CN109593140A - A kind of crispy gracilaria polysaccharide and its preparation method and application - Google Patents
A kind of crispy gracilaria polysaccharide and its preparation method and application Download PDFInfo
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- gracilaria
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- 241000206581 Gracilaria Species 0.000 title claims abstract description 95
- 150000004676 glycans Chemical class 0.000 title claims abstract description 78
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 78
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229960000935 dehydrated alcohol Drugs 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 15
- 230000001376 precipitating effect Effects 0.000 claims abstract description 14
- 239000012141 concentrate Substances 0.000 claims abstract description 10
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000502 dialysis Methods 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 6
- 230000010355 oscillation Effects 0.000 claims abstract description 5
- 238000003756 stirring Methods 0.000 claims abstract description 5
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 43
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 27
- 229960002949 fluorouracil Drugs 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 16
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 208000017897 Carcinoma of esophagus Diseases 0.000 claims description 11
- 235000009508 confectionery Nutrition 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
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- 239000003560 cancer drug Substances 0.000 claims description 3
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- 229910052731 fluorine Inorganic materials 0.000 claims description 2
- 239000011737 fluorine Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims 2
- 229940035893 uracil Drugs 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
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- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 230000000973 chemotherapeutic effect Effects 0.000 abstract 2
- 238000004108 freeze drying Methods 0.000 abstract 1
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- 210000004027 cell Anatomy 0.000 description 31
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 16
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- 241000699666 Mus <mouse, genus> Species 0.000 description 10
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- 238000005259 measurement Methods 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 6
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- 206010014025 Ear swelling Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
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- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical group CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical class ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
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- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Pharmacology & Pharmacy (AREA)
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Abstract
The invention belongs to biomedicine technical fields, and in particular to a kind of crispy gracilaria polysaccharide and its preparation method and application.The preparation method of the crispy gracilaria polysaccharide is the following steps are included: crisp river hedge is extracted after dehydrated alcohol is pre-processed and is dried using hot water, in triplicate, extracting solution merging obtains concentrate after being concentrated in vacuo, solution of trichloroacetic acid is added while stirring into concentrate, it is stood overnight at 4 DEG C, it is centrifuged off precipitating, obtain supernatant, then supernatant being adjusted to neutrality and carrying out alcohol precipitation to concentration of alcohol is 50~60%, it collects to stand after dehydrated alcohol oscillation washing is added after precipitating and be centrifuged, crispy gracilaria polysaccharide is made after dialysis freeze-drying.The present invention also provides application of the crispy gracilaria polysaccharide in terms of anti-inflammatory or anti-tumor chemotherapeutic medicine synergist.Crispy gracilaria polyoses producing method simple and stable of the present invention, the crispy gracilaria polysaccharide of preparation effect in terms of anti-inflammatory or anti-tumor chemotherapeutic medicine synergy is obvious, is suitble to promote and apply.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of crispy gracilaria polysaccharide and its preparation method and application.
Background technique
Crispy gracilaria is a kind of large-scale economy class cultivating seaweed of Gracilaria, and main product is in southeast such as China Zhejiang, Fujian, Jiangsu
Coastal area, be mainly distributed at present Guangdong Province Nan'ao it is coastal cultivate successfully and can large area plantation.Fresh crispy gracilaria color
For aubergine or light red, plump succulence, matter is crisp, darkens after dry.Due to early stage crisp river hedge yield rareness always not by
Pay attention to, therefore it is seldom to the research of crisp river hedge both at home and abroad, it is continuously increased recently as the yield of crispy gracilaria, the work of crisp river hedge
The research of property ingredient and biological activity also gradually causes the concern of people.Studies have shown that crispy gracilaria rich in various active polysaccharide,
Phycoerythrin and the necessary nutrient of dietary fiber and human body etc. have immunoregulatory activity, antitumor, anti-oxidant, disease-resistant
The various biologicals such as poison activity and medicinal health effect, wherein active polysaccharide attracts as a kind of effective component of high-efficiency low-toxicity
The extensive concern of researchers.Therefore how from crispy gracilaria polysaccharide component is easily effectively extracted with regard to particularly critical.It is more
The extracting method that carbohydrate content generallys use includes Hot water extraction, diluted acid and diluted alkaline extraction method, and new process includes enzyme process, ultrasound
Wave auxiliary, microwave-assisted extraction and ultrafiltration etc..
Such as Ju Yaoyao et al. is optimized by extraction process of the response surface experiments to crispy gracilaria polysaccharide (GCP), and
Its inhibiting effect to tumour cell is had studied, for the extracting method used for conventional Hot water extraction, the crispy gracilaria of preparation is more
Sugar can effectively inhibit the production of tumour cell, but the property of the crispy gracilaria polysaccharide is unstable, and response surface optimization is tested
Complex process.The crispy gracilaria polysaccharide extracted is further researched and developed into Pharmaceutical Polysaccharides or health care product polysaccharide, is beneficial to
The complex treatment and rehabilitation of inflammation and tumour patient, while making that the poison of the common anti-inflammatory anti-tumor drug of cell toxicant class is not secondary
With therefore, crispy gracilaria polysaccharide is worth further research and development, improves the economic use value of crispy gracilaria.
Based on current crispy gracilaria polysaccharide, there is the scarce of complex process, stability difference and repeatability difference in terms of preparation
It falls into, and the effect in terms for the treatment of inflammation and tumour, it is desirable to provide a kind of preparation process is simple, can be mass-produced, prepare
The good crispy gracilaria polysaccharide and its preparation method and application of product stability good quality.
Summary of the invention
The present invention is directed to overcome the shortcomings of the prior art, provides a kind of crispy gracilaria polysaccharide and preparation method thereof and answer
With the preparation method is simply reproducible, and the crispy gracilaria polysaccharide of preparation has significant antiphlogistic effects, and can be used as anti-swollen
The synergist of tumor medicine improves the sensibility of anti esophageal cancer chemotherapeutics significantly.
To achieve the goals above, technical scheme is as follows:
A kind of preparation method of crispy gracilaria polysaccharide, comprising the following steps:
S1, crisp river hedge are pulverized and sieved weighing, are pre-processed using dehydrated alcohol after rinsing and draining after drying, drying,
Obtain crispy gracilaria dried powder;
S2, crispy gracilaria dried powder is impregnated with pure water, solid-to-liquid ratio is 1:(7~15), 3~4h is extracted at 80~90 DEG C,
It is intermittently stirred during extraction, in triplicate, filtered extracting solution merges, and extracting solution is dense through rotavapor under vacuum
Concentrate is obtained after contracting, and the trichloroacetic acid that mass concentration is 3~7% is added while stirring into concentrate, stood at 4 DEG C
Night, 25~35min of centrifugation remove precipitating, obtain supernatant;
S3, the supernatant is adjusted to neutrality using sodium hydroxide, be added dehydrated alcohol to concentration of alcohol be 50~
60%, 4 DEG C stand overnight after be centrifuged 25~35min, collect precipitating, will precipitating be added dehydrated alcohol oscillation washing after stand from
Crispy gracilaria Thick many candies are made in triplicate in the heart.
S4, the crispy gracilaria Thick many candies are dissolved with pure water, it, will in the bag filter that molecule interception is 8~10kDa
Bag filter is immersed in pure water, 4 DEG C of dialysed overnights, repeats dialysis 5~10 times, when the pure water pH that dialyses is neutral, stops dialysis,
Dialyzate in collecting bag;
S5, dialyzate is freezed for 24 hours in -20 DEG C of refrigerators, is then lyophilized with vacuum freeze drier, it is more that crispy gracilaria is made
Sugar is stored in drier.
Further, it uses dehydrated alcohol to carry out pretreated operation in the step S1 to heat to be condensed back,
It is repeated twice.
Further, the solid-to-liquid ratio is 1:10.
Further, the Extracting temperature is 85 DEG C, and the extraction time is 3.5h.
Further, the addition volume of trichloroacetic acid is 0.3~0.5 times of the volume of the concentrated liquid in the step S1, described
The mass concentration of trichloroacetic acid is 5%.
Further, the centrifugal condition is 6000g, 4 DEG C.
Further, it is 55% that dehydrated alcohol to concentration of alcohol is added in the step S3.
Further, the molecule interception of bag filter is 9kDa in the step S4.
The present invention also provides the crispy gracilaria polysaccharide that the preparation method of the crisp river hedge polysaccharide obtains.
The present invention also provides the crispy gracilaria polysaccharide applications in preparing anti-inflammatory drugs.
The present invention also provides application of the crispy gracilaria polysaccharide in preparation treatment oesophagus cancer drug.
The present invention also provides a kind of pharmaceutical composition for treating the cancer of the esophagus, described pharmaceutical composition includes the crisp river of the present invention
Li polysaccharide and 5 FU 5 fluorouracil.
Further, the weight ratio of the crispy gracilaria polysaccharide and 5 FU 5 fluorouracil is (1~5): 1.
Further, the weight ratio of the crispy gracilaria polysaccharide and 5 FU 5 fluorouracil is 2.5:1
Compared with prior art, the invention has the following advantages:
(1) the crisp river hedge polysaccharide preparation process of the present invention is simple, and mild condition time-consuming is short, and yield is high, and stability is good, is suitble to big
Large-scale production.
(2) crispy gracilaria polysaccharide of the present invention can inhibit the ear swelling of mouse significantly, show good antiphlogistic effects, simultaneously
Crispy gracilaria polysaccharide to esophageal cancer cell ECA109 grow and be proliferated have certain inhibiting effect, by chemotherapeutics 5 FU 5 fluorouracil with
Crispy gracilaria polysaccharide, which is used in combination, can significantly improve 5 FU 5 fluorouracil to the lethality of esophageal cancer cell ECA109, improve chemotherapeutics
The anticancer sensibility of 5 FU 5 fluorouracil, can be used as a kind of novel anti-inflammatory anti-tumor medicinal preparation or sensitizer is applied to biological medicine
And in cancer-preventing health product, there is very high Development volue and application prospect
Detailed description of the invention
Fig. 1 is the measurement knot of influence of the crispy gracilaria polysaccharide to the survival rate of RAW264.7 macrophage and person monocytic cell
Fruit;
Fig. 2 is inhibited proliferation measurement result of the crispy gracilaria polysaccharide to esophageal cancer cell ECA109;
Fig. 3 is inhibited proliferation measurement result of 4 pharmaceutical composition of embodiment to esophageal cancer cell ECA109.
Specific embodiment
It is as described below, it is merely preferred embodiments of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
5 FU 5 fluorouracil (5-Fu) national drug standard H10980069 of the present invention is purchased from Beijing XieHe medicine Factory.
Embodiment 1, crispy gracilaria polysaccharide of the present invention and preparation method thereof
1 crispy gracilaria polysaccharide of the embodiment of the present invention and preparation method thereof, comprising the following steps:
S1, fresh crispy gracilaria pulverize and sieve after drying after rinsing and draining, and carry out condensing reflux using dehydrated alcohol and add
Heat treatment, is repeated twice, and filters, and filter residue drying obtains crispy gracilaria dried powder;
S2, the crispy gracilaria dried powder is impregnated, solid-to-liquid ratio 1:7 using pure water, 3h is extracted at 80 DEG C, during extraction
It is intermittently stirred, in triplicate, filtered extracting solution merges, and extracting solution obtains after rotavapor under vacuum is concentrated
The trichloroacetic acid that the mass concentration that volume is 0.3 times of the volume of the concentrated liquid is 3% is added into concentrate while stirring for concentrate,
It is stood overnight after mixing at 4 DEG C, removes precipitating after being centrifuged 30min, obtain supernatant;
S3, the supernatant is adjusted to neutrality using sodium hydroxide, it is 50%, 4 that dehydrated alcohol to concentration of alcohol, which is added,
Be centrifuged 30min after DEG C standing overnight, collect precipitating, will precipitating dehydrated alcohol oscillation washing is added after stand centrifugation, in triplicate,
Crispy gracilaria Thick many candies are made.
S4, the crispy gracilaria Thick many candies are dissolved with pure water, in the bag filter that molecule interception is 8kDa, will be dialysed
Bag is immersed in pure water, 4 DEG C of dialysed overnights, repetition dialysis 8 times, when the pure water pH that dialyses is neutral, stops dialysing, in collecting bag
Dialyzate;
S5, dialyzate is freezed for 24 hours in -20 DEG C of refrigerators, is then lyophilized with vacuum freeze drier, it is more that crispy gracilaria is made
Sugar is stored in drier.
Embodiment 2, crispy gracilaria polysaccharide of the present invention and preparation method thereof
2 crispy gracilaria polysaccharide of the embodiment of the present invention and preparation method thereof, comprising the following steps:
S1, fresh crispy gracilaria pulverize and sieve after drying after rinsing and draining, and carry out condensing reflux using dehydrated alcohol and add
Heat treatment, is repeated twice, and filters, and filter residue drying obtains crispy gracilaria dried powder;
S2, the crispy gracilaria dried powder is impregnated, solid-to-liquid ratio 1:10 using pure water, extracts 3.5h at 85 DEG C, extracts
Period is intermittently stirred, and in triplicate, filtered extracting solution merges, and extracting solution is after rotavapor under vacuum is concentrated
Concentrate is obtained, three chloroethenes that the mass concentration that volume is 0.4 times of the volume of the concentrated liquid is 5% are added while stirring into concentrate
Acid is stood overnight at 4 DEG C after mixing, is removed precipitating after being centrifuged 30min, is obtained supernatant;
S3, the supernatant is adjusted to neutrality using sodium hydroxide, it is 55%, 4 that dehydrated alcohol to concentration of alcohol, which is added,
Be centrifuged 30min after DEG C standing overnight, collect precipitating, will precipitating dehydrated alcohol oscillation washing is added after stand centrifugation, in triplicate,
Crispy gracilaria Thick many candies are made.
S4, the crispy gracilaria Thick many candies are dissolved with pure water, in the bag filter that molecule interception is 9kDa, will be dialysed
Bag is immersed in pure water, 4 DEG C of dialysed overnights, repetition dialysis 8 times, when the pure water pH that dialyses is neutral, stops dialysing, in collecting bag
Dialyzate;
S5, dialyzate is freezed for 24 hours in -20 DEG C of refrigerators, is then lyophilized with vacuum freeze drier, it is more that crispy gracilaria is made
Sugar is stored in drier.
The pharmaceutical composition of embodiment 3, the present invention treatment cancer of the esophagus
The embodiment of the present invention 3 treats the pharmaceutical composition of the cancer of the esophagus, including the crisp of 2 preparation method of embodiment of the present invention preparation
Gracilaria gigas Harvey polysaccharides and 5 FU 5 fluorouracil.
The weight ratio of the crispy gracilaria polysaccharide and 5 FU 5 fluorouracil is 1:1.
The pharmaceutical composition of embodiment 4, the present invention treatment cancer of the esophagus
The embodiment of the present invention 4 treats the pharmaceutical composition of the cancer of the esophagus, including the crisp of 2 preparation method of embodiment of the present invention preparation
Gracilaria gigas Harvey polysaccharides and 5 FU 5 fluorouracil.
The weight ratio of the crispy gracilaria polysaccharide and 5 FU 5 fluorouracil is 2.5:1.
The pharmaceutical composition of embodiment 5, the present invention treatment cancer of the esophagus
The embodiment of the present invention 5 treats the pharmaceutical composition of the cancer of the esophagus, including the crisp of 2 preparation method of embodiment of the present invention preparation
Gracilaria gigas Harvey polysaccharides and 5 FU 5 fluorouracil.
The weight ratio of the crispy gracilaria polysaccharide and 5 FU 5 fluorouracil is 5:1.
The toxicity test of test example one, crispy gracilaria polysaccharide
1, test material: the crispy gracilaria polysaccharide of the preparation of embodiment 2, source of mouse macrophage cell line (RAW264.7), people are single
Nucleus cell strain (THP-1), pancreatin, Australia fetal calf serum, 1640 culture mediums and DMEM culture medium;
2, cytotoxicity assay test method: is carried out using CCK8 method, comprising the following steps:
S1, RAW264.7 source of mouse macrophage cell line pass on training with the DMEM culture medium containing 10% Australia fetal calf serum
It supports to logarithmic growth phase, is digested with the pancreatin that concentration is 0.25%, fresh culture centrifugation is added and abandons supernatant, then plus fresh cultured
Cell suspending liquid is made in base, counts, is configured to 10 × 106The RAW264.7 source of mouse macrophage suspension of a/L;
S2,1640 culture medium secondary cultures of the person monocytic cell's cell strain (THP-1) containing 10% Australia fetal calf serum
To logarithmic growth phase, supernatant is abandoned in centrifugation, fresh culture is added, cell is resuspended, count, be configured to 10 × 106The cell of a/L is outstanding
20ng/mLPMA induction is added into human monocytemacrophage suspension in liquid;
S3, the RAW264.7 source of mouse macrophage suspension or human monocytemacrophage suspension that 100 μ L are added into 96 orifice plates,
After static waiting 5min, culture plate horizontal transfer is placed in 37 DEG C, 5%CO2Incubator adhere-wall culture is for 24 hours;
S4, adhere-wall culture for 24 hours after 96 orifice plates abandon supernatant, be separately added into the concentration gradient of 90 μ L embodiment 2 prepare
Crispy gracilaria polysaccharide solution (0,5,10,20,40,80,160 and 320 μ g/mL), after incubator is incubated for 48h, with PBS with the ratio of 1:1
After example dilution CCK-8 solution, 10 μ L CCK-8 dilute solutions are added to culture plate, are used after culture plate is incubated for 1h in incubator
Microplate reader measures the absorbance at 450nm.
3, test result is as shown in table 1.
Toxicity test result of the 1 crispy gracilaria polysaccharide of table to RAW264.7 source of mouse macrophage and person monocytic cell
By upper table 1 and Fig. 1 it is found that the crispy gracilaria polysaccharide for preparing of embodiment 2 in concentration is within the scope of 0~20 μ g/mL to small
Mouse RAW264.7 macrophage does not have cytotoxic effect, is being not have within the scope of 0~20 μ g/mL to person monocytic cell in concentration
Cytotoxic effect.
Test example two, the measurement of crispy gracilaria polysaccharide anti-inflammatory activity
1, test material: the crispy gracilaria polysaccharide of Examples 1 to 2 preparation, male mice 40;
2, test method, comprising the following steps:
Male Kunming strain mice 40 is taken only to be randomly divided into blank control group (physiological saline group), aspirin group, embodiment 1
~2 groups, every group 10, each group intragastric administration on mice is administered 7 days, once a day, dosage 0.2mL/10g, fasting before last dose
It can't help water 12h, after last dose 0.5h, the wide front and back two sides of every mouse right ear uniformly applies dimethylbenzene 0.3mL and causes scorching, and left ear is opposed
According to cervical dislocation puts to death mouse after 1h, cuts bilateral auricle along auricle base line, is swept away with diameter 6mm punch in the same position of ear
Two ear auricles, are weighed with electronic balance respectively immediately, indicate the swelling of ear inflammation with the difference of two auricle weight of left and right, and count
It calculates swelling inhibiting rate (%), the calculation formula of swelling inhibiting rate (%) is as follows:
Swelling inhibiting rate (%)=(blank control group be averaged swelling-administration group be averaged swelling)/blank group is averagely swollen
Expansibility × 100%;
3, test result: as shown in table 2.
The anti-inflammatory activity measurement result (x ± s, n=10) of 2 crispy gracilaria polysaccharide of table
Remarks: examining through t, compared with the control group, P < 0.05.
By upper table 2 it is found that compared with blank control group, crispy gracilaria polysaccharide prepared by the embodiment of the present invention 1~2 can be significant
Ground inhibits the ear swelling of mouse, shows good antiphlogistic effects, wherein best with the effect of embodiment 2, for it is of the invention most
Good embodiment illustrates that crispy gracilaria polysaccharide prepared by the present invention shows good anti-inflammatory activity.
Test example three, crisp river hedge polysaccharide anti esophageal cancer determination of activity
1, test material: crispy gracilaria polysaccharide, the esophageal cancer cell ECA109, pancreatin, Australia tire ox blood of the preparation of embodiment 2
Clearly, 1640 culture mediums, 5 FU 5 fluorouracil.
2, test method:
(1) crispy gracilaria polysaccharide detects esophageal cancer cell ECA109 survival rate, comprising the following steps:
1640 culture medium 37 DEG C of S1, cell culture: the esophageal cancer cell ECA109 containing 10% Australia fetal calf serum and
5% CO2It when concentration culture to logarithmic growth phase, is digested with 0.25% pancreatin, fresh culture centrifugation is added and abandons weight after supernatant
It is outstanding, it counts, is configured to 10 × 107The cell suspension of a/L, inoculating cell to 96 porocyte culture plates, it is thin that 100 μ l are added in every hole
Born of the same parents' suspension, cell number are 1 × 105, 37 DEG C, 5%CO2Incubator is incubated for for 24 hours;
S2, dosing: adhere-wall culture for 24 hours after, add containing concentration gradient crispy gracilaria polysaccharide solution (0,25,50,100,
200 μ g/mL) crispy gracilaria polysaccharide culture medium, every group of 3 multiple holes, 37 DEG C, 5%CO2Incubator is incubated for for 24 hours;
After S3, incubation for 24 hours, 5 μ L CCK-8 solution are added in every hole, and culture plate is placed in incubator and is incubated for 1h, are used
Microplate reader measures the absorbance at 450nm, and calculates cell opposite proliferation rate.
(2) crisp river hedge polysaccharide detects the antitumor synergistic effect of clinical chemotherapy medicine 5 FU 5 fluorouracil, comprising the following steps:
S1, by esophageal cancer cell ECA109 with 5 × 106/ L's is inoculated in fresh 1640 culture medium (containing 89%
1640 culture mediums, 10% Australia fetal calf serum FBS, the mycillin of 1% 1U/mL) in culture, in 37 DEG C, saturated humidity
Under the conditions of, it is placed in 5%CO2It is cultivated in incubator, liquid or passage 1 time is changed within every 2~3 days, by cell culture to logarithmic growth phase
Cell;
S2, the cell of above-mentioned logarithmic growth phase is digested, PBS washing simultaneously cell counter counting with 0.25% pancreatin, is used
1640 culture medium diluting cells containing 10% fetal calf serum, being adjusted to cell density is 1 × 104The cell suspension of/mL, is inoculated in
In 96 well culture plates, every 200 μ L of hole makes every hole cell density 2 × 103A, every group sets 3 parallel multiple holes, while setting 4 holes
In only plus 10%FBS RMPI 1640 culture medium, not celliferous blank group, for returning to zero;
S3, by above-mentioned 96 orifice plate under 37 DEG C, saturated humidity, be placed in 5%CO2Cell incubator in be incubated overnight, to
After cell is adherent, 20 μ g/mL are added to change every hole in the form of liquid 5 FU 5 fluorouracil and the combination of 3~5 anti esophageal cancer drug of embodiment
Object, and set negative control hole;
S4, after 48 hours, according to CCK8 kit specification, the training containing 10%CCK-8 is added in every Kong Yihuan liquid form
Base 200L is supported, continues to cultivate 2.5h, each hole light absorption value A of automatic elisa reading instrument Detection wavelength 450nm, versus cell is calculated and increases
Grow rate.
3, test result is as shown in Table 3 and Table 4.
3 crispy gracilaria polysaccharide of table is to esophageal cancer cell ECA109 survival rate measurement result
4 anti esophageal cancer pharmaceutical composition of table is to esophageal cancer cell ECA109 survival rate measurement result
Project | Survival rate |
Negative control group | 99.8 |
5 FU 5 fluorouracil | 70.3* |
Embodiment 3 | 46.4* |
Embodiment 4 | 40.7* |
Embodiment 5 | 44.9* |
Note:*Indicate that difference is extremely significant (P < 0.01) compared with negative control group.
(1) You Shangbiao 3 it is found that the crispy gracilaria polysaccharide for preparing of embodiment 2 that the cancer of the esophagus is reduced in a manner of concentration dependant is thin
The survival rate of born of the same parents ECA109 illustrates that crispy gracilaria polysaccharide of the present invention has certain inhibiting effect to the cancer of the esophagus;
(2) You Shangbiao 4 is it is found that compared with negative control group, be used alone 5 FU 5 fluorouracil to esophageal cancer cell
ECA109 has certain inhibiting effect, compared with 5 FU 5 fluorouracil group, 3~5 crispy gracilaria polysaccharide of embodiment and 5 FU 5 fluorouracil medicine
Compositions group obviously increases the inhibiting effect of esophageal cancer cell ECA109, wherein it is best with the inhibitory effect of embodiment 4, be
Highly preferred embodiment of the present invention illustrates that crispy gracilaria polysaccharide of the present invention increases the tumor killing effect of 5 FU 5 fluorouracil, urinates 5- fluorine phonetic
Pyridine plays synergistic effect.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. a kind of preparation method of crispy gracilaria polysaccharide, which comprises the following steps:
S1, crisp river hedge are pulverized and sieved weighing, are pre-processed using dehydrated alcohol after rinsing and draining after drying, drying obtains
Crispy gracilaria dried powder;
S2, crispy gracilaria dried powder is impregnated with pure water, solid-to-liquid ratio is 1:(7~15), 3~4h is extracted at 80~90 DEG C, is extracted
Period is intermittently stirred, and in triplicate, filtered extracting solution merges, and obtains concentrate after extracting solution vacuum concentration,
The trichloroacetic acid that mass concentration is 3~7% is added while stirring into concentrate, is stood overnight at 4 DEG C, is centrifuged 25~35min
Precipitating is removed, supernatant is obtained;
S3, the supernatant is adjusted to neutrality using sodium hydroxide, it is 50~60%, 4 that dehydrated alcohol to concentration of alcohol, which is added,
Be centrifuged 25~35min after DEG C standing overnight, discard supernatant liquid, collect precipitating, will precipitating be added it is quiet after dehydrated alcohol oscillation washing
Centrifugation is set, in triplicate, crispy gracilaria Thick many candies are made.
S4, the crispy gracilaria Thick many candies are dissolved with pure water, in the bag filter that molecule interception is 8~10kDa, will be dialysed
Bag is immersed in pure water, 4 DEG C of dialysed overnights, repeats dialysis 5~10 times, when the pure water pH that dialyses is neutral, is stopped dialysis, is collected
Dialyzate in bag;
S5, dialyzate is freezed for 24 hours in -20 DEG C of refrigerators, is then lyophilized with vacuum freeze drier, crispy gracilaria polysaccharide is made, is protected
It is stored in drier.
2. the preparation method of crispy gracilaria polysaccharide as described in claim 1, which is characterized in that use dehydrated alcohol in the step S1
Pretreated operation is carried out to be condensed back heat treatment, is repeated twice.
3. the preparation method of crispy gracilaria polysaccharide as described in claim 1, which is characterized in that the solid-to-liquid ratio in the step S2 is 1:
10。
4. the preparation method of crispy gracilaria polysaccharide as described in claim 1, which is characterized in that the Extracting temperature in the step S2 is
85 DEG C, extraction time 3.5h.
5. the preparation method of crispy gracilaria polysaccharide as described in claim 1, which is characterized in that trichloroacetic acid adds in the step S2
Enter 0.3~0.5 times that volume is the volume of the concentrated liquid, the mass concentration of the trichloroacetic acid is 5%.
6. the crispy gracilaria polysaccharide obtained of the preparation method of crispy gracilaria polysaccharide as described in Claims 1 to 5 is any.
7. crispy gracilaria polysaccharide application in preparing anti-inflammatory drugs as claimed in claim 6.
8. application of the crispy gracilaria polysaccharide as claimed in claim 6 in preparation treatment oesophagus cancer drug.
9. a kind of pharmaceutical composition for treating the cancer of the esophagus, which is characterized in that described pharmaceutical composition includes as claimed in claim 6
Crispy gracilaria polysaccharide and 5 FU 5 fluorouracil.
10. the pharmaceutical composition of the treatment cancer of the esophagus as claimed in claim 8, which is characterized in that the crispy gracilaria polysaccharide and 5- fluorine
The weight ratio of uracil is (1~5): 1.
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