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 PDF

Info

Publication number
CN109593140A
CN109593140A CN201811640069.5A CN201811640069A CN109593140A CN 109593140 A CN109593140 A CN 109593140A CN 201811640069 A CN201811640069 A CN 201811640069A CN 109593140 A CN109593140 A CN 109593140A
Authority
CN
China
Prior art keywords
crispy
gracilaria
polysaccharide
preparation
gracilaria polysaccharide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811640069.5A
Other languages
Chinese (zh)
Other versions
CN109593140B (en
Inventor
赵振岭
王飞
王一飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Jinan Biomedicine Research and Development Base Co Ltd
Original Assignee
Guangzhou Jinan Biomedicine Research and Development Base Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Jinan Biomedicine Research and Development Base Co Ltd filed Critical Guangzhou Jinan Biomedicine Research and Development Base Co Ltd
Priority to CN201811640069.5A priority Critical patent/CN109593140B/en
Publication of CN109593140A publication Critical patent/CN109593140A/en
Application granted granted Critical
Publication of CN109593140B publication Critical patent/CN109593140B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Materials Engineering (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

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

A kind of crispy gracilaria polysaccharide and its preparation method and application
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.
CN201811640069.5A 2018-12-29 2018-12-29 Gracilaria crispatus polysaccharide and preparation method and application thereof Active CN109593140B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811640069.5A CN109593140B (en) 2018-12-29 2018-12-29 Gracilaria crispatus polysaccharide and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811640069.5A CN109593140B (en) 2018-12-29 2018-12-29 Gracilaria crispatus polysaccharide and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN109593140A true CN109593140A (en) 2019-04-09
CN109593140B CN109593140B (en) 2021-04-16

Family

ID=65965470

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811640069.5A Active CN109593140B (en) 2018-12-29 2018-12-29 Gracilaria crispatus polysaccharide and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN109593140B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112494347A (en) * 2020-12-22 2021-03-16 汕头大学 Skin care water emulsion and preparation method thereof
CN115746160A (en) * 2022-11-30 2023-03-07 南方海洋科学与工程广东省实验室(广州) Method for reducing viscosity of polysaccharide

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1154137A (en) * 1968-02-27 1969-06-04 Krauss Maffei Ag Treating Marine Algae.
CN101475651A (en) * 2009-01-16 2009-07-08 伍曾利 Preparation of Gracilaria gigas Harvey polysaccharides
CN101613419A (en) * 2008-06-23 2009-12-30 广东海洋大学 The method of ultrasonic acid extraction of sulfated polysaccharide from gracilaria tenuistipitata
CN103694374A (en) * 2013-12-18 2014-04-02 青岛福创环境科技有限公司 Method for extracting agar, fucoidin and protein from gracilaria as raw material by using enzyme process
CN106699913A (en) * 2016-11-29 2017-05-24 福建农林大学 Method for efficiently preparing gracilaria sulfated polysaccharides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1154137A (en) * 1968-02-27 1969-06-04 Krauss Maffei Ag Treating Marine Algae.
CN101613419A (en) * 2008-06-23 2009-12-30 广东海洋大学 The method of ultrasonic acid extraction of sulfated polysaccharide from gracilaria tenuistipitata
CN101475651A (en) * 2009-01-16 2009-07-08 伍曾利 Preparation of Gracilaria gigas Harvey polysaccharides
CN103694374A (en) * 2013-12-18 2014-04-02 青岛福创环境科技有限公司 Method for extracting agar, fucoidin and protein from gracilaria as raw material by using enzyme process
CN106699913A (en) * 2016-11-29 2017-05-24 福建农林大学 Method for efficiently preparing gracilaria sulfated polysaccharides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
鞠瑶瑶: ""脆江蓠多糖的提取分离及抗肿瘤活性研究"", 《中国学术期刊数据库》 *
鞠瑶瑶等: ""响应面试验优化脆江蓠多糖提取工艺及其对肿瘤细胞的抑制作用"", 《食品科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112494347A (en) * 2020-12-22 2021-03-16 汕头大学 Skin care water emulsion and preparation method thereof
CN115746160A (en) * 2022-11-30 2023-03-07 南方海洋科学与工程广东省实验室(广州) Method for reducing viscosity of polysaccharide
CN115746160B (en) * 2022-11-30 2024-04-02 南方海洋科学与工程广东省实验室(广州) Method for reducing viscosity of polysaccharide

Also Published As

Publication number Publication date
CN109593140B (en) 2021-04-16

Similar Documents

Publication Publication Date Title
CN105085711B (en) A kind of preparation method and applications of chitosan oligosaccharide
CN105816430B (en) A kind of preparation method with antitumor action radix tetrastigme polyoses grain
CN105017438B (en) A kind of Radix et Rhizoma Gynurae divaricatae polysaccharide and its application in preparing for immunological regulation and anti-tumor drug and functional food
CN106064993A (en) The culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers., bioconversion mycelium, the mycelial extract of bioconversion and application thereof
CN109593140A (en) A kind of crispy gracilaria polysaccharide and its preparation method and application
KR100770775B1 (en) Functional drink for immune- enhancing and Manufacturing method thereof
CN105664140A (en) Glycopeptide composition as well as preparation method and application thereof
CN106421208B (en) Pharmaceutical composition with chemical liver injury resistance function and preparation method thereof
CN104277136A (en) Yellow river beach date polysaccharides, and extraction and refining method and application thereof
CN105796587B (en) Caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application
KR102251825B1 (en) Composition to inhibit liver damage using mushroom mycelia germinated on naked barley medium and Manufacturing method thereof
CN104489172A (en) Acanthopanax health tea for improving immunity and preparation method thereof
CN102754833A (en) Healthcare product with function of enhancing immunity and preparation method thereof
CN105533745A (en) Ginseng and honeysuckle tablets and production method thereof
CN107173805A (en) It is a kind of that there is the health food for preventing and treating lung cancer function
CN107375065A (en) It is a kind of that there is the composition and preparation method for safeguarding and recovering the effect of skin Tiny ecosystem
CN106389477A (en) Preparation method and application of whole-cell vegetable oil extracts of gordona terrae
CN101711790A (en) Wild Juglans mandshurica bark water extract used for curing liver cancer
CN105012366B (en) A kind of bright moon grass polysaccharide and its application in preparing for immunological regulation and anti-tumor drug and functional food
CN108635445A (en) The application of rodgersflower rhizome flavones, Cortex Phellodendri flavones and Radix Angelicae Sinensis polysaccharide compound in preparing veterinary drug
CN104353057A (en) Hypertension-reducing healthy product containing bird cherry anthocyanin
CN105106950B (en) A kind of immunologic adjuvant of live vaccine, preparation method and applications
Cui et al. Antitumor and immunoregulation effects and mechanism of n-butanol fraction from Zanthoxylum avicennae in H22 mice
CN109497511A (en) A kind of fungal extract composition and its preparing the application in functional food and health care product
CN102068537B (en) The preparation of Pericarpium Citri Reticulatae Radix Glycyrrhizae prevents and treats the health food of nasopharyngeal carcinoma and the production method of medicine

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant