CN107029240A - Purposes of the dendritic macromole polyamide amine in rutaecarpin - Google Patents
Purposes of the dendritic macromole polyamide amine in rutaecarpin Download PDFInfo
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Abstract
The invention discloses purposes of the dendritic macromole polyamide amine in rutaecarpin, the absorption enhancement of slightly solubility traditional Chinese medicine ingredients is acted on, dendritic macromole polyamide be G4 for PAMAM co OEG or G4 for PAMAM NH2, the concentration W/V of the two is 0.1%.The present invention is advantageous in that：We are from varying levels such as animal, cells, layer by layer deeply, systematic research PAMAM Dendrimers toxicity, promote absorption, confirm that PAMAM Dendrimers can safely, effectively promote the absorption of Chinese medicine refractory components, therefore it can be used as sorbefacient in food, medicine field, and be widely used, high-efficiency low-toxicity, especially in medicine field, PAMAM Dendrimers can improve the bioavilability of medicine, new pharmaceutic adjuvant can be further developed into, so as to improve the utilization present situation of Chinese medicine at present.
Description
Technical field
The present invention relates to a kind of new application of material, and in particular to dendritic macromole polyamide-amine (PAMAM
Dendrimers new application), belongs to food and medicine technical field.
Background technology
Dendritic macromole polyamide-amine (PAMAM Dendrimers) is relatively broad tree-shaped big point of Recent study
One of son, it is made up of initiation core, monomeric repeating unit and end group, G0~G10 for relative molecular mass be 517~
539000, molecular size is 1~13nm, and there is cavity compound with regular structure, exquisiteness, inside, and algebraically, functional group are controllable, low algebraically
(below G3.0) molecular configuration is more loose, and middle algebraically (G4.0~7.0) forms porous spherical stereochemical structure, high algebraically
(G8.0~10.0) surface is almost the packed structures of closing.Gather polar functional group on PAMAM Dendrimers surface, this
A little polar functional groups can be acted on various biomembranes, change the characteristic of film, so as to strengthen the permeability of medicine.PAMAM
Dendrimers hydrophobic cores and the cavity of dendritic network can be coated with drug molecule, so as to improve the dissolving of medicine
Degree.
But, it is not that the PAMAM Dendrimers of any algebraically are suitable for carrying medicine.The PAMAM of low algebraically
Dendrimers configurations are loose, easily tangled between molecule, easily leakage, the PAMAM of high algebraically after medicine is encapsulated
Dendrimers configurations are close, and medicine is difficult to be encapsulated, and are difficult release again once encapsulated, only medium algebraically (4~7 generation)
PAMAM Dendrimers are adapted to contain drug molecule, and viscosity is big, stability is strong and good biocompatibility.
PAMAM Dendrimers are also in one of most widely used dendritic macromole of biomedical sector.For
The genetic fragment that phosphate group formation compound in DNA transmission and gene therapy, with DNA molecular main chain is loaded is loaded into thin
Karyon, so as to reach gene therapy purpose.1993, Haensler and Szoke prime reports utilized PAMAM
DNA is transfected into the achievement in research in different mammals by denreimers.For mri contrast agent, in clinical research
In, and the residence time of the heavy metal ion such as gadolinium formation complex compound extension in the blood vessels, so that well in blood vessel, tissue, device
Radiography is positioned in official.J.Bulte researchs are found, can be used to after the PAMAM denreimers injection rat brains for the mark that is magnetized are interior
Follow the trail of their activities in intracerebral.For new drug carrier, because its have drugloading rate height, non-immunogenicity, hereditary-less toxicity,
Easily by advantages such as blood-brain barriers, one of study hotspot of pharmaceutical field is had become.Najlah is by insoluble drug methoxy naphthalene
Propionic acid acetyl amine key and PAMAM Dendrimers covalent bonds, empirical tests are better than original shape medicine with reference to the hydrophily of rear medicine
And stability is preferable.
It can be seen that, PAMAM Dendrimers are a kind of quite potential new drug carriers.
The absorption of medicine enters link primary after body as medicine and its formulation, refers to medicine from medicine-feeding part to blood
The process of fluid circulation transhipment, the mainly epithelium by the position such as intestines and stomach and alveolar, skin, schneiderian membrane and cornea is thin
What born of the same parents were carried out, except intravascular administration, this process will be passed through after medicinal application.Wherein, oral administration route be using most convenient,
Most extensively, the administering mode received by people is easiest to, traditional Chinese medicine convenient administration mode is also complied with.But extract oral is given
The studies have shown that with bioavilability is absorbed after medicine due to the complexity of traditional Chinese medicine ingredients, the diversity of physicochemical property is brought, such as
Poorly water-soluble, liposoluble constituent dissolution rate are poor, methods of glycosides facile hydrolysis etc., so as to influence oral administration biaavailability.In addition, in
The backwardness and formulation design of pharmaceutically dosage form and the foundation for preparing shortage science, are also to cause oral preparation of Chinese traditional medicinal bioavilability low
The reason for.Clinical efficacy difference is even not as the present situation of decoction matches after this is administered with some extract oral formulations.
Low bioavilability preparation not only have impact on Chinese Herbs, and also result in the waste of high amount of drug and its resource.
Therefore, strengthen basic research, change the present situation that form of Chinese drug still falls behind very much, research is adapted to the high bioavilability of character of traditional Chinese medicine
Delivery system preparation, has given play to the curative effect and advantage of Chinese medicine, the need for being not only clinical application, is also that Chinese medicine is medical at home and abroad
The demand of its powerful competitiveness, the even more modernization of Chinese medicine and international requirement are shown in product market.
At present, the approach for improving drug bioavailability mainly has two：
One approach is to change pharmaceutical physicochemistry property, improves its permeable membrane ability or improves its dissolution characteristics, such as micro mist
Change technology, solid dispersions technique, inclusion technique, pro-drug technology of preparing etc.；
Another approach is to improve the characteristic of film to improve the film permeability of medicine, or efflux pump suppression, to prevent body
To the use of the outer row, i.e. sorbefacient that absorb the drug.
The application of sorbefacient is the blood concentration and bioavilability simple effective method the most for improving medicine, its
It has been widely used in Western medicine research.However, in view of the diversity and complexity of effective component of chinese medicine, and often compound is given
Medicine, it is difficult to find the sorbefacient simultaneously effective to Chinese medicine Multiple components.Therefore, grinding in terms of relevant Chinese medicine sorbefacient
Study carefully and be rarely reported.
In addition, most sorbefacient promote drug absorption while, also the tissue to body medicine-feeding part and
Cell membranes in tissue brings toxic side effect.Therefore, the field of Chinese medicines search out it is a kind of safely, effectively, widely used absorption enhancement
Agent is extremely urgent.
The content of the invention
It is an object of the invention to provide the purposes in rutaecarpin of dendritic macromole polyamide-amine, research tree
Purposes of the dendritic macromolecules polyamide-amide (PAMAM Dendrimers) as sorbefacient in food, medicine field, especially
It is the purposes in the field of Chinese medicines.
To study PAMAM Dendrimers rush absorption, from the built vertical Changbai Mountain of early stage conventional Chinese medicine sample among the people
A kind of representative traditional Chinese medicine monomer active ingredient is have chosen in product storehouse --- rutaecarpin is model drug, analysis model medicine
Physicochemical property, using PAMAM Dendrimers from internal, external two kinds of angles improve model drug absorption characteristic.
The present invention is advantageous in that：
We are from varying levels such as animal, cell, molecules, layer by layer deeply, systematic research PAMAM Dendrimers
Toxicity, promote absorption, it was confirmed that PAMAM Dendrimers can safely, effectively promote the suction of Chinese medicine refractory components
Receive, therefore it can use in food, medicine field as sorbefacient, and be widely used, high-efficiency low-toxicity, especially exist
Medicine field, PAMAM Dendrimers can improve the bioavilability of medicine, can further develop into new medicinal
Auxiliary material, so as to improve the utilization present situation of Chinese medicine at present.
Brief description of the drawings
Fig. 1 is the comparison figure of different dosing group Caco-2 cell survival rates；
Fig. 2 is that different dosing group transhipment amount changes over time situation；
Fig. 3 is different dosing group Papp value changes situations；
Fig. 4 is that rutaecarpin blood concentration changes over time situation；
Fig. 5 is the comparison figure of different dosing group intestines irrigating solution total protein concentration；
Fig. 6 is the comparison figure of different dosing group intestines irrigating solution LDH activity.
Embodiment
Make specific introduce to the present invention below in conjunction with the drawings and specific embodiments.
First, Caco-2 cell models
Caco-2 cell models come from people's clone's colon cancer cell, have similar structure and function with intestinal epithelial cell,
With the structures such as microvillus and related enzyme systems, therefore it can be used to absorption of the aids drug in small intestine.
Caco-2 cells were inoculated in after Transwell plate culture certain times, Caco-2 cells can constantly adherent growth,
Division, differentiation, and ultimately form continuous cell monolayer.
2nd, PAMAM Dendrimers toxicity research
Tetrazolium bromide (MTT) colorimetric method is a kind of common method for detecting cell survival conditions, and cell is widely used at present
In terms of toxicity test, biotic factor Activity determination, screening anti-tumor medicine, economical and practical, sensitivity is high.
PAMAM-co-OEG：It is grafted the dendritic macromole polyamide-amine (PAG) of three polyethylene glycol.
PAMAM-NH2：It is grafted the dendritic macromole polyamide-amine of amine.
We are using 0.1% (w/v) G4 of tetrazolium bromide (MTT) experiment detection for PAMAM-co-OEG (PAG), 0.1% (w/v)
G4 is for PAMAM-NH2, 0.5% (w/v) G4 for PAMAM-co-OEG (PAG), 0.5% (w/v) G4 for PAMAM-NH2To Caco-2
The influence of cell survival rate, while setting negative control group (blank cultures), positive controls (3%Triton X-100) and adjusting
Zero group (acellular), and the survival rate of negative control group is set to 100%.
Testing result：It is respectively for the PAG Caco-2 cell survival rates treated with 0.1% (w/v), 0.5% (w/v) G4
95.3 ± 5.13% and 74.4 ± 4.22%, with 0.1% (w/v), 0.5% (w/v) G4 for PAMAM-NH2Treated Caco-2
Cell survival rate is respectively 78.2 ± 4.15% and 64.6 ± 5.74%, and the Caco-2 cell survival rates of positive controls are 50.1
± 4.02%, as shown in Figure 1.
As a result show：0.5% (w/v) G4 is for PAMAM-NH2With 0.5% (w/v) G4 for PAG to Caco-2 cell survival rates
Influence is larger, although Caco-2 cell survival rates are all higher than 3%TritonX-100 groups, but appoints and be so not suitable for follow-up reality
Test；0.1% (w/v) G4 is for PAMAM-NH2, 0.1% (w/v) G4 do not produce significantly for PAG to Caco-2 cell survival rates
Influence, the two can be used in follow-up experiment, and 0.1% (w/v) G4s of the G4 for PAG relatively with concentration for PAMAM-NH2More
Safety.
3rd, model drug
We have done careful chemical analysis to Changbai Mountain conventional Chinese medicine among the people, establish Changbai Mountain conventional Chinese medicine among the people
A point sample library is studied, and the targeting including the major diseases such as antitumor, hepatic injury, rheumatological disease has been done to sample in storehouse
Bioactivity screening, it is excellent to have selected many traditional Chinese medicine monomer active ingredients with good biological activity.
These traditional Chinese medicine monomer active ingredients are classified by chemical property and design feature, what is filtered out all kinds of represents into
Dividing includes：Saponins, flavonoids, alkaloids, Anthraquinones, lignin, Coumarins.
We choose a most class (alkaloids) physicochemical property of most common, quantity and absorption characteristic is all representative
Traditional Chinese medicine monomer active ingredient --- rutaecarpin is model drug.
Rutaecarpin：The principal alkaloid constituents of rutaceae evodia rutaecarpa, molecular formula is C19H17N3O, molecular weight is
303, with a variety of pharmacological effects, such as by caspase or other Control factors inducing apoptosis of tumour cell, by discharging CGRP
Prevent heart allergy reaction, by reducing the Secretion regulation blood pressure of aldosterone, suppress Escherichia coli, kill ascaris suum, diuresis,
Anti-inflammatory, analgesia etc..
4th, the PAMAM Dendrimers researchs for promoting absorption external to effective component of chinese medicine
To investigate rush absorptions of the PAMAM Dendrimers to effective component of chinese medicine from external angle, we are with research
The dissolubility and permeability of drug absorption are core, the physicochemical property of research model medicine, using above studying the toxicity that draws
Less PAMAM Dendrimers are combined with different type model drug, across the Caco-2 cell membrane transporter amount of analysis model medicine
And the situation of change of cell TEER values.
All data are usedRepresent, using the processing datas of SPSS 20.0 and between ASSOCIATE STATISTICS credit analysis, group compare
Relatively use to compare in one-way analysis of variance, group and examined using LSD-t, P<0.05, which represents difference, has statistical significance.
1st, the HPLC chromatogram condition of model drug
Rutaecarpin chromatographic condition：
| Chromatographic column | AgilentTC-C18 (250mm × 4.6mm, 5 μm) |
| Guard column | SEP-C18(8.0×10mm) |
| Mobile phase | Methanol：Acetonitrile：Water=20：38：42 |
| Detection wavelength | 260nm |
| Flow velocity | 1mL·min-1 |
| Column temperature | 30℃ |
| Sample size | 20μL |
By above-mentioned chromatographic condition sample introduction, the retention time of rutaecarpin is about 11min or so, and chromatographic peak point is symmetrical, baseline
Steadily, no hangover.
2nd, the solubility criteria curve of model drug
Rutaecarpin reference substance solution is obtained into 3,6,12,24,48 μ gmL with methanol dilution-1Series concentration standard items are molten
Liquid, by rutaecarpin chromatographic condition sample introduction, carries out linear regression to concentration (c) with peak area (A), obtains calibration curve equation
For：
A=30.119c+1.3683 (R2=0.9999).
As a result show：Rutaecarpin is in 3~48 μ gmL-1In the range of linear relationship it is good.
3rd, the solubility experiment of model drug
Reference《Chinese Pharmacopoeia》Version dissolubility assay method in 2015：Precision weighs rutaecarpin standard items 10.0mg and is placed in
In tool plug centrifuge tube, 5mL distilled water (pH value 6.8) is added, 25 DEG C ± 2 DEG C of constant temperature is vibrated after 30s, 30min per 5min
5000r·min-110min is centrifuged, supernatant 4mL is taken in 12000rmin-110min is centrifuged, then takes supernatant to cross 0.22 μm of filter
20 μ L sample introductions are taken after film, dilution certain multiple, the solubility of rutaecarpin is analyzed under HPLC and 6 repeating groups are set.
FDA recommend the deliquescent general standard of judgement compound be：D0=(M0/V0)/CS, wherein M0Medicament is given for maximum
Measure (mg), V0For 250mL, CSFor solubility (mgmL-1), work as D0It is high solubility pharmaceuticals, D when≤10>It is low-solubility when 1
Medicine.《Chinese Pharmacopoeia》The criterion of approximate solubility as defined in (version in 2015) is：Solubility (S)>0.01g·mL-1For
Highly dissoluble, 0.000 1gmL-1＜ S≤0.01gmL-1For low-solubility.Reference《Chinese Pharmacopoeia》Evodia rutaecarpa give medicament
Measure as 2~5g, the total amount of regulation rutaecarpin and Rutaecarpine must not be less than 0.15%, i.e. Wu Zhu in evodia rutaecarpa quality standard
The maximum dosage of cornel alkali is 7.5mg.
Solubility experiment result shows：Rutaecarpin is under the conditions of pH value 6.8, and solubility is 0.21 ± 0.01 μ gmL-1,
Therefore rutaecarpin D0For 142.86.
It is comprehensive《Chinese Pharmacopoeia》With FDA standard, it is low-solubility composition to determine rutaecarpin.
4th, the permeability standards curve of model drug
Rutaecarpin reference substance solution is obtained into 0.01,0.0125,0.025,0.05,0.1 μ gmL with methanol dilution-1System
Row concentration standards solution, by rutaecarpin chromatographic condition sample introduction, carries out linear regression to concentration (c) with peak area (A), obtains
Calibration curve equation is：
A=37.933c-0.0623 (R2=0.9992).
As a result show：Rutaecarpin is in 0.01~0.1 μ gmL-1In the range of linear relationship it is good.
5th, the permeability experiment of model drug
Caco-2 cells (experiment cell algebraically used is in 40~60 generations) are taken out from incubator, when cell growth is to being paved with
During blake bottle 80%~90%, centrifuged after being digested with pancreatin, plus fresh culture is diluted to 2 × 105Individual mL-1Cell suspension
It is inoculated in TranswellTMOn plate, nutrient solution is changed every other day and cell TEER values are measured, after cultivating 10 days, liquid is changed daily, electricity is surveyed
Resistance, and Caco-2 cellular morphologies are observed under the microscope.After culture 21 days, Transwell is taken outTMAP sides and BL sides are abandoned in plate, suction
Nutrient solution, slowly clean Transwell with Hank ' the s liquid for being preheated to 37 DEG C in advanceTMThe small indoor and outdoor of plate 2 times, last time adds
Enter to be placed in incubator after Hank ' s liquid and be incubated 20min.Take out TranswellTMPlate, measures and records cell TEER values, choosing
Select more than 500 Ω cm2Cell tested.
100 μ gmL are diluted to 37 DEG C of Hank ' s liquid is preheated to by appropriate rutaecarpin storing solution-1,
TranswellTMThe AP sides of plate add the rutaecarpin solution 0.5mL diluted, and fresh Hank ' s liquid is added in BL sides
1.5mL, the after medicine is added the 15th, 30,60,90,120min draws 200 μ L samples in 1.5mL centrifuge tubes from BL sides respectively
In and supplement after Hank ' the s liquid of equal volume, 2h, measure again and record cell TEER values.Different time points are analyzed in HPLC
The sample concentration of sampling, the transhipment amount of pharmaceutical units time is calculated according to standard curve, and apparent infiltration is calculated according to following equation
Coefficient (Papp)：
Papp=(dQ/dt)/(AC0)
Wherein, dQ/dt is transmembrane transport amount (the μ gs of pharmaceutical units time-1)；A is the area of carrier film
(1.12cm2)；C0For the initial concentration of test medicine；PappUnit is cms-1。
6th, PAMAM Dendrimers promote absorption experiment in vitro
First, Caco-2 cell models are built；Then, 100 μ gmL are added in the AP sides of Caco-2 cells-1Containing 0.1%
(w/v) G4 is for PAG, PAMAM-NH2Model drug (rutaecarpin) solution 0.5mL, added in the BL sides of Caco-2 cells new
Fresh Hank ' s liquid 1.5mL；Finally, the concentration of specimens that different time points are sampled, comparison model medicine (evodia rutaecarpa are analyzed with HPLC
Alkali) transmembrane transport amount and apparent permeability coefficients (Papp) value change.
7th, the permeability experiment of model drug and PAMAM Dendrimers promote absorption experimental result in vitro
Rutaecarpin permeability experiment is carried out according to experimental method above and PAMAM Dendrimers promote to absorb in vitro
Effect experiment, respectively at the TEER values that cell is detected before administration and after administration 120min, the results are shown in Table 1；Different time points Wu Zhu
Cornel alkali transhipment amount result is shown in Fig. 2；PappValue changes result is shown in Fig. 3.
Resistance change (n=3) before and after the transhipment of the different dosing group of table 1
From the resistance change result of table 1, PAMAM-NH2Cell TEER value changes are larger before and after administration group transhipment,
It is probably, because terminal amino group has considerable influence to the close contiguity of cell membrane, to have one to the integrality of Caco-2 cell membranes
Determine the destruction of degree.And PAG administration group cell TEER value changes are smaller, it may be possible to because the glycols branch of its surface grafting
Shaped polymer reduces the amino quantity of exposure outside, alleviates the toxicity to cell to the parcel of amino.
Experimental result, PAG administration groups and PAMAM-NH are transported in Fig. 22Rutaecarpin in administration group 120min
The increase more obvious than blank rutaecarpin group of transhipment amount, therefore 0.1% (w/v) PAG and 0.1% (w/v) PAMAM-NH2To evodia rutaecarpa
Alkali transhipment has a certain degree of facilitation.
Apparent permeability coefficients PappIt is commonly used to judge the power of the approach drug absorption abilities such as oral administration, PappValue>2×
10-6cm·s-1To absorb preferable composition.The P in Fig. 3appKnowable to value changes result, the P of blank rutaecarpin groupappIt is worth and is
(1.77±0.110)×10-6cm·s-1, therefore be hypotonicity medicine；The P of 0.1% (w/v) PAG administration groupsappIt is worth and is
(2.65±0.179)×10-6cm·s-1, 0.1% (w/v) PAMAM-NH2The P of administration groupappIt is worth for (2.72 ± 0.215) × 10-6cm·s-1, respectively with the P of blank rutaecarpin groupappValue is compared to 1.50 and 1.54 times of increase.
8th, conclusion
Learnt according to solubility and permeability experiment result, rutaecarpin belongs to low solubility hypotonicity medicine.One
Determine in degree, the dissolubility of drug molecule can influence it to enter the coefficient of dispersion of solution state, and then influence its permeability.
PAMAM Dendrimers promote absorption experiment result and shown in vitro, 0.1% (w/v) PAG and 0.1% (w/v)
PAMAM-NH2There are a certain degree of facilitation, comprehensive PAMAM Dendrimers to the transmembrane transport of Chinese medicine refractory components
In vitro toxicity is tested and cell TEER value results learn that PAG compares PAMAM-NH2It is safer, it is more suitable for being tested, and then develop
Into novel sorbefacient.
5th, researchs of the PAG to rush absorption in effective component of chinese medicine body
Further to investigate the less PAG of toxicity above drawn from internal angle to different type effective component of chinese medicine
Promote absorption, we are next using rat in body intestinal loop administration experimental method in situ, and relatively real reflection PAG is in enteron aisle
Absorbing state, detects that total protein content and lactic acid dehydrogenase activity in small intestine irrigating solution analyze PAG toxicity in vivo after experiment.
1st, experimental animal
Male SD rat, 200~220g of body weight, sub-cage rearing is in SPF grades of Animal Houses, per 6, cage, main environment index
Control range is：20~26 DEG C of room temperature, day and night temperature≤4 DEG C, relative humidity 40~70%, time/hour of minimum air changes 15,
Illumination light and shade 12h replaces.Periodic replacement bedding and padding, keep clean environment clean, it is ensured that the sufficient diet drinking-water of animal and activity are certainly
By, it is to avoid excessive noise and other interference.
2nd, prepared in body intestinal loop in situ
42 male SD rats (200~220g) are randomly divided into 7 groups, every group 6.Fasting 16h, can't help water before experiment.
10% chloraldurate solution anesthesia (0.5mL100g is injected intraperitoneally-1), rat completely, is lain on the back solid by rat anesthesia after a few minutes
Due on plank, alcohol disinfecting, belly median line opening exposes intestines, and what is be connected with the rear end pylorus of stomach is duodenum,
Away from being jejunum after duodenum about 1~3cm, about 20cm jejunal segments are isolated, T-shaped opening is cut at two sections respectively, silicone tube is inserted
And fixed with aseptic operation line, gently intestinal contents is rinsed well with the physiological saline for being preheated to 37 DEG C in advance, efflux is treated
Become after clarification, with air-discharging method by the residual liquid emptying in intestinal loop.Intestinal loop is put back into abdominal cavity, and covering is soaked with physiology above
The gauze moisturizing of salt solution, irradiates to keep rat temperature under light.
3rd, in body intestinal loop administration in situ
Prepare and contain drug solns, experimental rat gives the rutaecarpin solution with or without 0.1% (w/v) PAG, 3% respectively
TritonX-100 solution (positive controls).Dosage is 100mgkg-1, dosage is 5mLkg-1.Medicine is sucked
In syringe, gently squeeze into the intestinal loop for having prepared completion, check that whether decoction is full of intestinal segment, and start timing.Respectively to
Before medicine and administration 15,30,60,90,120,150,180,240min after from taking blood from jugular vein, take 0.3mL whole bloods to be placed in and used liver
In the treated centrifuge tube of plain sodium, 10000rmin-15min is centrifuged, upper plasma is separated, -20 DEG C of preservations are stand-by.In experiment not
Jugular vein blood collection experiment is carried out to positive controls, intestines toxicity test is only carried out.
4th, determination of plasma concentration
Blood plasma is taken out from -20 DEG C of refrigerators, is thawed, every part of sample presses blood plasma：Methanol=1：3 ratio protein precipitation, is vortexed
Mix 1min, ultrasonic 10min, 10000rmin-1High speed centrifugation 10min, takes supernatant, enters by the chromatographic condition above recorded
Sample, analyzes the blood concentration of different time points each sample.
All data are usedRepresent, using the processing datas of SPSS 20.0 and between ASSOCIATE STATISTICS credit analysis, group compare
Relatively use to compare in one-way analysis of variance, group and examined using LSD-t, P<0.05, which represents difference, has statistical significance.
5th, experimental result
Rat gives rutaecarpin solution and rutaecarpin solution containing 0.1% (w/v) PAG respectively in body intestinal loop in situ
Afterwards, the blood concentration result of different time points is shown in Fig. 4.
From Fig. 4 blood concentration result of variations, the blood concentration at each time point containing 0.1% (w/v) PAG administration groups is bright
It is aobvious to be higher than blank rutaecarpin group, therefore 0.1% (w/v) PAG has a certain degree of promotion to make to the body absorption of rutaecarpin
With.
In summary, the blood concentration at experimental group each time point containing PAG is obviously higher than the control group without PAG, PAG
It is remarkably improved the body absorption of Chinese medicine refractory components.
6th, PAG toxicity in vivo is detected
1st, the preparation of small intestine irrigating solution
Take after the completion of blood experiment, by small intestine Chinese medicine liquid emptying, cut, finally put to death rat.The small intestine cut, one end is put
In the sterile dry centrifuge tubes of 10mL, other end connection 5mL syringes are slowly injected into 4 DEG C of PBS (2.5mL100g-1) solution,
After lavation terminates, irrigating solution is fully blown and beaten to uniform, 4000rmin under 4 DEG C of environment-110min is centrifuged, supernatant is taken in -20
DEG C preservation is stand-by.
2nd, total protein concentration is detected
Detect that total protein concentration analyzes the toxicity of experiments in vivo, this method has been reported.This experiment uses Beijing health for public affairs
Protein concentration in the BCA kits detection sample of department's production.In the basic conditions, bivalent cupric ion can be reduced into by protein
Univalent copper ion, the copper ion being reduced can exist higher with Solution A effects generation purple compound and at 562nm
Absorbance.According to the linear relationship between absorbance and protein concentration, by measuring absorbance, and then albumen is calculated
Concentration.
According to BCA protein quantification kit operational manuals, BSA standard items are diluted to 2000,1000,500 with PBS,
250,125,62.5 μ gmL-1Totally 6 concentration；A liquid in BCA kits and B liquid are pressed 50：1 ratio is mixed, and configures BCA works
Make liquid；Taking the BSA standard items and testing protein sample that 25 μ L have diluted to be added in 96 orifice-plate microporosities respectively, (each sample does 3
Parallel group), 200 μ L BCA working solutions are added per hole, 30min is incubated under the conditions of 37 DEG C are placed in after mixing, is cooled to after room temperature,
OD (absorbance) value in every hole is determined at 572nm with ELIASA.Using BSA standard concentrations as abscissa, corresponding OD values are
Ordinate, draws standard curve, and according to the total protein concentration of standard curve calculating sample.
3rd, lactic dehydrogenase Concentration Testing
The toxicity of LDH (lactic dehydrogenase) concentration analysis experiments in vivo is detected, this method has also been reported.This experiment is used
The lactic dehydrogenase enzyme concentration that Nanjing is built up in the LDH kits detection sample of research institute's production.LDH is to be present in body respectively to organize
A kind of important enzyme in organ, when lesion occurs for body, LDH contents will increase in blood, and LDH can be catalyzed lactic acid generation third
Ketone acid, then brown-red complex is generated with DNPH reaction, and then pass through colorimetrically analysing enzyme activity.In body
The change of LDH contents, the lesion situation of body can be reflected indirectly.
Associative operation is carried out according to LDH testing cassetes specification, method is shown in Table 2.
The LDH detection methods of table 2
LDH concentration in sample is calculated according to the following formula：
4th, experimental result
All data are usedRepresent, using the processing datas of SPSS 20.0 and between ASSOCIATE STATISTICS credit analysis, group compare
Relatively use to compare in one-way analysis of variance, group and examined using LSD-t, P<0.05, which represents difference, has statistical significance.
(1) total protein concentration
Drawing BCA protein quantification calibration curve equations is：A=0.9976c+0.0867 (R2=0.9991), 62.5~
2000μg·mL-1In the range of linear relationship it is good.
Rat is after body intestinal loop administration 4h in situ, and total protein concentration testing result is shown in Fig. 5 in each administration group small intestine irrigating solution.
It is poor containing 0.1% (w/v) PAG groups and blank model drug group total protein concentration from Fig. 5 total protein concentration results
It is different unobvious, 3%TritonX-100 positive controls and blank model drug group total protein concentration significant difference, positive control
Group toxicity is big to damage serious to small intestine, and PAG groups small toxicity damages smaller to small intestine.
(2) lactic dehydrogenase enzyme concentration
Rat is after body intestinal loop administration 4h in situ, and LDH content detection results are shown in Fig. 6 in each administration group small intestine irrigating solution.
From Fig. 6 LDH activity testing results, containing 0.1% (w/v) PAG groups and blank model drug group LDH activity difference
Not substantially, 3%TritonX-100 positive controls and blank model drug group LDH activity significant difference, positive controls LDH
Active high explanation small intestine infringement is serious, and PAG groups damage smaller to small intestine.
5th, conclusion
Rat intestinal instillation can be used for the Position Research to enteron aisle, and PAG promotees absorption research and intestines in this experiment
Toxicity detection result shows that 0.1% (w/v) PAG safe and effective can promote the absorption of Chinese medicine refractory components.
Total protein content and LDH enzymatic activitys are relatively low in intact animal small intestine irrigating solution, and total protein oozes out from blood plasma,
LDH is cytoplasm enzyme, when some reasons make permeability of cell membrane increase or cell death, and both materials, which substantial amounts of can dissolve, to be released
Put into extracellular fluid, therefore total protein content and LDH activity can reflect the extent of damage of cell membrane in small intestine irrigating solution.This reality
(w/v) PAG is smaller on intestines irrigating solution total protein content and LDH activity influence 0.1% in testing, comprehensive Absorption test above
As a result, PAG can be developed further into new type of safe, efficient sorbefacient.
7th, conclusion
We systematic research PAMAM Dendrimers toxicity, promote to absorb and made from animal, cell varying level
With, it was confirmed that PAMAM Dendrimers can safely, effectively promote the absorption of Chinese medicine refractory components, therefore it can make
For sorbefacient use in food, medicine field, and be widely used, high-efficiency low-toxicity, especially in medicine field, PAMAM
Dendrimers can improve the bioavilability of medicine, can further develop into new pharmaceutic adjuvant, so that in improving
The utilization present situation of medicine at present.
It should be noted that the invention is not limited in any way for above-described embodiment, all use equivalent substitutions or equivalent change
The technical scheme that the mode changed is obtained, all falls within protection scope of the present invention.
Claims (7)
1. purposes of the dendritic macromole polyamide-amine in rutaecarpin, it is characterised in that：Dendritic macromole polyamide is made
The absorption enhancement of slightly solubility drug ingredient is acted on for sorbefacient.
2. purposes of the dendritic macromole polyamide-amine according to claim 1 in rutaecarpin, it is characterised in that
The medicine is Chinese medicine.
3. purposes of the dendritic macromole polyamide-amine according to claim 2 in rutaecarpin, it is characterised in that
The monomer active ingredient of the Chinese medicine is rutaecarpin.
4. purposes of the dendritic macromole polyamide-amine according to claim 1 in rutaecarpin, it is characterised in that
The dendritic macromole polyamide is G4 for PAMAM-co-OEG.
5. purposes of the dendritic macromole polyamide-amine according to claim 4 in rutaecarpin, it is characterised in that
The G4 is 0.1% for PAMAM-co-OEG concentration W/V.
6. purposes of the dendritic macromole polyamide-amine according to claim 1 in rutaecarpin, it is characterised in that
The dendritic macromole polyamide is G4 for PAMAM-NH2。
7. purposes of the dendritic macromole polyamide-amine according to claim 6 in rutaecarpin, it is characterised in that
The G4 is for PAMAM-NH2Concentration W/V be 0.1%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690591A (en) * | 2020-06-19 | 2020-09-22 | 贵州中医药大学 | Diuretic active drug cell screening model and establishment method and application thereof |
| CN111686076A (en) * | 2020-06-30 | 2020-09-22 | 陕西中医药大学 | Adriamycin-loaded polymer micelle and preparation method and application thereof |
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2017
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690591A (en) * | 2020-06-19 | 2020-09-22 | 贵州中医药大学 | Diuretic active drug cell screening model and establishment method and application thereof |
| CN111690591B (en) * | 2020-06-19 | 2022-03-15 | 贵州中医药大学 | Diuretic active drug cell screening model and establishment method and application thereof |
| CN111686076A (en) * | 2020-06-30 | 2020-09-22 | 陕西中医药大学 | Adriamycin-loaded polymer micelle and preparation method and application thereof |
| CN111686076B (en) * | 2020-06-30 | 2022-03-15 | 陕西中医药大学 | Adriamycin-loaded polymer micelle and preparation method and application thereof |
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