CN109045085A - Nettle extract and the application on the antianaphylactic drug of preparation treatment - Google Patents
Nettle extract and the application on the antianaphylactic drug of preparation treatment Download PDFInfo
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Abstract
The present invention provides nettle extract, the extract includes the extract after 85-95 DEG C of hot water extract, mass concentration 70-80% ethanol extract and alcohol extracting after petroleum ether, ethyl acetate and extracting n-butyl alcohol.The present invention evaluates its antiallergic activity eventually by the inhibiting rate size to hyaluronidase.The nettle antiallergic activity ingredient obtained using technical solution of the present invention is dissolved out more completely in hot ethanol, and after petroleum ether, ethyl acetate and extracting n-butyl alcohol, purity improves and hyaluronic acid enzyme inhibition rate is caused to increase.
Description
Technical field
The present invention relates to a kind of extracts of nettle, and are applied to preparation and treat in antianaphylactic drug, belong to life
Object pharmaceutical technology field.
Background technique
Nettle (Urtica fissa E Pritz) belongs to Urticaceae (Urticaceae) nettle of Angiospermae subordinate
Belong to (Urtica L.) plant, be herbaceos perennial, dilute shrub has seta, is distributed mainly on China Anhui, Hunan, lake
The ground such as north, Henan, Shaanxi, Gansu, Sichuan, Guizhou and Sichuan, herb can be used as medicine.With expelling endogenous wind to relieve convulsion, promoting blood circulation dehumidifying and
The effects of antidoting against snake bite.According to the literature, the chemical component of nettle mainly has flavonoids, organic acid, phenols, polysaccharide, tonka-bean
Plain class, lignin, sterols and protein abundant etc..Nie Lu can be used for treating the study found that positive Yi nationality, distributed over Yunnan, Sichuan and Guizhou drug nettle
The anaphylaxis dermatosis such as papule, eczema, nettle rash, curative effect are very significant.Foreign study found that nettle genus plants extract can press down
Make the expression of relevant to allergic rhinitis receptor and enzyme.
Summary of the invention
Technical solution of the present invention has potential inhibiting effect to allergic reaction in view of nettle extract, provides a kind of nettle
The extract of antianaphylaxis components
The present invention uses alcohol extracting to nettle herb dry powder and fresh nettle respectively and water mentions；Cold water is mentioned have been referred to hot water
Solvent gradually extracting method analyzes the heat resistance and dissolubility of nettle antiallergy substance.Specifically include following content:
Nettle extract, the hot water extract that the extract is 85-95 DEG C.
Nettle extract, the extract are mass concentration 70-80% ethanol extract, and the Extracting temperature is
65-75℃。
Above-mentioned nettle extract, the extract be nettle dry powder or nettle herb dry powder, wherein extract with it is described
5-95 DEG C of hot water extract or mass concentration 70-80% ethanol extract mass ratio be 1:10-50.
Further preferred scheme be the extract be after the extraction of mass concentration 70-80% ethanol extract again successively
The lyophilized preparation after petroleum ether extraction, ethyl acetate extraction, extracting n-butyl alcohol.
The nettle extract is treated the application on antianaphylactic drug in preparation by technical solution of the present invention.Specifically
It is that its antiallergic activity is evaluated to the inhibiting rate size of hyaluronidase by each component.
The nettle antiallergic activity ingredient obtained using technical solution of the present invention is dissolved out more completely in hot ethanol, and
After petroleum ether, ethyl acetate and extracting n-butyl alcohol, purity improves and hyaluronic acid enzyme inhibition rate is caused to increase.Then root
According to single-factor orthogonal experiment scheme, the optimal extract process of nettle is finally obtained.
Detailed description of the invention
Fig. 1 nettle extract causes the influence of itch to mouse D-40, wherein A: nettle extract is to mouse
The influence of itch frequency；B: influence of the nettle extract to mouse itch number.
Ear skin indigo plant dye figure during Fig. 2 Balb/c mouse models of passive skin irritability.
The influence of Balb/c mouse passive cutaneous anaphylaxis after Fig. 3 nettle extract excites sensitization.
Influence of Fig. 4 nettle extract to IgE and Histamine concentrations in Balb/c mice serum after sensitization excitation.
Wherein, A: influence of the nettle extract to IgE concentration in Balb/c mice serum after sensitization excitation；B nettle is extracted
Influence of the object to Histamine concentrations in Balb/c mice serum after sensitization excitation.
Influence of Fig. 5 nettle extract to RBL-2H3 cell-proliferation activity.
The influence that Fig. 6 various concentration nettle extract discharges RBL-2H3 cell activation β-HEX.
Influence of Fig. 7 various concentration nettle extract to RBL-2H3 cell activation histamine release.
Fig. 8 various concentration nettle extract intensifies rear RBL-2H3 cell activation degranulation p-Lyn/Lyn, p- to sensitization
The gray value result of Syk/Syk protein expression.A: it is de- that various concentration nettle extract intensifies rear RBL-2H3 cell activation to sensitization
The gray value result of particle p-Lyn/Lyn protein expression；B various concentration nettle extract intensifies rear RBL-2H3 cell to sensitization
Activate the gray value result of degranulation p-Syk/Syk protein expression.
Fig. 9 various concentration nettle extract intensifies rear RBL-2H3 cell activation degranulation p-Lyn/Lyn, p- to sensitization
The statistical result of Syk/Syk protein expression.A: it is de- that various concentration nettle extract intensifies rear RBL-2H3 cell activation to sensitization
The statistical result of particle p-Lyn/Lyn protein expression；B various concentration nettle extract intensifies rear RBL-2H3 cell to sensitization
Activate the statistical result of degranulation p-Syk/Syk protein expression.
Specific embodiment
Nettle: originating in Yichang City, Hubei Province Yuanan County, meets Chinese Pharmacopoeia 2005 through Yichang Medicines and Chemical Reagents Inspection Bureau's detection
Version one (examines number: 20061188).
It is as follows to implement the instrument that technical solution of the present invention is used:
22 type centrifuge of Suprafuge (Heraeus company)；Evaporator RE-52A (Shanghai Yarong Biochemical Instrument Plant)；
KQ-250B type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；AB204-N assay balance (Switzerland Mettler
Toledo company)；721E visible spectrophotometer (Shanghai Spectrum Apparatus Co., Ltd.)；KDM type regulating temperature electrothermal cover (Shandong Juancheng
Yong Guang instrument plant)；Crusher for Chinese herbal medicine (China)；DK-S26 electric-heated thermostatic water bath (the upper limited public affairs of the macro experimental facilities of Nereid
Department)；(200 μ L, 1000 μ L are each one) for microscale sampler.
It is as follows to implement the reagent that technical side of the invention uses:
The concentrated sulfuric acid (analysis is pure), phenol (analysis is pure), glucose (analysis is pure), 95% ethyl alcohol (medical grade).Self-control is double to steam
Water.
The extraction of nettle effective active composition is as follows:
Sample nettle is cleaned, takes part nettle fresh goods to be placed in 4 DEG C of refrigerators and saves backup, separately take and be partially disposed in convection oven
In 60 DEG C drying.Crushed with high speed disintegrator, after crossing 20 meshes, dry powder be placed in drier and save backup.
Nettle dry powder hot water extraction
Precision weighs 30g dry powder, and cooling through 90 DEG C of hot water extraction 1h, solid-liquid ratio 1:40, filtering, filter residue repeats above-mentioned
Operation, after merging filtrate twice, is concentrated, and freeze-drying obtains nettle dry powder hot water extraction object, as sample A.
Nettle fresh leaf hot water extraction
Precision weighs fresh nettle 200g, and through 90 DEG C of hot water extraction 1h, solid-liquid ratio 1:15 again repeats filter residue after filtering
Aforesaid operations, concentrated frozen is dry after merging extracting solution twice, nettle fresh leaf hot water extraction object is obtained, as sample B.
Nettle dry powder hot ethanol refluxing extraction
Precision weighs 30g dry powder, through refluxing extraction 1h, solid-liquid ratio 1:40 under the conditions of 70 DEG C of 75% ethyl alcohol, after filtering again
Filter residue is repeated into aforesaid operations, concentrated frozen is dry after merging extracting solution twice, nettle dry powder hot ethanol extract is obtained, as
Sample C.
The extraction of nettle dry powder cold water
Precision weighs 30g dry powder, extracts for 24 hours through cold water, solid-liquid ratio 1:40, by extracting solution concentrated frozen after removing filter residue
It is dry, nettle dry powder cold water extract is obtained, as sample D.
Nettle alcohol extracting thing is through different solvents separating and extracting
Precision weighs nettle dry powder 60g, 75% ethyl alcohol the refluxing extraction 2h at 70 DEG C, solid-liquid ratio 1:40, cooling, mistake
Filter repeats above-mentioned steps, merges extracting solution twice, ethanol extract is obtained after concentration, through petroleum ether extraction to petroleum ether layer
It is colourless, petroleum ether layer, low pressure concentration are obtained, it is sample E that freeze-drying, which obtains petroleum ether extract,.By sample E a part again through second
Acetoacetic ester extraction obtains ethyl acetate layer, low pressure concentration until ethyl acetate layer is colourless, and freeze-drying obtains acetic acid ethyl ester extract,
For sample F.By sample F a part again through extracting n-butyl alcohol, until n-butanol layer is colourless, n-butanol layer, extract liquor concentration, freezing are obtained
N-butyl alcohol extract is obtained after drying, is sample G.- 20 DEG C of warp of sample G a part are freeze-dried, is sample H.
The method that hyaluronidase inhibits in vitro
Hyaluronidase participates in the generation of type Ⅰ hypersensitivity, has very strong correlation with allergic reaction and inflammatory reaction,
The drug that can influence mast cell degranulation reaction can have an impact the activity of hyaluronidase.Therefore the present invention is with transparent
It is anti-to go out nettle extract with this optimal screening for judgment criteria of the matter acid enzyme extracorporeal inhibiting rate as nettle extract antiallergic activity
The highest part of activity.With steps are as follows:
(1) preparation of solvent
Hyaluronidase solution: 0.01g hyaluronidase is added to 4mL hac buffer, is configured to ultimate density
For the hyaluronidase solution of 1250 μ g/mL.
Sodium hyaluronate solution: 0.005g Sodium Hyaluronate is added in 10mL hac buffer, after completely dissolution
Obtain 0.5 μ g/mL sodium hyaluronate solution.
Hac buffer (pH=5.6): taking 2300 μ L glacial acetic acids in volumetric flask, and deionized water is added and is diluted to
200mL takes 4.8mL as solution A after mixing；5.44g acetic acid sodium reagent is taken, deionized water dissolving is added and is settled to 200mL,
45.2mL is measured after mixing as B solution；A, B solution are mixed, deionized water is added and is settled to 100mL.It is accurately surveyed with PH instrument
Determine pH value, and adjusts pH value to 5.6 with solution A or B.
Ehrlich's reagent: the p- dimethylaminobenzaldehyde of 0.8g is dissolved in 15mL concentrated hydrochloric acid and 15mL dehydrated alcohol.
Acetylacetone,2,4-pentanedione solution: 7mL acetylacetone,2,4-pentanedione is dissolved in the sodium carbonate liquor of 100mL1.0mol/L to (this solution need to show
With current).
(2) allergic reaction method is as follows:
By the CaCl of 0.1mL2(0.25mmol/L) solution and hyaluronic acid enzyme solution 0.5mL are put into test tube, 37 DEG C of incubations
20 minutes, 0.5mL sample solution is added into test tube and is incubated for 20 minutes at 37 DEG C.0.5mL sodium hyaluronate solution is added
And in 37 DEG C of incubation 30min.Then room temperature stands 5min, adds 0.1mL0.4mol/L NaOH solution and 0.5mL acetylacetone,2,4-pentanedione molten
Liquid heats 15min, then ice bath 5min in boiling water, and 1.0mL Erlich reagent is added and is diluted with 3.0mL dehydrated alcohol.It puts
It sets 20 minutes until its color changes.Absorbance value is measured with microplate reader, and calculates its hyaluronic acid enzyme inhibition rate.Specifically
The sample solution that scheme is divided into 4 groups: A group is substituted with acetate buffer solution, the sample solution and hyaluronidase solution in B group with
Hac buffer substitution, C group is normal test solution absorbance；Hyaluronidase solution is replaced in D group with hac buffer
Generation.
Hyaluronic acid enzyme inhibition rate=((A-B)-(C-D))/(A-B) × 100%
The result obtained by adopting the above technical scheme is as follows:
Sample A, sample B, sample C, sample D, sample E, sample F, sample G and sample H are obtained with above-mentioned extracting method, then
Experimental evaluation its antiallergic activity inhibited in vitro by hyaluronidase, it is as follows to obtain result:
1 nettle active constituent of table hyaluronic acid enzyme inhibition rate (N=3)
Note:#Indicate that the p < 0.01 compared with sample A group, * indicate p > 0.05 compared with sample A group.
The inhibiting rate of the hyaluronidase of drug and its anti-allergic effects in the technical solution that hyaluronidase inhibits in vitro
It is positively correlated.According to international standard, anti-allergic effects are divided into strong, medium, weak and without anti-allergic effects, corresponding inhibition
Rate P is respectively P >=70%, P >=50%, 30%≤P < 50%, P < 30%.The external suppression result of hyaluronidase is shown: sample
The inhibiting rate of product A and sample B quite (p > 0.05), illustrate fresh nettle in the drying process without loss antiallergy component.Sample
Inhibiting rate (p < 0.01) of the inhibiting rate greater than sample A of product C, sample H, and the inhibiting rate very little of the cold water extraction of sample D, only
Be 32.6%, show nettle antianaphylaxis components 75% hot ethanol in dissolve out relatively completely.The inhibiting rate highest of H sample is
65.9%.Orthogonal test method is as follows
On the basis of the experiment of single factor result of concentration of alcohol A, Extracting temperature B and solid-liquid ratio C, three factors have been carried out
Three horizontal orthogonal tests.
2 nettle extraction process factor level table of table
As seen from table, this experiment is three levels, three factors, therefore using orthogonal arrage L9 (34) come experiment arrangement.
3 L of table9(34) orthogonal experiments
4 analysis of variance table of table
As can be known from the above table, influence sequence of each factor to comprehensive score is A > C > B, then (the K value point from the point of view of each factor level
Analysis), A factor is K1>K2>K3, B factor is K2>K1>K3, C factor is K3>K2>K1, extraction conditions are with A1B2C3It is preferred.In conjunction with life
The actual conditions of production determine the optimal extract process of pearl gracilis polysaccharide are as follows: solid-liquid ratio 8: 1, extraction time are 2 times, are mentioned
Taking the time is 3 hours.
5 concentration of alcohol of table to nettle extract hyaluronic acid enzyme inhibition rate influence (N=3)
Note: * indicates the p < 0.05 compared with other each groups
As a result: nettle extract concentration of alcohol be 75%-95% concentration range in inhibiting rate it is higher, and amplitude of variation compared with
It is small, therefore concentration of alcohol Application Range is determined as between 75%-95%.
6 temperature of table to nettle extract hyaluronic acid enzyme inhibition rate influence (N=3)
Note: * indicates the p < 0.05 compared with other each groups
As a result: the hyaluronic acid enzyme inhibition rate of nettle extract increases as the temperature rises, and in 70-90 DEG C of range
Interior variation is little, therefore Extracting temperature is chosen between 70-90 DEG C.
7 solid-liquid ratio of table to nettle extract hyaluronic acid enzyme inhibition rate influence (N=3)
Note: * indicates the p < 0.05 compared with other each groups
As a result: the hyaluronic acid enzyme inhibition rate of nettle extract is higher when solid-liquid ratio is 1:40 or more, therefore solid-liquid ratio selects
It takes between 1:40-1:50.
The extraction of the nettle antianaphylaxis components carried out by adopting the above technical scheme and the evaluation of antiallergic activity
H sample hyaluronic acid enzyme inhibition rate highest shows that nettle antiallergic activity ingredient dissolves out more completely in hot ethanol,
And after petroleum ether, ethyl acetate and extracting n-butyl alcohol, purity improves and hyaluronic acid enzyme inhibition rate is caused to increase.
As shown in Tables 3 and 4, influence of each factor to nettle extract inhibiting rate is descending are as follows: A > B > C, A factor, that is, second
Determining alcohol has a significant impact inhibiting rate, A2 > A3 > A1, and B, C factor, that is, temperature and solid-liquid ratio influence less, to examine on inhibiting rate
Consider the condition question of technical solution, selects A2B2C1, it may be assumed that 85% concentration of alcohol, 80 DEG C of temperature and 1:40 solid-liquid ratio.
The most strong antiallergic activity component filtered out by hyaluronidase body outer suppressioning experiment, experiment is established in vivo
Balb/c mouse D-40 model, the penetrating model of capillary and whole body actively and passively skin allergy experimental evaluation
The antiallergic activity of nettle extract, influence of the analysis experiment in vitro detection nettle extract to RBL-2H3 cell-proliferation activity,
RBL-2H3 cell degranulation model is established, the variation of histamine and β-hexosaminidase burst size, Western Blot inspection are detected
Nettle extract is surveyed to Fc ε RI signal path GAP-associated protein GAP Syk/P-Syk and Lyn/P-Lyn after the activation of RBL-2H3 cell sensitization
The influence of expression.The specific method is as follows:
It is as follows to implement the instrument that technical solution of the present invention is used:
Inverted microscope (CKX41), Image-ProPlus 7.0C be multi-functional and image analysis system, GZX-9070MEB
Air blast drying box, Automation-tissue-dehydrating machine, all-wave length microplate reader (Mutiskan Spectrum), inverted microscope
(CKX41)。
It is as follows to implement the reagent that technical solution of the present invention is used:
CCK8 kit, 0.25% pancreatin, DNP-HSA, anti-DNP-IgE monoclonal antibody, mouse Histamine ELISA reagent
Box, mouse ovalbumin specific IgE kit, MEM high glycoform culture medium, Syk antibody, P-Syk antibody, Lyn antibody, P-
Lyn antibody, Sodium Hyaluronate etc..
The mouse D-40 that technical solution of the present invention carries out causes itch scheme as follows
The Babl/c mouse that weight is 20 ± 2g is chosen, half male and half female is randomly divided into blank control group, model group, chlorine thunder
He determine control group (1.3mg/kg), nettle extract high (624mg/kg), in (312mg/kg), low dose group (156mg/kg),
Every group 6.Each group presses prescribed dose stomach-filling 0.1mL/10g respectively, and blank control group gives the physiological saline of same dose.Continuously
Administration 7 days, after the last administration 30min, the D-40 injection 0.1mL/ of every group of mouse tail vein injection 0.04%
10g, difficult to tackle with the fore paw of mouse, rear solid end scratches body, and it is indication that mouth, which stings whole body, records itch number and the duration of mouse.Knot
Fruit is as shown in Figure 1, NE can effectively inhibit mouse itch caused by D-40, and in a certain range be in concentration dependant
Property, effect is very significant in high dose, similar to positive control drug Loratadine.
The mouse low molecule mouse vasopermeability scheme that technical solution of the present invention carries out is as follows
The Babl/c mouse that weight is 20 ± 2g is chosen, half male and half female is randomly divided into blank control group, model group, chlorine thunder
He determine control group (1.3mg/kg), nettle extract high (624mg/kg), in (312mg/kg), low dose group (156mg/kg),
Every group 6.Each group presses prescribed dose stomach-filling 0.1mL/10g respectively, and blank control group gives the physiological saline of same dose.Continuously
Administration 7 days, after the last administration 30min, 1% Azo-Blue solution of the equal tail vein injection 0.1mL/10g dosage of each group mouse, with
Intracutaneous injection 0.1mL concentration is 1% histamine phosphate at mouse web portion center depilation afterwards.Cervical dislocation is put to death small after 30 minutes
Mouse separates skin of abdomen, and the blue dye skin of same size is taken out with punch, is put into test tube after shredding, adds 4mL's
70% acetone soln is stayed overnight in 60 DEG C of water-baths after mixing, is centrifuged 10min under 2000r/min revolving speed after 12h, supernatant is taken to exist
Its OD value at 610nm, comparison among groups are surveyed on all-wave length multi-function microplate reader, and find out inhibiting rate.The results are shown in Table 5,
Nettle extract can be substantially reduced histamine induced mice capillary permeability in basic, normal, high dosage, make in high dose
With very significantly, effect and Claritin Loratadine are similar.
8 nettle extract of table to histamine sensitized mice capillary permeability influence (N=6)
Note:#Indicate that the p < 0.01 compared with normal group, * * indicate the p < 0.01 compared with model group
The mouse low molecule mouse systemic models of passive skin irritability scheme that technical solution of the present invention carries out is as follows
Sero-fast preparation: taking female sd inbred rats 6, subcutaneous administration 20mL/mL concentration ovalbumin and isometric alum
Adjuvant sensitization, dosage 0.5mL.It is administered every other day, continuous 3 times.The 11st day after last sensitization, with 5% chloraldurate 0.7mL/
100g intraperitoneal injection of anesthesia, the abdominal aorta arterial blood extracting after its anesthesia are centrifuged 15min in the case where revolving speed is 4000r/min, take
Layer serum, is sub-packed in EP pipe, -20 DEG C save backup.Sensitization: the Babl/c mouse that selection weight is 20 ± 2g, half male and half female,
Be randomly divided into blank control group, model group, Loratadine control group (1.3mg/kg), nettle extract high (624mg/kg), in
(312mg/kg), low dose group (156mg/kg), every group 6.Each group presses prescribed dose stomach-filling 0.1mL/10g, control group respectively
Give the physiological saline of same dose.It is administered every other day, continuously three times, concentration is being used to be 5% chloraldurate after the last administration
After 0.1mL/10g mouse anesthesia, the sensitized serum of every 20 μ l of mouse right ear intracutaneous injection, while left ear injection except for the normal group
Same amount of normal saline marks at sensitization.Normal group two ears of mouse inject the physiological saline of 20 μ l.
Excitation: gastric infusion, dosage are the same again after for 24 hours.After 1h is administered, in addition to blank control group, every mouse tail is quiet
Arteries and veins injects 0.2mLOVA and Azo-Blue mixed solution (OVA of 20mg/mL and 1% isometric Azo-Blue), is remembered with camera
Record the variation of mouse ear skin color.Mouse is put to death after 1h, and takes same area size skin with punch, it is desirable that same position
And there is color, it is infiltrated in formamide solution, is stayed overnight in 64 DEG C of water-bath constant temperature, after 12h under 2500r/min revolving speed immediately
It is centrifuged 15min, takes supernatant, is placed in all-wave length multi-function microplate reader OD value of the measurement under 650nm wavelength, and does and compares between group
Compared with.As a result as shown in Figures 2 and 3, nettle extract have the function of inhibition OVA caused by passive cutaneous anaphylaxis, and
Effect of high dosage is significant, but slightly weak compared to positive drug Loratadine.
The mouse low molecule mouse systemic active skin allergy scheme that technical solution of the present invention carries out is as follows
Sensitization: choose weight be 20 ± 2g Babl/c mouse, half male and half female, be randomly divided into blank control group, model group,
Loratadine control group (1.3mg/kg), nettle extract high (624mg/kg), in (312mg/kg), low dose group (156mg/
Kg), every group 6.OVA and alum adjuvant mixed solution (OVA of 1mg/mL and equal bodies is once subcutaneously injected in the next day that each group mouse is equal
The mixing of product alum) 0.2mL, amount to three times, blank control group gives physiological saline.
Excitation: from last sensitization the 10th day, corresponding drug 0.2mL/ is administered only in every group of intragastric administration on mice, is administered every other day,
Amount to three times.After last dose 30min, the OVA solution 0.2mL sensitization of mouse tail vein injection 4mg/mL, blank control group is given
It is replaced with physiological saline.Subsequent eyeball takes blood, is centrifuged 10 minutes under 3000r/min revolving speed, after taking upper serum, uses mouse
ELISA kit surveys histamine and OVA specific IgE content in serum.As a result as shown in Figure 4 and Figure 5, nettle extract can be bright
It is aobvious to reduce OVA specific IgE and Histamine concentrations level in mice serum, and effect trend is close, is within the scope of a certain concentration
Dose dependent.Thus infer that nettle extract can effectively inhibit the mouse systemic active anaphylaxis as caused by OVA.
The external scheme that technical solution of the present invention carries out is as follows
The influence that nettle extract discharges RBL-2H3 cell activation β-HEX
The RBL-2H3 cell for taking logarithmic phase to grow, is made cell suspension and is counted after digesting, and adjustment cell concentration is
2.5×105A/mL cell mixing and is inoculated into flat 24 orifice plate in MEM complete medium, and every hole is inoculated with 1mL.Inoculation
When continuous mixed cell suspension, the cell quantity to ensure every hole inoculation is roughly the same.24 orifice plates are put into constant incubator
It incubates.Normal group, model group, nettle extract concentration gradient group (100 μ g/mL, 200 μ g/mL, 400 μ g/mL), the positive are set
Medicine group (Loratadine of 5ng/mL), normal group without sensitization, excitation and drug-treated, model group by sensitization and excitation but
Without drug-treated, NE group and positive drug group need to be handled through sensitization, excitation and relative medicine.Model group and nettle extract
Group and the positive drug control group anti-DNP IgE monoclonal antibody sensitization of 0.2 μ g/mL.Next day, by group of cells use containing pair
The MEM serum free medium of drug is answered to be incubated for 1h.After being incubated for 1h, culture medium is sucked out and is rinsed cell 3 times with PBS buffer solution.It connects
Get off for the DNP-HSA PBS buffer solution that 200 μ l concentration are 30ng/mL to be added in group of cells except for the normal group and excite.Just
The sterile saline of Chang Zuyong equivalent replaces.After 24 orifice plates are incubated for 1 hour in the incubator, supernatant is taken.By supernatant
It is placed in 96 orifice plates, then 50 μ l chromophoric solutions are added in every 50 μ l of hole.After being incubated for 1 hour in the incubator, dripped into each hole
200 μ l stop baths are added to react with color development stopping.OD value is detected at all-wave length microplate reader 405nm wavelength.
The inhibiting rate of each group β-HEX release is calculated using formula: β-HEX inhibiting rate (%)=[1- (TOD-COD)/
(MOD-COD)] × 100%.As a result as shown in figure 5, the nettle extract of 50,100,200,400 μ g/mL to RBL-2H3 without increasing
Grow toxic effect.Can select within this range the nettle extract of 100,200,400 μ g/mL as drug effect cell it is low,
Middle and high dosage.
The influence that nettle extract discharges RBL-2H3 cell activation histamine and β-HEX
The RBL-2H3 cell for taking logarithmic phase to grow, is made cell suspension and is counted after digesting, and adjustment cell concentration is
2.5×105A/mL cell mixing and is inoculated into flat 24 orifice plate in MEM complete medium, and every hole is inoculated with 1mL.Inoculation
When continuous mixed cell suspension, the cell quantity to ensure every hole inoculation is roughly the same.24 orifice plates are put into constant incubator
It incubates.Normal group, model group, nettle extract concentration gradient group (100 μ g/mL, 200 μ g/mL, 400 μ g/mL), the positive are set
Medicine group (Loratadine of 5ng/mL), normal group without sensitization, excitation and drug-treated, model group by sensitization and excitation but
Without drug-treated, NE group and positive drug group need to be handled through sensitization, excitation and relative medicine.Model group and nettle extract
Group and the positive drug control group anti-DNP IgE monoclonal antibody sensitization of 0.2 μ g/mL.Next day, by group of cells use containing pair
The MEM serum free medium of drug is answered to be incubated for 1h.After being incubated for 1h, culture medium is sucked out and is rinsed cell 3 times with PBS buffer solution.It connects
Get off for the DNP-HSA PBS buffer solution that 200 μ l concentration are 30ng/mL to be added in group of cells except for the normal group and excite.Just
The sterile saline of Chang Zuyong equivalent replaces.24 orifice plates are placed in incubator and are incubated for 1h, 4000r/min is centrifuged after 5min again
Secondary collection supernatant detects histamine content in supernatant by rat histamine ELISA kit.Supernatant is placed in 96 orifice plates
In, then 50 μ l chromophoric solutions are added in every 50 μ l of hole.After being incubated for 1 hour in the incubator, it is whole that 200 μ l are added dropwise into each hole
Only solution is reacted with color development stopping.As a result as shown in fig. 6, nettle extract can obviously inhibit RBL-2H3 cell β-HEX after activation
Release, effect is in a certain range concentration dependent, and effect and positive drug Loratadine are similar in high dose.Such as figure
Shown in 7, nettle extract can obviously inhibit the release of sensitization RBL-2H3 cell histamine, effect in a certain range in concentration according to
Lai Xing, and acted on significantly in middle and high dosage.
Influence of the nettle extract to P-Lyn/Lyn, P-Syk/Syk protein expression in the RBL-2H3 cell after activation
Protein extraction: the RBL-2H3 cell for taking logarithmic phase to grow is made cell suspension and is counted after digesting, and adjustment is thin
Born of the same parents' concentration is 5 × 105A/mL cell mixing and is inoculated into flat 6 orifice plates in MEM complete medium, and every hole is inoculated with 2mL.
Continuous mixed cell suspension when inoculation, the cell quantity to ensure every hole inoculation are roughly the same.6 orifice plates are put into constant incubator
Middle incubation.Normal group, model group, nettle extract concentration gradient group (100 μ g/mL, 200 μ g/mL, 400 μ g/mL), sun are set
Property medicine group (PP210 μM of Lyn inhibitor, OXSI-20.1 μM of Syk inhibitor), normal group without sensitization, excitation and drug at
Reason, model group by sensitization and excitation but without drug-treated, nettle extract group and positive drug group need to through sensitization, excitation with
And relative medicine processing.Model group and nettle extract group and the positive drug control group anti-DNP IgE Dan Ke of 0.2 μ g/mL
Grand antibody sensitized.Group of cells is used the MEM serum free medium containing corresponding drug to be incubated for 1h by next day.After being incubated for 1h, it is sucked out
Culture medium is simultaneously rinsed cell 3 times with PBS buffer solution.Next the DNP-HSA PBS buffer solution for being 30ng/mL by 200 μ l concentration
It is added in group of cells except for the normal group and excites.The sterile saline of normal group equivalent replaces.After the completion of excitation, discard
Cell supernatant, the interior PBS1mL that 4 DEG C of pre-coolings are added of Tissue Culture Flask are washed cell 2 times, are swept cell and cell suspension is made, use
Cell suspension is transferred in 1.5mL EP pipe by liquid-transfering gun, during which ice bath 30min is blown and beaten, it is ensured that cell is complete repeatedly with pipettor
Cracking.12000rpm is centrifuged 10min at 4 DEG C.Supernatant is collected, as the total liquid of albumen.
Determination of protein concentration: protein concentration is surveyed with BCA determination of protein concentration kit, specific method is said referring to kit
Bright book.
Protein electrophoresis: cleaning glass plate, after glass plate dries, by a focussing glass and a flat glass plate composition one
It is right, be the gap of encapsulating between glass plate, be put into gel maker, insertion chock fixes glass plate, check bottom whether be aligned with
Exempt from leak adhesive.1. selecting 8% separation gel and 5% concentration glue.Separation gel is poured into height appropriate, can be used before encapsulating
Comb tries, and liquid level about 5-8mm is advisable on comb tooth distance separation glue.Then pure water is slowly even added in gap
Until filling, process not break up glue.2. every kind of sample that total amount is 50 μ g is drawn and is carefully added to poly- according to concentration
In the hole of acrylamide gel.Each sample retains a sample well, and 3 μ L protein labels are added as target protein transferring film
The instruction of efficiency；3. gelling agent is placed in electrophoresis tank, enough electrophoresis solutions are added after electrophoresis.Sample addition electrophoresis hole is gone forward side by side
Row electrophoresis.Gel voltage 75V is concentrated, with 120V separation gel.Electrophoresis has just been run through to bromophenol blue just stops electrophoresis, transferring film.
Transferring film: removing glass plate and pry open, and cuts film segment according to the molecular weight of destination protein, and be transferred to transferring film buffer
In.Cutting out pvdf membrane keeps its size almost identical as film segment and is soaked in methanol to activate.Meanwhile by transferring film clip, sponge
Pad, filter paper are also placed in transfering buffering liquid.It opens clip and is sequentially placed sponge, filter paper, pvdf membrane and glue, close transferring film clip
It is transferred in transferring film slot, 200mA constant current transferring film (about 1h).Immune response: 1. after transferring film use 5% skim milk, seal
Close 1h.2. diluting primary antibody (5% skim milk of TBST dissolution, phosphorylated protein use the 5%BSA of TBST dissolution), 4 DEG C of incubations
Overnight.3. being washed three times on TBST at room temperature decolorization swinging table, each 5min.4. secondary antibody dilutes 3,000 times with TBST, in room temperature
It is lower to be incubated for 30 minutes, and washed three times on decolorization swinging table with TBST at room temperature, 5 minutes every time.
Chemiluminescence: mixing the A liquid of ECL and B liquid in darkroom in equal volume, and the albumen of pvdf membrane is placed on exposure up
Between light casket double-layer films, ECL mixed liquor is added, after 2-3min, residul liquid-removing is gone to start to expose.Film after exposure develops
And fixing.
Gel image analysis: being scanned archive for film, arranges discoloration, and with the light of related software processing target band
Density value.As a result as shown in Figure 8,9, the nettle extract of 400 μ g/mL can be substantially reduced in Fc ε RI signal path Lyn and
The phosphorylation level of Syk.
Hyaluronic acid enzyme inhibition rate as index, is made optimization to the extraction process of nettle extract by the present invention.Using
To nettle herb dry powder and fresh nettle using alcohol extracting and water mentions, cold water mentions and hot water mentions and the organic solvent gradually side of extraction
Method mentions to identify the heat resistance and dissolubility of nettle antiallergy substance using experiment of single factor and optimization of orthogonal test nettle
The extraction process of object is taken, the strongest part of nettle antiallergic activity is thus filtered out.
Animal body parts, this research have selected multiple classical I type anaphylaxis models, have evaluated nettle extract from many aspects
Antiallergic activity: D-40 cause itch the result shows that, nettle extract is to itch caused by D-40
There is obvious inhibiting effect, is mainly manifested in and can significantly reduce licking body number and licking the body duration for mouse；Histamine induction
Mouse capillary permeability, which increases result and can be seen that nettle extract, effectively lowers capillary caused by allergic reaction
Permeability increases；OVA inducing systemic active anaphylaxis the result shows that, nettle extract can obviously lower in allergy mice serum
Histamine and OVA specific IgE concentration；The prompt of passive cutaneous anaphylaxis result, nettle extract can effectively inhibit OVA
Local skin allergic reaction caused by sensitization excites.
Cellular portions, the present invention are successfully established RBL-2H3 in-vitro culture model, and detect nettle extract to antigen induction
RBL-2H3 cell degranulation influence.RBL-2H3 cell possesses almost identical with mast cell and basophilic granulocyte
Degranulation approach is crosslinked with IgE high-affinity receptor (Fc ε RI), discharge a series of pre-synthesis and it is new and at medium, arouse
One strong immune allergic reaction.This cell strain has that easily culture, character be stable, the advantages such as single, is analyzed in vitro
The ideal alternative model of all kinds of physiology relevant to mast cell, pathology, mechanism of drug action etc..The present invention passes through CCK- first
8 cell Proliferation toxicity are verified nettle extract and are acted on RBL-2H3 cell no cytotoxicity, to exclude toxic effect interference knot
Fruit.Again by the release of histamine and β-hexosaminidase in RBL-2H3 cell after detection activation, further demonstrates nettle and mention
Object is taken to significantly inhibit mast cell degranulation reaction.Activation is detected finally by the method for Western blot
The phosphorylation level of RBL-2H3 cell Fc ε RI signal path middle and upper reaches Protein S yk and Lyn afterwards, as a result, it has been found that nettle extract
The phosphorylation of Syk and Lyn can obviously be inhibited.
Claims (5)
1. nettle extract, which is characterized in that the hot water extract that the extract is 85-95 DEG C.
2. nettle extract, which is characterized in that the extract is mass concentration 70-80% ethanol extract, the extraction
Temperature is 65-75 DEG C.
3. nettle extract according to claim 2 or 3, which is characterized in that the extract is nettle dry powder or nettle
Numb herb dry powder, wherein extract and 5-95 DEG C described in claim 2 or 3 of hot water extract or mass concentration 70-80% second
The mass ratio of alcohol extracting thing is 1:10-50.
4. nettle extract according to claim 2, which is characterized in that the extract is to extract through claim 3
It is successively extracted again through petroleum ether extraction, ethyl acetate afterwards, lyophilized preparation after extracting n-butyl alcohol.
5. nettle extract according to claim 1-4 treats the application on antianaphylactic drug in preparation.
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Cited By (1)
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| CN108578446A (en) * | 2018-04-26 | 2018-09-28 | 太阳树（厦门）生物工程有限公司 | A kind of production method of low lead nettle extract |
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