic acid from regression equation.
1.3.2, the result:
Try to achieve the uronic acid quality percentage composition of house lizard glycopeptide according to regression equation: 3.08%.
2, the mode of connection of monose---methylate
2.1, materials and methods:
2.1.1, methylation reaction:
Take by weighing house lizard glycopeptide 8mg, add the 2ml dmso solution.Add exsiccant sodium hydroxide 20mg, ultrasonic 10min leaves standstill 90min under the room temperature.Under ice bath, dropwise add methyl iodide 1ml, become bright yellow solution until reactant.Return to room temperature then, continue ultrasonic 10min, underpressure distillation eliminates excessive methyl iodide, with chloroform 3ml dissolving, is transferred in the separating funnel, adds isopyknic water then, washs 3 times, removes water layer.Methylate repeatedly 7 times, analyze through IR, at 3500cm
-1The hydroxyl absorption peak of left and right sides Qiang Erkuan disappears substantially, and the methyl peak at 2900cm place is when significantly strengthening, and the hydroxyl on the prompting polysaccharide is methylated.
2.1.2, methylated polysaccharide depolymerization, hydrolysis, the preparation alditol acetate derivative.
Methylated sample is added 90% formic acid (V/V) 4ml, tube sealing, 100 ℃ of hydrolysis 6h add 2～3mL methyl alcohol in reaction flask, and 45 ℃ of following concentrating under reduced pressure evaporates to dryness repeat above operation to eliminate excessive formic acid.Add 4mol/L trifluoroacetic acid 4ml dissolving again, sealing, 105 ℃ of hydrolysis 4h add 2～3mL methyl alcohol in reaction flask, and 45 ℃ of following concentrating under reduced pressure evaporates to dryness repeat above operation to eliminate excessive trifluoroacetic acid.Sample adds tetrahydro boron sodium 20mg after the hydrolysis, distilled water 2ml, and room temperature reduction 3h, the neutralization of 25% acetate adds 2～3mL methyl alcohol in reaction flask, and 45 ℃ of following concentrating under reduced pressure evaporates to dryness repeat above operation to eliminate excessive acetic acid.Residuum is removed moisture at 80 ℃ of heating 15min.Add the 3ml diacetyl oxide, 100 ℃ of reacting by heating 1h add 2ml toluene in reaction solution, and the back 45 ℃ of unreacted aceticanhydrides of following pressure reducing and steaming that vibrate so repeat repeatedly to eliminate aceticanhydride.Then the sample after the acetylize is dissolved in the chloroform, adds isopyknic distilled water wash chloroform layer again 3 times, eliminate water layer, the derivative of preparation alditol acetate, reaction product is carried out gas chromatography mass spectrometry analysis (GC-MS) with the dissolving of 0.1ml chloroform.
2.2, the result:
2.2.1 total ion current figure is referring to Fig. 8.
2.2.2, methylation analysis such as following table:
| The methylated sugar alcohol acetic ester of part | Connection type | Ms fragment (m/z) |
| The methylated rhamnosyl of 2- | 1,3, the rhamnosyl that 4-connects | 58，87，97，115，117，129，139，159，171273 |
| 2, the methylated rhamnosyl of 4- | 1, the rhamnosyl that 3-connects | 45，58，74，87，99，101，118，129，161，173， 189，234 |
| The methylated rhamnosyl of 4- | 1,2, the rhamnosyl that 3-connects | 59，71，85，8799，115，127，129，142，159， 189，201，261 |
| 2, the methylated Fucose of 3- | 1, the Fucose that 4-connects | 57，73，86，98，103，115，127，140，200 |
| 2,3, the methylated Fucose of 4- | The Fucose that 1-connects | 58，71，87，99，101，113，117，131，161 |
| 3, the methylated Fucose of 4- | 1, the Fucose that 2-connects | 59，71，87，99，113，117，129，159，173，189， 233 |
| 2,3, the methylated seminose of 6- | 1, the seminose that 4-connects | 58，71，81，87，99，101，113，117，129，131，1 42，143，161，173，233 |
| 2, the methylated glucose of 6- | 1,3, the glucose that 4-connects | 58，87，117，129，143，185，305 |
| 2,3,4, the methylated glucose of 6- | The glucose that 1-connects | 59，71，87，101，117，129，145，161，205 |
| 2, the methylated semi-lactosi of 3- | 1,4, the semi-lactosi that 6-connects | 58，87，99，191，117，131，161，203，263 |
| 2,3, the methylated semi-lactosi of 6- | 1, the semi-lactosi that 4-connects | 58，71，87，99，101，113，117，129，131，161， 173，233 |
| 3, the methylated glucosamine of 6- | 1, the glucosamine that 4-connects | 74，87，99，113，116117，129，142，158，173， 202，233 |
(7) protein is formed and content analysis
1, materials and methods:
Get an amount of house lizard glycopeptide, add hydrochloric acid, tube sealing, 110 ℃ of hydrolysis 24h concentrate, and regulating the pH value is 6.2-6.3, measures amino acid through automatic analyzer for amino acids and forms and content.
2, result:
| The amino acid title | House lizard glycopeptide (μ g/mg) |
| Aspartic acid | 29.9 |
| Threonine | 7.12 |
| Serine | 8.58 |
| L-glutamic acid | 32.68 |
| Glycine | 27.26 |
| L-Ala | 8.56 |
| Xie Ansuan | 2.58 |
| Methionine(Met) | 0.9 |
| Isoleucine | 4.94 |
| Leucine | 4.86 |
| Tyrosine | 1.48 |
| Phenylalanine | 0.9 |
| Methionin | 13.74 |
| Histidine | 3.96 |
| Arginine | 11.2 |
| Proline(Pro) | 4.62 |
| Total amount | 163.28 |
In the table: show that the house lizard glycopeptide contains 16 seed amino acids, amino acid whose quality percentage composition is 16.33%.
(8) type of cardohydrata-peptide linkage---β elimination reaction
1, materials and methods:
Take by weighing house lizard glycopeptide 4mg, be dissolved in sodium hydroxide-1mol/L tetrahydro boron sodium of 2ml0.3mol/L, add a toluene, 45 ℃ of hydrolysis 24h, add in 25% acetate (V/V) and dilute alkaline soln with termination reaction, get 0.5ml and detect amino acid whose composition and content, the sample aqueous solution of preparation same concentrations is as blank.
2, result:
House lizard glycopeptide amino acid before and after the β elimination reaction forms and content compares (μ g/mg)
| The amino acid title | House lizard glycopeptide (not hydro-oxidation sodium) | House lizard glycopeptide (hydro-oxidation sodium) |
| Aspartic acid | 29.9 | 27.62 |
| Threonine | 7.12 | 3.64 |
| Serine | 8.58 | 6.32 |
| L-glutamic acid | 32.68 | 29.46 |
| Glycine | 27.26 | 25.98 |
| L-Ala | 8.56 | 6.88 |
| Xie Ansuan | 2.58 | 2.16 |
| Methionine(Met) | 0.9 | 0.88 |
| Isoleucine | 4.94 | 4.22 |
| Leucine | 4.86 | 4.02 |
| Tyrosine | 1.48 | 1.14 |
| Phenylalanine | 0.9 | 0.6 |
| |
0 | 1.16 |
| Methionin | 13.74 | 13.24 |
| Histidine | 3.96 | 3.62 |
| Arginine | 11.2 | 8.38 |
| Proline(Pro) | 4.62 | 5.98 |
The house lizard glycopeptide is after the β elimination reaction, and in every milligram of house lizard glycopeptide, the content of Threonine drops to 3.64mg by 7.12mg, and the content of aminobutyric acid rises to 1.16mg by 0mg.Prompting house lizard glycopeptide Jian Alto type contains O one connection.
(9) mensuration of sulfate
1, materials and methods:
Gelatin: the 2g gelatin is dissolved in the 400ml water at 60-70 ℃, 4 ℃ of preservations;
Bariumchloride gelatin reagent: the 0.5g bariumchloride is dissolved in the 100ml gelatin solution, 4 ℃ of preservations;
Trichoroacetic acid(TCA): 8% aqueous solution (W/V);
The standard substance of vitriol: 400ug/ml;
All utensils are used deionized water rinsing after washing with HNO3 again.
The accurate standard substance 0,0.05,0.1,0.2,0.3 of drawing, 0.4ml, water complements to 0.4ml, adds the 0.35ml trichoroacetic acid(TCA) respectively, 0.25ml bariumchloride gelatin reagent, mixing leaves standstill 15min, measures turbidity at the 360nm place.Precision takes by weighing house lizard glycopeptide 2mg, and 6mol/L hydrochloric acid hydrolysis 6h adds 45 ℃ of evaporated under reduced pressure of methyl alcohol repeatedly, and each use the 1ml water dissolution residue, house lizard glycopeptide absorption 0.4ml * 2 part.Get 1 part and add 0.35ml trichoroacetic acid(TCA), 0.25ml bariumchloride gelatin reagent; 1 part adds the 0.35ml trichoroacetic acid(TCA) in addition, 0.25ml gelatin reagent, and mixing leaves standstill 15min, measures turbidity at the 360nm place.Absorbancy when the absorbancy when having bariumchloride to exist deducts no bariumchloride and exists is eliminated the influence of the uv-absorbing substance that contains in hydrolyzed solution with this contrast.Content and its corresponding absorbancy according to the standard substance sulfate are drawn out typical curve, draw regression equation.According to the absorbancy of house lizard glycopeptide, try to achieve the content of sulfate from regression equation.
2, result:
Try to achieve the sulfate quality percentage composition of house lizard glycopeptide according to regression equation: 14.7%
House lizard glycopeptide and gecko polysaccharide contrast activation analysis
1, gecko polysaccharide and house lizard glycopeptide are to the influence of people's liver cancer SMMC-7721 cell proliferation
The influence of gecko polysaccharide to human hepatocellular carcinoma BEL-7402 cell's propagation observed in our original research, finds that gecko polysaccharide can suppress human hepatocellular carcinoma BEL-7402 cell's propagation.The influence to people's liver cancer SMMC-7721 cell proliferation of gecko polysaccharide and house lizard glycopeptide is observed in this research simultaneously.
1.1, gecko polysaccharide is to the influence of people's liver cancer SMMC-7721 cell proliferation
1.1.1, materials and methods:
The SMMC-7721 cell is available from preclinical medicine institute of China Concord Medical Science University cell centre; The DMEM high glucose medium, U.S. GIBCO company product; Gecko polysaccharide is with the dissolving of PBS damping fluid, and the ultrafiltration removal of impurities is mixed with the storing solution of 2mg/ml.
The SMMC-7721 cell is at 5%CO
2Cultivate in the incubator, substratum is the high sugar of DMEM that contains 10% inactivated fetal bovine serum, contains 1% pair anti-(penicillin and Streptomycin sulphate).Went down to posterity once in per 3～4 days, take the logarithm vegetative period cell be used for the experiment.12 orifice plates every hole inoculation 0.5 * 10
4/ ml cell (2ml culture system) inoculated 84 holes altogether.Inoculate dosing in back 24 hours and cultivate, set up separately gecko polysaccharide height (200 μ g/ml), in (100 μ g/ml), low dose group (10 μ g/ml) and PBS negative control group.Respectively at after the dosing 30 minutes, 1～6 day every group collect 3 porocytes respectively, trypan blue dyeing, living cell counting and dead cell are made growth curve, calculate cell survival rate.Calculate cell survival rate with following formula: cell survival rate (%)=(viable cell/viable cell+dead cell) * 100%.Statistical method adopts two-way analysis of variance, and all (version:16.0, Chicago USA) finish statistics by statistical package SPSS.
1.1.2, the result:
Cell proliferation has obvious restraining effect to people's liver cancer SMMC-7721 for gecko polysaccharide high dosage, middle dosage group, and cell proliferation does not have restraining effect but low dose group is to people's liver cancer SMMC-7721, referring to Fig. 9.
1.2, the house lizard glycopeptide is to the influence of people's liver cancer SMMC-7721 cell proliferation
1.2.1, materials and methods:
The SMMC-7721 cell is available from preclinical medicine institute of China Concord Medical Science University cell centre; The DMEM high glucose medium, U.S. GIBCO company product; The house lizard glycopeptide is with the dissolving of PBS damping fluid, and the ultrafiltration removal of impurities is mixed with the storing solution of 2mg/ml.
The SMMC-7721 cell is at 5% CO
2Cultivate in the incubator, substratum is the high sugar of DMEM that contains 10% inactivated fetal bovine serum, contains 1% pair anti-(penicillin and Streptomycin sulphate).Went down to posterity once in per 3～4 days, take the logarithm vegetative period cell be used for the experiment.12 orifice plates every hole inoculation 0.5 * 10
4/ ml cell (2ml culture system) inoculated 84 holes altogether.Inoculate dosing in back 24 hours and cultivate, set up separately house lizard glycopeptide height (200 μ g/ml), in (100 μ g/ml), low dose group (10 μ g/ml) and PBS negative control group.Respectively at after the dosing 30 minutes, 1～6 day every group collect 3 porocytes respectively, trypan blue dyeing, living cell counting and dead cell are made growth curve, calculate cell survival rate.Calculate cell survival rate with following formula: cell survival rate (%)=(viable cell/viable cell+dead cell) * 100%.Statistical method adopts two-way analysis of variance, and all (version:16.0, Chicago USA) finish statistics by statistical package SPSS.
1.2.1, the result:
All cell proliferation has obvious restraining effect to people's liver cancer SMMC-7721 for house lizard glycopeptide high dosage, middle dosage and low dose group.Above result shows that the house lizard glycopeptide is better than gecko polysaccharide to the restraining effect of people's liver cancer SMMC-7721 cell proliferation, referring to Figure 10.
2, gecko polysaccharide and house lizard glycopeptide are to the influence of people's liver cancer SMMC-7721 emiocytosis interleukin 8 (IL-8)
2.1, gecko polysaccharide is to the influence of people's liver cancer SMMC-7721 emiocytosis interleukin 8 (IL-8)
2.1.1, materials and methods:
The SMMC-7721 cell is available from preclinical medicine institute of China Concord Medical Science University cell centre; The DMEM high glucose medium, U.S. GIBCO company product; Gecko polysaccharide is with the dissolving of PBS damping fluid, and the ultrafiltration removal of impurities is mixed with the storing solution of 2mg/ml.
The SMMC-7721 cell is at 5% CO
2Cultivate in the incubator, substratum is the high sugar of DMEM that contains 10% inactivated fetal bovine serum, contains 1% pair anti-(penicillin and Streptomycin sulphate).Went down to posterity once in per 3～4 days, take the logarithm vegetative period cell be used for the experiment.12 orifice plates every hole inoculation 0.5 * 10
4/ ml cell (2ml culture system) inoculated dosing in back 24 hours and cultivated, set up separately gecko polysaccharide height (200 μ g/ml), in (100 μ g/ml), low dose group (10 μ g/ml) and PBS negative control group.120h collects and respectively organizes cell conditioned medium after dosing, and is centrifugal, gets supernatant.Detect the expression of IL-8 with double antibody sandwich method.The concentration gradient of the standard substance of IL-8 is set to 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 7.8pg/ml, 0pg/ml, parallel 2 holes of establishing of each dosage group.Parallel 3 holes of establishing of each dosage group of sample.The absorbancy corresponding with it according to each concentration of standard substance (OD value) matched curve equation is again with its concentration of OD value substitution Equation for Calculating of sample.Statistical method adopts one-way analysis of variance, and all (version:16.0, Chicago USA) finish statistics by statistical package SPSS.
2.1.2, the result:
Gecko polysaccharide is to the influence of people's liver cancer SMMC-7721 emiocytosis IL-8
| Grouping | Mean ± standard deviation |
| The blank group | 249.337±13.052 |
| High dose group (200 μ g/ml) | 224.577±15.275 |
| Middle dosage group (100 μ g/ml) | 229.420±5.082 |
| Low dose group (10 μ g/ml) | 249.940±17.811 |
P＝0.108
Show that gecko polysaccharide does not have restraining effect to people's liver cancer SMMC-7721 emiocytosis IL-8.
2.2, the house lizard glycopeptide is to the influence of people's liver cancer SMMC-7721 emiocytosis interleukin 8 (IL-8)
2.2.1, materials and methods:
The SMMC-7721 cell is available from preclinical medicine institute of China Concord Medical Science University cell centre; The DMEM high glucose medium, U.S. GIBCO company product; The house lizard glycopeptide is with the dissolving of PBS damping fluid, and the ultrafiltration removal of impurities is mixed with the storing solution of 2mg/ml.
The SMMC-7721 cell is at 5% CO
2Cultivate in the incubator, substratum is the high sugar of DMEM that contains 10% inactivated fetal bovine serum, contains 1% pair anti-(penicillin and Streptomycin sulphate).Went down to posterity once in per 3～4 days, take the logarithm vegetative period cell be used for the experiment.12 orifice plates every hole inoculation 0.5 * 10
4/ ml cell (2ml culture system) inoculated dosing in back 24 hours and cultivated, set up separately house lizard glycopeptide height (200 μ g/ml), in (100 μ g/ml), low dose group (10 μ g/ml) and PBS negative control group.120h collects and respectively organizes cell conditioned medium after dosing, and is centrifugal, gets supernatant.Detect the expression of IL-8 with double antibody sandwich method.The concentration gradient of the standard substance of IL-8 is set to 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 7.8pg/ml, 0pg/ml, parallel 2 holes of establishing of each dosage group.Parallel 3 holes of establishing of each dosage group of sample.The absorbancy corresponding with it according to each concentration of standard substance (OD value) matched curve equation is again with its concentration of OD value substitution Equation for Calculating of sample.Statistical method adopts one-way analysis of variance, and all (version:16.0, Chicago USA) finish statistics by statistical package SPSS.
2.2.2, the result:
The house lizard glycopeptide is to the influence of people's liver cancer SMMC-7721 emiocytosis IL-8
| Grouping | Mean ± standard deviation |
| The blank group | 269.183±33.716 |
| High dose group (200 μ g/ml) | 185.773±11.470●▲ |
| Middle dosage group (100 μ g/ml) | 202.543±7.665○ |
| Low dose group (10 μ g/ml) | 230.400±21.341 |
P＝0.006
● compare P=0.001 with the blank group
Zero with blank group relatively 1, P=0.005
▲ compare P=0.032 with low dose group
Illustrate that house lizard glycopeptide high dosage, middle dosage group all have obvious restraining effect to people's liver cancer SMMC-7721 emiocytosis IL-8.Above result shows that the house lizard glycopeptide has the function of the inhibition people liver cancer SMMC-7721 emiocytosis IL-8 that gecko polysaccharide do not possess.
Application Example 1:
The house lizard glycopeptide is made the injection type that meets the pharmaceutics requirement.
Contain the preparation of the injection liquid of house lizard glycopeptide:
The house lizard glycopeptide, dissolved in distilled water filters, and dialyses 3 times, and adjustment concentration is 10mg/ml, degerming, depyrogenation, aseptic subpackaged is the 2ml/ bottle.
Application Example 2:
The house lizard glycopeptide is made the oral dosage form that meets the pharmaceutics requirement.
Contain the preparation of house lizard glycopeptide oral capsule:
The house lizard glycopeptide, the Capsules of packing into, every 50mg seals, gumming, bottling.
Contain the preparation of house lizard glycopeptide oral tablet:
The house lizard glycopeptide adds 2 parts in dextrin, 2 parts of starch, and 1 part of Icing Sugar, the hydroxide gel reaches 3% of total amount, and compressing tablet is the 50mg/ sheet, bottling.
The application of house lizard glycopeptide in the medicine of preparation treatment malignant tumour.
The application of house lizard glycopeptide in the medicine of preparation treatment liver cancer.
The application of house lizard glycopeptide in the medicine of preparation treatment disease of viral infection.
The application of house lizard glycopeptide in the medicine that preparation treatment HIV infects.
The application of house lizard glycopeptide in the preparation immunomodulator.
Above-mentioned detailed description of this house lizard glycopeptide and preparation method and its medical applications being carried out with reference to embodiment; be illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (9)
1, house lizard glycopeptide is characterized in that: weight-average molecular weight is greater than 2,000,000Da; Specific rotatory power is-60 °; The quality percentage composition of carbon is 23.39%, and the quality percentage composition of protium is 4.925%, and the quality percentage composition of nitrogen element is 6.51%; The sugar moieties of its composition contains rhamnosyl, Fucose, and seminose, glucose, semi-lactosi, glucosamine, mol ratio is: rhamnosyl: Fucose: seminose: glucose: semi-lactosi: glucosamine=1.334:3.363:3.375:6.983:10.480:0.019; The mode of connection of monose is: rhamnosyl is with 1,3-, 1,3,4-and 1,2, the form that 3-connects exists, and Fucose is with 1-, 1,4-and 1, the form that 2-connects exists, and seminose is with 1, the form that 4-connects exists, and glucose is with 1-, and 1,3, the form that 4-connects exists, and semi-lactosi is with 1,4-, 1,4, the form that 6-connects exists, and glucosamine is with 1, and the form that 4-connects exists; The quality percentage composition of uronic acid is 3.08%; Its mating type protein portion contains 16 seed amino acids, and the quality percentage composition is 16.33%; The type of cardohydrata-peptide linkage contains O-and connects; The quality percentage composition of sulfate is 14.7%.
2, preparation method according to the described house lizard glycopeptide of claim 1, it is characterized in that: with the house lizard drying, broken back goes out lipid-soluble substance with the organic solvent extracting, with the dregs of a decoction after the degreasing with by 1-10 times of hot water of house lizard quality under 30-100 ℃ of condition lixiviate 2-12 hour, the water extract filters back gradation adding ethanol to 15%-80% concentration (V/V) and precipitates, with resolution of precipitate in water, 1000-10000r/min centrifuging and taking supernatant, conventional sevage method is removed foreigh protein removing, dialysis, be dried to powder, be dissolved in again powder quality 30-180 doubly contain 0.5mol/L sodium-chlor, the 10mmol/L halfcystine, the phosphate buffered saline buffer of the 0.1mol/L of 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH6.5, add the papoid of powder quality 0.2%-2% behind the papoid and the 0.5-2 toluene doubly that add powder quality 0.5%-4%, 50 ℃ of-80 ℃ of water-bath 4h-48h, continue water-bath 4h-24h; Hydrolyzed solution concentrates the back with ethanol sedimentation, and collecting precipitation is used dehydrated alcohol successively, and washing with acetone is water-soluble again, filters, and conventional sevage method is removed foreigh protein removing, concentrate, and dialysis, drying gets the house lizard glycopeptide.
3, house lizard glycopeptide according to claim 1 is characterized in that: be made into the injection type that meets the pharmaceutics requirement.
4, house lizard glycopeptide according to claim 1 is characterized in that: be made into the oral dosage form that meets the pharmaceutics requirement.
5, the application of house lizard glycopeptide according to claim 1 in the medicine of preparation treatment malignant tumour.
6, the application of house lizard glycopeptide according to claim 1 in the medicine of preparation treatment liver cancer.
7, the application of house lizard glycopeptide according to claim 1 in the medicine of preparation treatment disease of viral infection.
8, the application of house lizard glycopeptide according to claim 1 in the medicine that preparation treatment HIV infects.
9, the application of house lizard glycopeptide according to claim 1 in the preparation immunomodulator.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100678979A CN101486755A (en) | 2009-02-20 | 2009-02-20 | House lizard, preparation and medical use |
| CN 201010104900 CN101768215B (en) | 2009-02-20 | 2010-01-19 | House lizard glycopeptide, method for preparing same and medicinal application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100678979A CN101486755A (en) | 2009-02-20 | 2009-02-20 | House lizard, preparation and medical use |
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| CN101486755A true CN101486755A (en) | 2009-07-22 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106866481A (en) * | 2016-12-30 | 2017-06-20 | 浙江海洋大学 | Green grass or young crops accounts for the preparation method and purposes of vitaminAD extract in fish guts |
| CN110346457A (en) * | 2018-04-04 | 2019-10-18 | 青岛大学附属医院 | A kind of detection method using monosaccharide composition in acid hydrolysis of microwave and anion-exchange chromatography-pulsed amperometric method analysis polysaccharide |
| CN112250737A (en) * | 2020-10-26 | 2021-01-22 | 北京市农林科学院 | Auricularia polytricha glycopeptide with lead-removing function and preparation method and application thereof |
-
2009
- 2009-02-20 CN CNA2009100678979A patent/CN101486755A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106866481A (en) * | 2016-12-30 | 2017-06-20 | 浙江海洋大学 | Green grass or young crops accounts for the preparation method and purposes of vitaminAD extract in fish guts |
| CN110346457A (en) * | 2018-04-04 | 2019-10-18 | 青岛大学附属医院 | A kind of detection method using monosaccharide composition in acid hydrolysis of microwave and anion-exchange chromatography-pulsed amperometric method analysis polysaccharide |
| CN112250737A (en) * | 2020-10-26 | 2021-01-22 | 北京市农林科学院 | Auricularia polytricha glycopeptide with lead-removing function and preparation method and application thereof |
| CN112250737B (en) * | 2020-10-26 | 2021-11-02 | 北京市农林科学院 | Auricularia polytricha glycopeptide with lead-removing function and preparation method and application thereof |
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