CN109485706A - A kind of cordate houttuynia glycoprotein and its preparation and application - Google Patents
A kind of cordate houttuynia glycoprotein and its preparation and application Download PDFInfo
- Publication number
- CN109485706A CN109485706A CN201811300319.0A CN201811300319A CN109485706A CN 109485706 A CN109485706 A CN 109485706A CN 201811300319 A CN201811300319 A CN 201811300319A CN 109485706 A CN109485706 A CN 109485706A
- Authority
- CN
- China
- Prior art keywords
- glycoprotein
- cordate houttuynia
- norovirus
- supernatant
- houttuynia
- 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
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Botany (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of cordate houttuynia glycoprotein and its preparation and application, cordate houttuynia glycoprotein sterling yield prepared by the present invention is 1.245-1.827%, total Sugar concentration 55.6-62.9%, uronic acid mass concentration 15.3-19.5%, protein quality concentration 9.52-10.87% in glycoprotein.Cordate houttuynia glycoprotein of the present invention is greater than or equal to 50mg/mL and is both greater than 85% to the inhibiting rate of people's norovirus GI, GII type and mouse norovirus MNV-1 effect 120min, can be used in preparing norovirus activity inhibitor.
Description
(1) technical field
The present invention relates to a kind of cordate houttuynia glycoprotein, in particular to a kind of cordate houttuynia glycoprotein and its preparation with anti-promise such as
Application in virus.
(2) background technique
Norovirus (Norovirus, NoV) belongs to the norwalk virus category of Caliciviridae (Caliciviruses), most
It is early to be found for the first time with the method for Electronic Speculum by scholars such as Kapikian in 1972.It is compiled generally according to NoV viral rna polymerase
The difference of code area's nucleotide or coat protein region amino acid sequence, can be classified as 5 different genomes (GGI, GGII,
GGIII, GGIV and GGV).Wherein GI, GII and GIV type main infection people, and GV type main infection mouse.Norovirus is infectious
It by force, is to cause one of most commonly encountered diseases pathogenic microorganism of acute human gastroenteritis, wherein GII type is that the prevalence that China has been reported that is excellent
Gesture strain can usually be propagated by the water source of pollution, food, article, air etc..Norovirus generally passes through excrement-oral instructions and broadcasts,
Transmissibility is strong, and winter-spring season is the high-incidence season of norovirus, easily infection population especially infant, causes diarrhea.Do not have at present
There is vaccine that can prevent norovirus, antiviral drugs also not special in treatment.
After the first norovirus cases of infection are reported from nineteen ninety-five by China, norovirus sense is repeatedly broken out in domestic many areas
Metachromia acute gastroenteritis case (epidemic situation is concentrated mainly on Guangdong and Zhejiang Province).On October 17th, 2016, Harbin Medical University
It is uncomfortable to be connected to 43 student response digestive systems, vomiting occurs, generates heat, the symptom of diarrhea, be diagnosed as norovirus infection afterwards.
Since in late January, 17, there are the diseases such as aggregation student's nausea and vomiting diarrhea in Zhejiang Jiaxing city Haining, sea salt two places some schools
Shape, is detected through local Disease Control and Prevention Center, is equally determined as norovirus infection.According to the epidemic disease of Zhejiang Province's health and Family Planning Committee
Feelings notification, 2016, our province retrieved norovirus infectious diarrhea case 281 altogether, involved 9613 people；Report epidemic outbreaks
7.
Food-borne promise such as disease break out it is most with eat contaminated water and food is related, how effectively to remove virus
Contact scar is a major issue concerning people's health.Polysaccharide compound refer to polyhydroxy aldehyde or polyhydroxyketone and
The general name of its condensation polymer and certain derivatives, is widely present in plant kingdom, such as cordate houttuynia, trollflower, motherwort, ginkgo
Polysaccharide compound all rich in inside leaf, hawthorn, sugarcane, propolis etc. is that a kind of have antibacterial, anti-oxidant, anti-inflammatory exempt from
The native compound of a variety of pharmacological activity such as epidemic disease.Studies have shown that many polysaccharide components have stronger antivirus action and secondary work
It is highly-safe with small.Nakano in 1997 etc. has significantly with the polysaccharide that 1% sodium carbonate is extracted from plant Rooibos leaf
HIV-resistant activity, Seungbo R et al. reported different natural drug components in 2015 to the antiviral effect of norovirus,
Wherein just there is polysaccharide plant.
Cordate houttuynia (Classification system: HouttuyniacordataThunb.), originates in each province on the south China Yangtze river basin." name
Doctor records " in Tang Su song say: " give birth to wetland, can also overgrow at the yin of mountain valley, leaf such as buckwheat and fertile, stem puniceus, river Zuoren is quite
Food, the marshland full of water weeds dish of Central Shanxi Plain meaning, leaf has stink smell, therefore is commonly called as: cordate houttuynia." cordate houttuynia polysaccharide has certain biological function and medicinal
Value.Meng Jiang etc. determines the polyoses content of different sources cordate houttuynia using phend-sulphuric acid, finds polyoses content in cordate houttuynia
Polyoses content for 15.73-20.29%, and different sources cordate houttuynia has a certain difference.Zhou Qiang is obtained using water extraction
A kind of cordate houttuynia neutral polysaccharide constituted by adjoining sugar of muttering, molecular weight 38000Da, by xylose, arabinose, glucose, half
Four kinds of monosaccharide compositions of lactose, molar composition ratio example is 2.21:1:2.79:2.Cordate houttuynia also has inhibiting effect to a variety of viruses.
Modern pharmacology research prove the steam distillation object of fresh cordate houttuynia to simple sore rash I type viral (HSV-I), influenza virus and
Human immunodeficiency I type viral (HIV-I) has direct inhibitory activity, and no cytotoxicity.The researchs such as Zheng Xuanhe have shown that cordate houttuynia
Also there is certain inhibiting effect for hemorrhagic fever viruse (EHFV).Experimental study shows that cordate houttuynia water extract has and inhibits SARS disease
The effect of poison.However inhibiting effect of the houttuynia extract to norovirus is had not been reported at present.
In conclusion the anti-norovirus effect of research cordate houttuynia polyoses extract and its effective ingredient, reduce promise such as disease
Poison infection ensures people's life health, and has great theoretical and practical significance to the control of norovirus.
(3) summary of the invention
It is an object of the present invention to provide a kind of cordate houttuynia glycoprotein and its preparation and application, cordate houttuynia glycoprotein of the present invention influences
The integrality of norovirus shell, suppressing virus replication related gene expression, can be used in preparing norovirus bacteriostatic agent and
It is used to prepare prevention or treatment norovirus anti-diarrhea drug.
The technical solution adopted by the present invention is that:
The present invention provides a kind of cordate houttuynia glycoprotein, and the glycoprotein is prepared as follows: (1) being gone fresh cordate houttuynia
Drying after miscellaneous, crushes and (preferably crosses 80 meshes), obtains Heartleaf Houttuynia Herb；
(2) it takes Heartleaf Houttuynia Herb to be added in distilled water, adds cellulase, carry out enzyme digestion reaction in 55 DEG C of constant temperature stirrings
(preferably 2-4h) after fully reacting, enzymolysis liquid is once centrifuged, obtains a supernatant primary sedimentation；It collects and resets and add on primary
Enter activated carbon to be stirred at room temperature colour fading (preferably 30min), secondary centrifuging obtains secondary supernatant secondary precipitation；Take secondary supernatant again
Trichloroacetic acid is added, stands overnight after shaking up, is centrifuged three times, obtains supernatant three times and precipitates three times；Take supernatant abandoning three times three times
Precipitating, in dialysing (preferably 48h) in bag filter by dialyzate of secondary distilled water, dialyzate is concentrated into 1/5 volume in bag taking, to
After ethyl alcohol progress alcohol precipitation is added in concentrate overnight, supernatant is abandoned in centrifugation, and precipitating as cordate houttuynia glycoprotein crude product obtains after purification
Obtain cordate houttuynia glycoprotein；The distilled water volume dosage is calculated as 50-60ml/g (preferably 55ml/g) with Heartleaf Houttuynia Herb quality；Institute
It states cellulase and Heartleaf Houttuynia Herb mass ratio is 0.001-0.15:1 (preferably 0.001:1)；The trichloroacetic acid additional amount with
Secondary supernatant volume is calculated as 20-30g/L, preferably 30g/L.
Further, step (1) drying condition is 50 DEG C, is dried to until capable of forming powder.
Further, step (2) cellulose enzyme activity be 25U/mg, the primary centrifugation, secondary centrifuging, three times be centrifuged and from
Heart condition is 8000 × g centrifugation 10min, and dialysis bag retention molecular weight is MW 14,000Da.
Further, step (2) ethyl alcohol volume additional amount is calculated as 60-70%, preferably 70%, the activity with the volume of the concentrated liquid
Charcoal additional amount is calculated as 3-5g/L, preferably 5g/L with a supernatant volume.
Further, step (2) method of purification are as follows: cordate houttuynia glycoprotein crude product is taken to be configured to concentration 100g/L with deionized water
Crude product solution, by DEAE-52 ion exchange column on crude product and resin quality ratio 1:30, be first washed with deionized water slough except neutrality
Polysaccharide, then with 0-0.5mol/L NaCl aqueous solution gradient elution, detected using Phenol-sulphate acid method, collect the stream containing desired polysaccharide
Liquid (preferably continuously collects eluent using the glass tube of 10mL specification, every pipe acquisition time is 5min, the elution in collecting pipe out
Liquid using Phenol-sulphate acid method detection polyoses content and merges the collection liquid in the continuous collecting pipe number of higher polyoses content, obtains
Polysaccharide collection liquid), then by the efflux containing desired polysaccharide through Sephadex G-50 (fine, 1.8x 135cm, Pharmacia)
Pillar desalination, and the efflux with specified molecular weight (30-50kDa) protein peak is collected under 280nm, freeze-drying, as
Cordate houttuynia glycoprotein.
The present invention also provides a kind of cordate houttuynia glycoprotein to prepare the application in norovirus activity inhibitor, described
Norovirus is people's norovirus GI type, people's norovirus GII type or mouse norovirus MNV-1, the norovirus activity suppression
Preparation is non-structural protein RdRp (NS7 gene), VPg (NS5 gene) expression inhibiting agent.
Cordate houttuynia (English name HERBA HOUTTUYNIAE) of the present invention is the herbal medicine that Chinese Pharmacopoeia is included, and herbal medicine comes
Source is the dry aerial parts of saururaceae plant houttuynia cordata (Classification system: Houttuynia cordata Thunb.).
Compared with prior art, beneficial effect of the present invention is mainly reflected in: cordate houttuynia glycoprotein sterling prepared by the present invention
Yield is 1.245-1.827%, total Sugar concentration 55.6-62.9%, uronic acid mass concentration 15.3- in glycoprotein
19.5%, protein quality concentration 9.52-10.87%.Cordate houttuynia glycoprotein of the present invention be greater than or equal to 50mg/mL to people's promise such as
The inhibiting rate of viral GI, GII type and mouse norovirus MNV-1 effect 120min are both greater than 85%, can be used in preparing promise such as disease
Cytotoxic activity inhibitor.
(4) Detailed description of the invention
The scanning electron microscope (SEM) photograph of Fig. 1 mouse norovirus MNV-1；A: arrow meaning is that cordate houttuynia glycoprotein handles provirus shape
State；B: arrow meaning is that cordate houttuynia glycoprotein handles restrovirus form.
The influence that Fig. 2 cordate houttuynia glycoprotein expresses mouse norovirus nonstructural protein gene；A:NS5 gene；B:NS7 base
Cause；C: the influence of various concentration cordate houttuynia glycoprotein and 2TU to NS5 and NS7 gene expression amount.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This: room temperature described in the embodiment of the present invention refers to 25-30 DEG C.
Embodiment 1: cordate houttuynia glycoprotein
(1) the fresh cordate houttuynia that will be bought from market is dried in 50 DEG C of baking ovens until capable of forming powder, powder after impurity elimination
It is broken, 80 meshes are crossed, sealing is placed on spare in drier respectively；
(2) it takes step (1) Heartleaf Houttuynia Herb 5g in 2L beaker, is added distilled water (cordate houttuynia (g): water (ml)=1:55)
275mL adds 5mg cellulase (enzyme activity 25U/mg), enzyme digestion reaction is carried out in 55 DEG C of constant temperature stirring 2h, by enzymolysis liquid 8000
× g is once centrifuged 10min, obtains a supernatant primary sedimentation, and addition activated carbon makes its concentration 5g/ after collecting a supernatant
L, is stirred at room temperature colour fading 30min, and 8000 × g secondary centrifuging 10min obtains secondary supernatant secondary precipitation；Take secondary supernatant again
Addition trichloroacetic acid is stood overnight after shaking up its final concentration of 30g/L；8000 × g is centrifuged 10min three times, obtains supernatant three times
It precipitates three times；Supernatant abandoning three times is taken to precipitate three times, in saturating by dialyzate of secondary distilled water in bag filter (MW 14,000Da)
48h is analysed, the outer sample (i.e. permeate) of bag filter is then concentrated to 1/5 volume, ethyl alcohol, which is added, makes ethyl alcohol volume dense eventually (v/v) degree be
70%, alcohol precipitation is centrifuged 10min in 8000 × g after overnight and abandons supernatant, and precipitating is cordate houttuynia glycoprotein crude product 1.132g.
(3) it takes a certain amount of cordate houttuynia glycoprotein crude product to be configured to the glycoprotein solution of concentration 100g/L with distilled water, presses
DEAE-52 ion exchange column on (glycoprotein: resin) mass ratio 1:30.It is washed with deionized water and sloughs except neutral polysaccharide, then with not
With (0-0.5mol/L) NaCl aqueous solution gradient elution of concentration, eluent is continuously collected using the glass tube of 10mL specification, often
Pipe acquisition time is 5min, and the eluent in collecting pipe is using Phenol-sulphate acid method detection polyoses content and by higher polyoses content
Collection liquid in continuous collecting pipe number merges, and obtains efflux containing desired polysaccharide, then the efflux containing desired polysaccharide is passed through
Sephadex G-50 (fine, 1.8x 135cm, Pharmacia) pillar desalination, collecting molecular weight at 280nm is 30-50kDa
Protein peak efflux, freeze-drying, as cordate houttuynia glycoprotein sterling (HCTE), 1g crude product can obtain 0.067g sterling.
Cordate houttuynia glycoprotein sterling quality yield is 1.347%, and the molecular weight of sterling is about 45kDa (High Performance Gel Permeation
Chromatography HPGPC), quality group become (Phenol-sulphate acid method) total reducing sugar 58.29%, (sulfate-carbazole) uronic acid 18.5%,
(Lowry method) protein 9.58%.
Embodiment 2: influence of the cordate houttuynia glycoprotein to norovirus activity inhibition and its protein coat integrality
1, test material
Virus: people norovirus GI type (NoV GI), people's norovirus GII type (NoV GII) are (by Zhejiang Province's disease prevention
Control centre provides), mouse norovirus MNV-1 (be purchased from Chinese Academy of Sciences's Culture Collection).
Trial drug: cordate houttuynia glycoprotein sterling prepared by embodiment 1 is dissolved in the water, and is prepared into 100mg/mL cordate houttuynia
Glycoprotein mother liquor.
Detection kit: (hair R- visits in Germany for QIAGEN virus RNA extraction kit, norovirus ELISA kit
Biopharm company)；One Step PrimescriptTM RT-PCR Kit is purchased from TaKaRa company.
Key instrument: ABI7500 fluorescence quantitative PCR instrument, Hitachi scanning electron microscope TM4000, microplate reader.
2, test method
(1) antivirus action time-dependent relation is tested: taking norovirus (NoV GI) and norovirus (NoV respectively
GII) 100 μ L of Feces of Patients With Diarrhea sample homogenization liquid supernatant, 100mg/mL cordate houttuynia glycoprotein mother liquor, which is added, keeps its final concentration of
10mg/mL tests every time while being arranged negative control and positive control pipe, and same amount of normal saline is added in negative control pipe, positive
50 μM of 2-TU (2- thio uridine) is added in control tube, and incubation time is respectively 0,5,15,30,60,120min at room temperature, collects
The sample of different time processing simultaneously detects viral residual quantity.
Mouse norovirus MNV-1 is tested under similarity condition.
(2) antivirus action concentration-dependent relation is tested: taking norovirus (NoV GI) and norovirus (NoV respectively
GII) 100 μ L of Feces of Patients With Diarrhea sample homogenization liquid supernatant is added 100mg/mL cordate houttuynia glycoprotein mother liquor and makes final concentration point
Not Wei 0,1,10,25,50mg/mL, control tube be added same amount of normal saline, every time test simultaneously be arranged negative control (in equal volume
Physiological saline) and positive control (50 μM of 2-TU) pipe, it is incubated for 30min at room temperature, obtains the sample of various concentration processing.
Mouse norovirus MNV-1 is tested under similarity condition.
3, sample detection
Take step (1), in (2) different condition processing sample, acted on using one-step method fluorescence quantitative PCR detection cordate houttuynia
Front and back viral nucleic acid variation: by different condition in QIAGEN virus RNA extraction kit specification method extraction step (1), (2)
Viral RNA in the sample of processing takes 5 μ L viral RNA templates to be added in reaction tube, using One Step PrimescriptTM
RT-PCR Kit (TaKaRa) carries out reverse transcription amplification (RT-PCR), is put into the progress of ABI7500 fluorescence quantitative PCR instrument, and to knot
Fruit fluorescence detection.After experiment, Ct value is small when being equal to 30, and measurement result is defined as effective positive.Ct value between 30 to 40,
2 times repeatable, if amplification curve has obvious increased logarithmic phase, measurement result is that effectively the positive, other situations are considered as testing result
It is negative.Positive quantitative result is voluntarily analyzed by computer software, calculates its initial copy value.
Take step (1), in (2) different condition processing sample, using RNaseA enzyme detection cordate houttuynia effect front and back virus
Coat protein integrality: 3.5 μ L RNA enzyme (7000Units/mL) nuclease-free water (v/v) 1:10 by volume is taken
It is respectively added to after dilution in 100 μ L steps (1) and (2) different samples, 37 DEG C are placed 1 hour free RNA of degradation, and 18U is added
Ribonuclease inhibitor (40U/ μ L, Sigma) after hatch at room temperature 30 minutes, extract nucleic acid RT-qPCR method detection
Viral level.After each experiment, rack and pipette are all made of RNase remover and thoroughly clean, and eliminate any remaining
RNase.It is control group that RNaseA enzymatic treatment, which is not added: control group Virus Sample directly proposes detection of nucleic acids.
Take step (1), in (2) different condition processing sample, it is outer that front and back virus is acted on using ELISA method detection cordate houttuynia
Shell antigen integrity: the negative control hole (PBS of equivalent is arranged in lath needed for taking out from the aluminium foil bag after equilibrium at room temperature 20min
Buffer), Positive control wells (norovirus of untreated destruction) and sample aperture (sample of step (1) and (2) preparation), yin
Property, Positive control wells are separately added into 50 μ LPBS buffers and unbroken norovirus sample；Treated for addition in sample aperture
50 μ L of sample to be tested.The addition of remaining reagent is operated according to people's norovirus detection kit specification.As a result judge: negative right
According to OD value less than 0.2, positive control OD value is greater than 0.8, and sample OD value is greater than threshold value, is determined as the positive, otherwise is feminine gender.It is positive
Intensity can judge according to the size of OD value.
Take step (1), in (2) different condition processing sample, front and back virus is acted on using scanning electron microscope analysis cordate houttuynia
Metamorphosis: Virus Sample is placed in S-4800II Flied emission scanning electricity through Gradient elution using ethanol, critical point drying, viscous sample, metal spraying
Virus capsid protein ultra microstructure is observed on mirror.
3. test result
Viral nucleic acid detection test result is shown in Tables 1 and 2.The test of cordate houttuynia glycoprotein antivirus action time-dependent relation
In, cordate houttuynia glycoprotein is acted on the dosage of 10mg/mL and people's norovirus NoV GI, NoV GII and mouse norovirus MNV-1
0,15,30,60,120min, the results are shown in Table 1, with the increase of inactivation time, virus quantity is constantly reduced.Cordate houttuynia glycoprotein is made
Reach maximum efficiency with inactivation between 30min to 1h, acts between 1h to 2h, inactivating efficacy increase is no longer obvious.Cordate houttuynia sugar
In the test of albumen antivirus action concentration-dependent relation, various concentration cordate houttuynia glycoprotein is tied with after norovirus effect 30min
Fruit such as table 2, in 0~50 (mg/mL) dosage range, compared with Normal group, cordate houttuynia glycoprotein has one to norovirus
Determine deactivation, and as concentration improves, effect is more obvious, cordate houttuynia glycoprotein concentration 50mg/mL effect is most significant, cordate houttuynia
Glycoprotein 50mg/mL is both greater than the inhibiting rate of people's norovirus GI, GII type and mouse norovirus MNV-1 effect 120min
85%.
Virus capsid protein integrity detection the results are shown in Table 3, RNaseA enzymatic treatment descendant's norovirus NoV GI, GII and
Mouse norovirus MNV-1 detection of nucleic acids rate significantly lowers, and illustrates that cordate houttuynia glycoprotein is likely to cause virus capsid protein
It destroys, RNaseA enzyme is degraded viral nucleic acid.ELISA testing result (table 4) is further illustrated with cordate houttuynia glycoprotein
The extension of action time, virus capsid protein damaged degree increase.
The scanning electron microscope analysis of mouse norovirus MNV-1 is shown in Fig. 1, and MNV-1 is through (A in Fig. 1) before the processing of cordate houttuynia glycoprotein
It was found that, processing provirus particle is completely rounded with (B in Fig. 1) after processing, and processing restrovirus shell mechanism is destroyed,
In irregular figure.
The temporal correlation of 1 cordate houttuynia glycoprotein antivirus action of table
HCTE cordate houttuynia glycoprotein, DW distilled water.
2 cordate houttuynia glycoprotein antivirus action concentration dependence of table
HCTE: cordate houttuynia glycoprotein, CK: negative control, 2TU:2- sulphur urine glycosides (2-thiouridine).
3 RNAase of table processing analysis protein coat integrity analysis
4 ELISA of table is reacted to virus surface characteristic site primer
Note: the definition by OD value less than 0.01 is "+", and definition of the OD value between 0.05-0.01 is " ++ ", and OD value exists
Definition between 0.1-0.05 is " +++ ".
4. conclusion
The above result shows that cordate houttuynia glycoprotein has significantly norovirus NoVGI/GII and mouse norovirus MNV-1
Inhibiting effect, and cordate houttuynia glycoprotein is in time and dose-dependence to the inhibiting effect of norovirus.Further pass through
ELISA test and scanning electron microscopic observation discovery, cordate houttuynia glycoprotein make coat protein structure generate variation and breakage occur.
Embodiment 3: cordate houttuynia glycoprotein inhibits duplication of the mouse norovirus in its host cell
1, test material
Virus and cell: mouse norovirus MNV-1 and RAW264.7 cell are (purchased from Chinese Academy of Sciences's Microbiological Culture Collection
The heart).
Trial drug: cordate houttuynia glycoprotein sterling prepared by embodiment 1 is dissolved in the water, and is prepared into 100mg/mL cordate houttuynia
Glycoprotein mother liquor.
Detection kit: (hair R- visits in Germany for QIAGEN virus RNA extraction kit, norovirus ELISA kit
Biopharm company)；One Step PrimescriptTM RT-PCR Kit is purchased from TaKaRa company.
Key instrument: ABI7500 fluorescence quantitative PCR instrument.
2, test method
Take a certain amount of 264.7 cell inoculation of RAW in 12 holes for containing basal medium (DMEM+10%PBS+1%PS)
Plate (cell density 1 × 106/ well) in, it is separately added into different final concentration (1,10,25,50mg/mL) cordate houttuynia glycoprotein and 1
×106PFU/mL mouse norovirus MNV-1 sucks supernatant after being incubated at room temperature 1 hour jointly with RAW264.7 cell, to remove
Cordate houttuynia glycoprotein and virion therein collect cell after basal medium incubation at room temperature 24 hours are then added again,
And MNV-1 viral RNA, the operation of specific steps by specification are extracted using QIAGEN Viral nucleic acid extraction reagent box.Viral RNA mould
Plate uses One Step PrimescriptTM RT-PCR Kit (TaKaRa) to carry out reverse transcription as cDNA.Distilled water DW and 2 sulphur
Uridine 2TU is respectively as negative control and positive control.
PCR detects mouse norovirus non-structural protein RdRp (NS7), VPg (NS5) and GAPDH gene primer respectively and is shown in Table
5.Amplification condition is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 25s, 60 DEG C of annealing 25s, 72 DEG C of extension 1min, 35 circulations；72℃
Extend 5min.After reaction with 1.5% agarose gel electrophoresis testing goal band.
5 mouse norovirus non-structural protein RdRp (NS7) of table, VPg (NS5) and GAPDH gene primer
Quantitative analysis is carried out to the expression of target fragment using one-step method real-time fluorescent quantitative PCR.5 μ LcDNA template (1-
100ng), 0.5 μ L primer, the SYBR quantitative fluorescent PCR premix of 25 μ L, which merges a certain amount of deionized water of addition, arrives final volume
50μL.Quantitative pcr amplification condition is 95 DEG C of denaturation 15s, and 60 DEG C of annealing extend 1min, 40 circulations.The level of gene expression is pressed
The relative value of drug-treated sample and untreated sample is expressed: RQ=2-ΔΔCT。
3. test result
Cordate houttuynia glycoprotein is respectively with 1,10,25, the dosage of 50mg/mL and mouse norovirus MNV-1 (1 × 106PFU/
ML) in RAW 264.7 cell (cell density 1 × 106/ well) in be incubated for jointly 1 hour, cordate houttuynia glycoprotein exists to MNV-1
Non-structural protein RdRp (NS7 gene), VPg (NS5 gene) and the result of GAPDH gene expression are shown in figure in the duplication of its host cell
2, after removal drug and MNV-1 virus, rejoins culture medium and be incubated for RAW264.7 cell 24 hours.When cordate houttuynia glycoprotein
In 10mg/mL, the expression of RAW264.7 intracellular virus NS7 and NS5 virus nonstructural protein is significantly reduced, as drug is dense
The raising of degree, inhibiting effect gradually increase, and when concentration reaches 50mg/mL, the inhibitory effect of cordate houttuynia glycoprotein is close to 2 sulphur urines
Glycosides 2TU (a kind of HIV suppression common drug).
4. conclusion
The above result shows that cordate houttuynia glycoprotein has duplication of the mouse norovirus MNV in host cell RAW264.7
Significant inhibiting effect.
Claims (9)
1. a kind of cordate houttuynia glycoprotein, it is characterised in that the cordate houttuynia glycoprotein is prepared as follows: (1) by fresh fish raw meat
It dries, crushes after careless impurity elimination, obtain Heartleaf Houttuynia Herb；
(2) it takes Heartleaf Houttuynia Herb to be added in distilled water, adds cellulase, carry out enzyme digestion reaction in 55 DEG C of constant temperature stirrings, instead
After answering completely, enzymolysis liquid is once centrifuged, a supernatant primary sedimentation is obtained；A supernatant addition activated carbon room temperature is collected to stir
Colour fading is mixed, secondary centrifuging obtains secondary supernatant secondary precipitation；It takes secondary supernatant to add trichloroacetic acid, was stood after shaking up
Night is centrifuged three times, obtains supernatant three times and precipitates three times；Take three times supernatant in saturating by dialyzate of secondary distilled water in bag filter
It analyses, dialyzate is concentrated into 1/5 volume in bag taking, and after ethyl alcohol progress alcohol precipitation is added into concentrate overnight, centrifugation, abandoning supernatant sinks
Forming sediment is cordate houttuynia glycoprotein crude product, and cordate houttuynia glycoprotein is obtained after purification；The distilled water volume dosage is with Heartleaf Houttuynia Herb
Quality is calculated as 50-60mL/g；The cellulase and Heartleaf Houttuynia Herb mass ratio are 0.001-0.15:1；The trichloroacetic acid adds
Enter amount and 20-30g/L is calculated as with secondary supernatant volume.
2. cordate houttuynia glycoprotein as described in claim 1, it is characterised in that step (1) the Heartleaf Houttuynia Herb partial size is 80 mesh
Sieve.
3. cordate houttuynia glycoprotein as described in claim 1, it is characterised in that step (2) the primary centrifugation, secondary centrifuging, three times
Centrifugation and centrifugal condition are 8000 × g centrifugation 10min.
4. cordate houttuynia glycoprotein as described in claim 1, it is characterised in that step (2) dialysis bag retention molecular weight is MW14,
000Da。
5. cordate houttuynia glycoprotein as described in claim 1, it is characterised in that step (2) ethyl alcohol volume additional amount is with the volume of the concentrated liquid
It is calculated as 60-70%, the active carbon additional amount is calculated as 3-5g/L with a supernatant volume.
6. cordate houttuynia glycoprotein as described in claim 1, it is characterised in that step (2) method of purification are as follows: take cordate houttuynia glycoprotein
Crude product is configured to the crude product solution of concentration 100g/L with deionized water, by DEAE-52 ion on crude product and resin quality ratio 1:30
Exchange column is first washed with deionized water and sloughs except neutral polysaccharide, then with 0-0.5mol/L NaCl aqueous solution gradient elution, using sulphur
The efflux containing desired polysaccharide is collected in the detection of acid phenol method, then will contain desired polysaccharide efflux through Sephadex G-50 pillar
The efflux of molecular weight 30-50kDa protein peak at 280nm, freeze-drying, as cordate houttuynia glycoprotein are collected in desalination.
7. cordate houttuynia glycoprotein described in a kind of claim 1 is preparing the application in norovirus activity inhibitor.
8. the use as claimed in claim 7, it is characterised in that the norovirus is people's norovirus GI type, people's norovirus
GII type or mouse norovirus MNV-1.
9. the use as claimed in claim 7, it is characterised in that the norovirus activity inhibitor is non-structural protein NS7 base
Cause or NS5 gene expression inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811300319.0A CN109485706B (en) | 2018-11-02 | 2018-11-02 | Houttuynia cordata glycoprotein and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811300319.0A CN109485706B (en) | 2018-11-02 | 2018-11-02 | Houttuynia cordata glycoprotein and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109485706A true CN109485706A (en) | 2019-03-19 |
| CN109485706B CN109485706B (en) | 2021-04-30 |
Family
ID=65693624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811300319.0A Active CN109485706B (en) | 2018-11-02 | 2018-11-02 | Houttuynia cordata glycoprotein and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109485706B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101390868A (en) * | 2007-09-19 | 2009-03-25 | 复旦大学 | Plant polysaccharide and preparation method and use thereof |
| CN102234671A (en) * | 2010-05-07 | 2011-11-09 | 中国农业科学院农产品加工研究所 | Method for extracting polysaccharide from longan pulp |
| WO2013055127A2 (en) * | 2011-10-12 | 2013-04-18 | 경희대학교 산학협력단 | Pharmaceutical composition for preventing and treating dementia, parkinson's disease or epilepsy, containing houttuynia cordata thunb. extract as active ingredient |
| CN105311048A (en) * | 2014-06-30 | 2016-02-10 | 复旦大学 | Application of houttuynia cordata polysaccharide in preparation of drugs for preventing and treating influenza A and viral pneumonia |
| CN106038849A (en) * | 2016-05-11 | 2016-10-26 | 南京泽朗医药科技有限公司 | Antiviral oral liquid |
| CN106046192A (en) * | 2016-07-20 | 2016-10-26 | 潜山县三新茶叶专业合作社 | Process for extracting pachyman from poria coccus wolf |
| CN107698689A (en) * | 2017-09-29 | 2018-02-16 | 贵州中科健生物医药有限公司 | A kind of method that polysaccharide is extracted from cordate houttuynia |
| CN108424470A (en) * | 2018-03-28 | 2018-08-21 | 徐州工程学院 | A kind of preparation method of mushroom stems polysaccharide |
-
2018
- 2018-11-02 CN CN201811300319.0A patent/CN109485706B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101390868A (en) * | 2007-09-19 | 2009-03-25 | 复旦大学 | Plant polysaccharide and preparation method and use thereof |
| CN102234671A (en) * | 2010-05-07 | 2011-11-09 | 中国农业科学院农产品加工研究所 | Method for extracting polysaccharide from longan pulp |
| WO2013055127A2 (en) * | 2011-10-12 | 2013-04-18 | 경희대학교 산학협력단 | Pharmaceutical composition for preventing and treating dementia, parkinson's disease or epilepsy, containing houttuynia cordata thunb. extract as active ingredient |
| CN105311048A (en) * | 2014-06-30 | 2016-02-10 | 复旦大学 | Application of houttuynia cordata polysaccharide in preparation of drugs for preventing and treating influenza A and viral pneumonia |
| CN106038849A (en) * | 2016-05-11 | 2016-10-26 | 南京泽朗医药科技有限公司 | Antiviral oral liquid |
| CN106046192A (en) * | 2016-07-20 | 2016-10-26 | 潜山县三新茶叶专业合作社 | Process for extracting pachyman from poria coccus wolf |
| CN107698689A (en) * | 2017-09-29 | 2018-02-16 | 贵州中科健生物医药有限公司 | A kind of method that polysaccharide is extracted from cordate houttuynia |
| CN108424470A (en) * | 2018-03-28 | 2018-08-21 | 徐州工程学院 | A kind of preparation method of mushroom stems polysaccharide |
Non-Patent Citations (1)
| Title |
|---|
| 陈琴，严成: "响应面优化酶法提取鱼腥草叶中多糖的工艺", 《湖北农业科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109485706B (en) | 2021-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Feng et al. | Antiviral activity of phosphorylated Radix Cyathulae officinalis polysaccharide against Canine Parvovirus in vitro | |
| Jansi et al. | Emerging paradigms of viral diseases and paramount role of natural resources as antiviral agents | |
| Chen et al. | Comparison of Bush Sophora Root polysaccharide and its sulfate's anti-duck hepatitis A virus activity and mechanism | |
| Malabadi et al. | Outbreak of Coronavirus (SARS-CoV-2) Delta variant (B. 1.617. 2) and Delta Plus (AY. 1) with fungal infections, Mucormycosis: Herbal medicine treatment | |
| Liu et al. | Effects of Hericium erinaceus polysaccharide on immunity and apoptosis of the main immune organs in Muscovy duck reovirus-infected ducklings | |
| CN112220799A (en) | Product for inhibiting virus and application | |
| Xu et al. | The complete genome analysis of two enterovirus 96 strains isolated in China in 2005 and 2009 | |
| Pang et al. | Antiviral effects of aqueous extract from Spatholobus suberectus Dunn. against coxsackievirus B3 in mice | |
| BRPI0814725B1 (en) | USE OF ANTIVIRAL COMPOSITION | |
| CN105418410A (en) | Emodin derivatives and application thereof in preparation of anti-HIV-1 medicines | |
| CN105456283A (en) | Application of macrolide antibiotics or medical salt thereof to preparation of medicine for resisting to hand-foot-and-mouth disease | |
| CN1330330C (en) | Antivirus medicinal composition, preparation method and use | |
| CN102552369B (en) | Lon icera japonica Thunb leaf extractive serving as anti-porcine reproductive and respiratory syndrome viral agent | |
| CN109485706A (en) | A kind of cordate houttuynia glycoprotein and its preparation and application | |
| Guo et al. | Inhibition of enterovirus 71 infection by polysaccharides extracted from Picochlorum sp. 122 via the AKT and ATM/ATR signaling pathways | |
| Marbawati et al. | Effects of curcumin and pentagamavunon-0 against dengue-2 virus Infection in vero cells; an in vitro study | |
| CN112043688A (en) | A composition for preventing and/or treating coronavirus infection | |
| CN115105502B (en) | Application of compound containing stephania plant alkaloid in preparation of cat infectious peritonitis medicine | |
| CN101044857A (en) | Application of compounding preparation in medicine for treating human Aids hepatitis and skin diseases | |
| CN101695511A (en) | Pomegranate rind extract and production method and application thereof | |
| CN1122107A (en) | Antiviral powder, antiviral extract, and pharmaceutical preparation containing said powder and/or said extract | |
| Balamurugan et al. | Potential Effect of Acacia arabica. on Peste des Petits Ruminants. Virus Replication | |
| CN112022867A (en) | Application of geraniin in preparing medicine for resisting novel coronary virus | |
| Klangbud et al. | The efficacy of the traditional Thai remedy “Ya-Ha-Rak” against dengue virus type 2 | |
| CN110075117A (en) | Heparin sodium is as the purposes for inhibiting grass carp reovirus infection 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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210406 Address after: 330045 No. 1101, Zhimin Avenue, Nanchang Economic and Technological Development Zone, Jiangxi Province Applicant after: JIANGXI AGRICULTURAL University Address before: No. 3399, Binsheng Road, Binjiang District, Hangzhou City, Zhejiang Province Applicant before: ZHEJIANG CENTER FOR DISEASE CONTROL AND PREVENTION |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |