CN106377500A - Andrographolide nano suspension and preparation method thereof - Google Patents
Andrographolide nano suspension and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutical preparations, and provides an andrographolide nano suspension and a preparation method thereof. The andrographolide has low solubility in water, can be easily excreted by P-gp, has high metabolism rate, and thus, has lower dissolution rate and lower in-vivo bioavailability. In order to enhance the dissolution rate and bioavailability of the drug, a P-gp inhibitor TPGS and an ionic stabilizer SDS are selected to prepare the andrographolide nano suspension by using a medium grinding technique; by using the particle size and distribution as evaluating indicators, a Box-Behnken center combined degisn test optimized preparation technique is utilized to prepare the andrographolide nano suspension; and freeze-drying is used for solidifying. After carrying out the characterization, the research in short-term stability, Caco-2 permeability and in-vitro dissolution, and the in-vivo pharmacokinetic and pharmacodynamic research, the results indicate that the andrographolide nano suspension can increase the dissolution rate of andrographolide and enhance the oral-administration bioavailability and anti-inflammatory action.
Description
Technical field
The present invention relates to pharmaceutical technology field is and in particular to a kind of andrographolide nano suspension and preparation method.
Background technology
Andrographolide (andrographolide) is to extract, in acanthaceous plant Herba Andrographitis, the principle active component obtaining
One of, it is diterpene ginkgolide, in Folium Andrographis, content is up to 1.84%.Modern study finds that andrographolide has extensively
General pharmacologically active, it has the effect such as preferable anti-inflammatory, anti-infective, anticancer, liver protection.However, its hydrophobicity is stronger, in water
Solubility low (0.07mg/mL) (Chen LL, Wang ZH.Investigation of basic physical and
chemical properties of andrographolide[J].Pharm Today,2010,20(1):41-43.), easy quilt
Arrange outside P-gp, metabolism is fast, leads to its low dissolution rate and relatively low vivo biodistribution availability (2.67%) (Ye L, Wang T, Tang
L et al.Poor oral bioavailability of a promising anticancer agent
andrographolide is due to extensive metabolism and efflux by P-glycoprotein
[J].J Pharm Sci,2011,100(11):5007-17.), thus limiting its clinical practice.
Improve insoluble drug bioavilability and can adopt various medicaments technology, by forming soluble-salt, change
Crystal formation or make amorphous noncrystal, add surfactant wetting or Micellar Solubilization, micronizing or nanosizing increase surface area,
Forming compound with phosphatide increases hydrophily, forms the solid dispersions of water-solubility carrier, cyclodextrin molecular inclusion, emulsification, micro-
Emulsification etc. preparation technique, make solution, supensoid agent, tablet, capsule, emulsion, micro emulsion, from formulations such as micro emulsion, micellar solutions.Though
So improve the bioavilability of andrographolide to a certain extent, but its low drugloading rate, low envelop rate, low stability limit
Its clinical practice.
Content of the invention
It is an object of the invention to provide a kind of andrographolide nano suspension and preparation method, to improve in Herba Andrographitis
The bioavilability of ester, stability.
Inventor passes through on the basis of single factor exploration, and application BBD- effect surface method optimizes andrographolide nanometer suspension
The preparation technology of agent, determines optimal preparation technology.Andrographolide nano suspension, Ran Houtong are obtained with optimal preparation technology
Cross freeze-drying to be solidified, improve its stability further.Comprehensive in-vitro evaluation has been carried out to said preparation, including particle diameter and
Particle diameter distribution, Zeta potential, SEM (SEM), X-ray diffraction (PXRD), differential scanning calorimetry (DSC),
Caco-2 permeability test, dissolution in vitro analysis, drug effect medicine move experiment it is intended to produce a kind of safer, stable, biological utilisation
Degree is high, be suitable to the andrographolide nanometer formulation of industrialized production.
A kind of andrographolide nano suspension of the present invention, is made up of andrographolide, stabilizer, and stabilizer is to have
The vitamin E polyethylene glycol succinic acid ester (TPGS) of P-gp inhibitory action and ionic stabilizer lauryl sodium sulfate (SDS).
Described andrographolide nano suspension is it is characterised in that formulation is freeze-dried powder.
Described andrographolide nano suspension, the step that preparation method includes following orders：(1) weigh in Herba Andrographitis
Ester, adds in the aqueous solution including TPGS and SDS；(2) aforesaid liquid is placed in refiner high speed shear；And then will be above-mentioned (3)
Suspension is poured in medium grinder, grinds, prepared andrographolide nano suspension；(4) addition of gained nano suspension is sweet
Dew alcohol, is respectively charged in cillin bottle；(5) it is placed in -80 DEG C of refrigerator pre-freezes, then is positioned in freeze drier, prepare punching
Lotus lactone nanosuspension frozen powder.
Described andrographolide nano suspension, described in preparation method step (1), the consumption of solvent should make in Herba Andrographitis
The concentration of ester is 0.5g/100ml.
Described andrographolide nano suspension, stabilizer in the aqueous solution of described TPGS and SDS of preparation method step (1)
TPGS and SDS concentration is calculated as 0.62%, respectively 0.30%, 0.32% with mass volume ratio, and such as 200mL water includes
0.600gTPGS, 0.640gSDS.
Described andrographolide nano suspension, in the described medium grinder of preparation method step (3), using 0.3-
0.4mm zirconia bead 850g, rotating speed is 1056rpm, and milling time is 68min.
Described andrographolide nano suspension, the described cryoprotection agent concentration of preparation method step (4) is with quality volume
Than the mannitol being calculated as 1%.
Described andrographolide nano suspension, the described pre-freezing temperature of preparation method step (5) is -80 DEG C, the pre-freeze time
12h.
Described andrographolide nano suspension, the described freeze-drying temperature of preparation method step (5) is to maintain when -30 DEG C
10h, maintains 5h, 0 DEG C of maintenance 2h, 25 DEG C of maintenance 2h when -10 DEG C.
Described andrographolide nano suspension, the step that preparation method includes following orders：(1) weigh in 1g Herba Andrographitis
Ester, adds and includes 0.600gTPGS, in the 200mL aqueous solution of 0.640gSDS；(2) aforesaid liquid is placed in refiner
16000rpm high speed shear 5min；(3) and then by above-mentioned suspension pour in medium grinder, using 0.3-0.4mm zirconium oxide
Globule 850g, grinds 68min, prepared andrographolide nano suspension under rotating speed 1056rpm；(4) gained nano suspension
Add the mannitol being calculated as 1% with mass volume ratio, be respectively charged in cillin bottle, every bottle of 5mL；(5) it is placed in -80 DEG C of refrigerator pre-freezes
12h, then be positioned in freeze drier, prepare andrographolide nanosuspension frozen powder.
Beneficial effect：When adding 1% mannitol in the present invention as freeze drying protectant, andrographolide nano suspension freezes
The RDI of dry powder is minimum, is 113 ± 1.14% (n=3), shows its particle diameter no significant difference before and after being lyophilized.
It is 215.6 ± 3.3nm that gained nano suspension of the present invention characterizes average grain diameter, polydispersity coefficient is 0.165 ±
0.02, Zeta potential is -36.9 ± 2.8mV (n=3).
In gained nanosuspension frozen powder of the present invention, andrographolide, SDS and TPGS are existed with unformed state.
Gained andrographolide nano suspension particle diameter of the present invention is 200nm, is in rod particle under electron scanning mirror more
Shape or bulk, size distribution is more uniform, no adhesion and clustering phenomena between particle.
Gained andrographolide nano suspension of the present invention is molten in different pH buffer in Dissolution experiments in vitro
Go out dissolution rate all to significantly improve.
Gained andrographolide nanosuspension frozen powder of the present invention tests short-term stability well, average grain diameter after a week
There is no significant difference.
Gained andrographolide nano suspension of the present invention is good in Caco-2 cell permeability, shows significantly higher
Penetrate through cell membrane, by P-gp inhibitor TPGS, the outer row function of P-gp can be reduced.
Gained andrographolide nano suspension of the present invention Cmax and AUC0 t after rat oral gavage administration is significantly increased.
The wherein nano suspension of andrographolide containing TPGS is higher than the andrographolide nano suspension bioavilability not containing TPGS.
Gained andrographolide nano suspension of the present invention has more preferable antiinflammatory action, real in rat toes swelling anti-inflammatory
In testing, compared with other drug forms of andrographolide, it mitigates mouse toes to gained andrographolide nano suspension of the present invention
Swelling and reduction serum NO, IL-1, TNF content, improve SOD active effect more preferably.
The pharmacokinetics of gained andrographolide nano suspension of the present invention and pharmacodynamics feature show its absorption in vivo
It is improved, antiinflammatory action strengthens, this low asking of bioavilability in human body and animal body by effectively solving andrographolide
Topic, is very beneficial for the natural products that this has multiple biological activities and goes further to clinic as medicine.
Specific embodiment
Form is described in further detail to the above of the present invention again by the following examples.
Embodiment 1:Box-Behnken center combination design tests preferred andrographolide nano suspension preparation technology
Major experimental material：
On the basis of single factor test, select to affect significant 4 factors to andrographolide nano suspension preparation technology, that is,
Stabilizer concentration (A), TPGS and SDS ratio (B), medium grinder rotating speed (C) and milling time (D) are independent variable, with particle diameter
Size and polydispersity coefficient carry out Box-Behnken center combination design, founding mathematical models optimization for dependent variable (response)
Preparation technology.
With the particle diameter of andrographolide nano suspension and polydispersity coefficient (PI) as evaluation index, stabilizer is dense for this experiment
Degree (A), TPGS and SDS ratio (B), medium grinder rotating speed (C) and milling time (D) investigate factor, using Box-Behnken
Effect surface method optimizes the condition of andrographolide nano suspension preparation method, and carries out forecast analysis.Test result indicate that it is steady
Determine agent concentration (A), medium grinder rotating speed (C) and milling time (D) notable on particle size impact, stabilizer concentration (A) is right
Coefficient of dispersion impact is notable, obtains andrographolide nano suspension by software analysis and with reference to the mathematical analysis of regression model
Optimum preparation technology：Stabilizer concentration is 0.62% (W/V), and medium grinder rotating speed is 1056rpm, and milling time is 68min.
Table 1-Box-Behnken center combination design form
Table 2-Box-Behnken center combination design experimental result
For checking the reliability of response phase method, andrographolide nano suspension is carried out using above-mentioned Optimal technique process
Preparation, repeats to test 3 times, the results are shown in Table.Test shows the preparation work obtaining using Box-Behnken design-response surface optimization
Skill conditional parameter accurately and reliably, favorable reproducibility.
Table 3- checking test result (n=3)
Experimental result more than synthesis, determines that the optimal preparation technology of andrographolide nano suspension is：Weigh 1g to wear
Heart lotus lactone, adds in 200mL water (including 0.600g TPGS, 0.620g SDS), refiner 16000rpm high speed shear
Then above-mentioned suspension is poured in medium grinder (0.3-0.4mm zirconia bead 850g), in rotating speed 1056rpm by 5min
Lower grinding 68min, prepared andrographolide nano suspension.
Embodiment 2：Weigh 1g andrographolide, add in 200mL water (including 0.600g TPGS, 0.620g SDS), even
Pulp grinder 16000rpm high speed shear 5min, then pours above-mentioned suspension into medium grinder (0.3-0.4mm zirconia bead
In 850g), grind 68min, prepared andrographolide nano suspension under rotating speed 1056rpm.Add 1% (W/V) mannitol,
It is respectively charged in cillin bottle, every bottle of 5mL.It is placed in -80 DEG C of refrigerator pre-freeze 12h, then be positioned in freeze drier, -30 DEG C of maintenances
10h, -10 DEG C of maintenance 5h, 0 DEG C of maintenance 2h, 25 DEG C of maintenance 2h, prepare andrographolide nanosuspension frozen powder.
Embodiment 3：Prepared by andrographolide nano suspension (without TPGS)：Weigh 1g andrographolide, add 200mL
Water (is added without TPGS, containing only 0.620g SDS), and remaining is pressed example 2 above process and prepares, and obtains andrographolide nanometer suspension
Agent freeze-dried powder (without TPGS) (standby).
Embodiment 4：The physical mixture preparation of andrographolide and stabilizer：Weigh 1g andrographolide, 0.600g
TPGS, 0.620g SDS is added in mortar and grinds uniformly, obtains andrographolide physical mixture (standby).
Embodiment 5：Particle diameter and Zeta potential
Measure PCS using laser particle analyzer and characterize the andrographolide nano suspension that embodiment 2 method prepares
Particle diameter and PI.The andrographolide nano suspension Sample Dilution that embodiment 2 method is prepared to suitable concentration,
It is measured with 90 ° of angle of scattering at 25 DEG C.Determine zeta potential value using identical Instrument measuring particle electrophoresis speed.All
Measurement all in triplicate, and records mean value.PCS measurement result shows that andrographolide nano suspension records its average grain diameter
For 215.6 ± 3.3nm, polydispersity coefficient is 0.165 ± 0.02, and Zeta potential is -36.9 ± 2.8mv (n=3).Illustrate to implement
The andrographolide nano suspension particle diameter that example 2 method prepares is less and particle diameter distribution is homogeneous.
Embodiment 6：Differential scanning calorimetric analysis
Physical mixed by SDS, TPGS, andrographolide bulk drug, andrographolide prepared by embodiment 4 and stabilizer
The andrographolide nanosuspension frozen powder that thing and embodiment 2 method prepare carries out differential scanning calorimetric analysis.Measure
Parameter：Heating rate is 10 DEG C/min, and temperature elevating range is 30-300 DEG C, and empty aluminium dish, as blank, measures
DSC figure understands, andrographolide bulk drug has melting peak at 240 DEG C, and SDS has two melting peaks and exists respectively
110 and 193 DEG C, there is melting peak at 40 and 175 DEG C in TPGS and mannitol respectively.Andrographolide and the physical mixed of stabilizer
All there is the melting peak that melting peak, respectively TPGS, SDS and andrographolide move to left at 40,106 and 180 DEG C in thing.In Herba Andrographitis
There is not the melting peak of andrographolide and stabilizer in ester nano suspension, analysis reason is for, after being prepared into nano suspension, wearing
Heart lotus lactone, SDS and TPGS are existed with unformed state.
Embodiment 7：X-ray diffraction is analyzed
Physical mixed by SDS, TPGS, andrographolide bulk drug, andrographolide prepared by embodiment 4 and stabilizer
The andrographolide nanosuspension frozen powder that thing and embodiment 2 method prepare is packed in planchet, respectively using Ni-
Wave filter, Cu Ka ray, the voltage of 40kV and the electric current of 25mA, swept with 2 °/min speed in the range of 3-40 ° of 2 θ
Retouch.
X- diffraction analysis figure understand, andrographolide 5.00 °, 9.94 °, 12.10 °, 14.90 °, 15.76 °, 17.54 °,
19.92 °, 22.72 °, 30.08 ° and 33.88 ° have diffraction maximum, illustrate that it is crystal structure.In the Herba Andrographitis of embodiment 4 preparation
In the physical mixture of ester and stabilizer, andrographolide crystal characteristic still suffers from, but peak intensity reduces, and analysis reason is probably
Stabilizer and drug interaction is added to affect the intensity at peak.In nano suspension, andrographolide crystal diffraction peak disappears,
Existed with unformed state, analysis reason is in media milling process, destroys the crystal structure of andrographolide.X- diffraction divides
Analysis result demonstrates DSC result further.
Embodiment 8：Scanning electron microscope analysis
Before detection, andrographolide nanometer suspension andrographolide bulk drug and embodiment 2 method being prepared
Agent freeze-dried powder sample spot invests on conductive tape and metal spraying 240s under vacuo.Select ESEM operation accelerating potential be
15kV, observes the shape of andrographolide bulk drug and andrographolide nanosuspension frozen powder under SEM
State.
Under SEM, andrographolide bulk drug particle diameter is 100 μm about.Andrographolide nanometer of the present invention
Supensoid agent particle diameter is 200nm, is in bar-shaped or block, size distribution is more uniform particle more, has no adhesion and poly- between particle
Collection phenomenon.The particle diameter difference of andrographolide bulk drug and nano suspension is larger, and the reduction of particle diameter is due to medium milling mistake
Cheng Zhong, impact force and shearing force destroy andrographolide bulk drug particle.
Embodiment 9：Dissolution in vitro is tested
Chromatographic condition：C18 chromatographic column (4.6mm × 250mm, 5 μm), mobile phase：Methanol-water (65:35)；
35 DEG C of column temperature；Flow velocity 1.0mL/min；Detection wavelength 225nm；Sample size：10μL.
The preparation of reference substance solution：Take andrographolide standard items appropriate, accurately weighed, it is placed in the volumetric flask of 10mL,
Plus methyl alcohol dissolving, and it is settled to scale, shake up, be configured to reference substance storing solution (123.4 μ g/mL).
Precision weigh andrographolide bulk drug, embodiment 4 preparation andrographolide and stabilizer physical mixture,
The andrographolide nanosuspension frozen powder that dripping pills of andrographolide and embodiment 2 method prepare (is equivalent to and wears in right amount
Heart lotus lactone 15mg), press《Chinese Pharmacopoeia》Version annex XC dissolution method the second method pertinent regulations are measured within 2015.Molten
Going out medium is pH 1.2, pH 4.5 and pH 6.8 buffer solution, and temperature is 37 ± 0.5 DEG C, and rotating speed is 75rpm.Respectively at 5,10,
Sampling 5mL (supplying the dissolution medium of equitemperature equivalent immediately) when 20,30,45,60min, 14000rpm centrifugation 10min. presses upper
State chromatographic condition to be measured, calculate the accumulation dissolution rate of different time each of which respectively.
Test result indicate that：The andrographolide of embodiment 4 preparation is dripped with the physical mixture of stabilizer, andrographolide
The andrographolide nano suspension of ball and embodiment 2 preparation is molten in different pH buffer compared with andrographolide bulk drug
Go out speed all to significantly improve.Drip with the physical mixture of stabilizer and andrographolide with the andrographolide of embodiment 4 preparation
Ball is compared, and dissolution rate in different pH buffer for the andrographolide nano suspension of the present invention also significantly improves.?
In 10min, in different pH buffer, andrographolide nano suspension dissolution rate is about 85%, andrographolide bulk drug
Dissolution rate is about 25%, and andrographolide physical mixture dissolution rate is about 65%, and the dissolution rate of dripping pills of andrographolide is about
65%.Andrographolide bulk drug in pH 1.2 dissolution medium, during dissolution time 60min, andrographolide nano suspension
Dissolution rate be respectively andrographolide bulk drug, andrographolide physical mixture and dripping pills of andrographolide 3.57,
1.43 and 1.37 times.In pH 4.5 dissolution medium, during dissolution time 60min, the dissolution rate of andrographolide nano suspension
It is respectively 3.79,1.28 and the 1.38 of andrographolide bulk drug, andrographolide physical mixture and dripping pills of andrographolide
Times.In pH 6.8 dissolution medium, during dissolution time 60min, the dissolution rate of andrographolide nano suspension is respectively punching
4.76,1.37 and 1.40 times of lotus lactone bulk drug, andrographolide physical mixture and dripping pills of andrographolide.In Vitro Dissolution
Degree test result indicate that, andrographolide nano suspension dissolution in different pH buffer of the present invention is preferable.
Embodiment 10：Short-term stability Journal of Sex Research
Procedure：By the ADG-NS (andrographolide nano suspension) for preparing embodiment 2 method tightly
Be sealed in glass container and be maintained at one) 40 DEG C, testing chamber for medicine stability (long case 222, the moral of 75% relative humidity
State) 1 week assessing its short-term stability.Select average grain diameter as the index of the stability of preparation, detect its second, the four He
The particle diameter of the 6th day.
Result shows：ADG-NS after freeze-drying average grain diameter after a week does not have significant difference, nano suspension
In nanoscale ADG keep stable.
Embodiment 11：The permeability test of Caco-2 cell
Caco-2 Premeabilisation of cells is tested and is provided directive function for testing internal potential source biomolecule availability.Transmembrane transport is tested
In, in Merlon six orifice plate crossed with tissue culture treated, every hole cell density is 1 × 10^5 for Caco-2 cell culture.
Cell, after inoculation after 21 days, merges in Quan Peizhong and is divided into enterocyte, forms monolayer.Use Millicell ERS
Voltohmyst (Millipore, Billerica, MA, USA) measures transepithelial electrical resistance (TEER) to reflect Caco-2 cell list
The integrality of molecular layer.More than 350 Ω cm2, TEER value shows that cell monolayer collects shaping.In an experiment, every hole Caco-2 cell
Monolayer in 37 DEG C of precultures 30min, then with the transhipment medium of pre-heated not drug containing (Hanks balanced salt solution,
HBSS) clean three times.When the blank transhipment medium of consistent volume is contained in every hole, will containing ADG (andrographolide) meal,
In the Herba Andrographitis that the andrographolide nano suspension (containing TPGS) that embodiment 2 method prepares is prepared with embodiment 3 method
Ester nano suspension (without TPGS) transhipment liquid is added in tip (AP).Press 0,30,60,90,120 minutes from substrate (BL)
Point removes sample (0.4 milliliter), and adds isopyknic fresh culture immediately.The measurement of experiment fore-and-aft survey resistance.Apparent
Infiltration coefficient (Papp, cel) is calculated using equation below.
Papp=(dQ/dt)/A × C0
DQ/dt is the compound line occurrence rate (μ G/S) in receiving terminal, C0It is for room initial concentration (ng/ml), A is individual layer
The surface area (cm2) of cell membrane.
The permeability test experimental data of table 4-Caco-2 cell
Compared with Herba Andrographitis bulk drug group,*p<0.05,**p<0.01；
Compared with andrographolide nano suspension group,#p<0.05,##p<0.01
Result shows：The presence of Verapamil can significantly improve the infiltration (P of ADG and ADG-NS without TPGS<；
0.01).Compared with ADG meal group, ADG-NS (with or without TPGS) all dramatically increases membrane passage (Papp) (P<；
0.01).The dissolution velocity of enhanced nano suspension and solubility can change the permeability to ADG for the enterocyte.Nanometer is mixed
One of distinguishing feature of suspension is exactly to have the potentiality improving ADG dissolution velocity, and this can change cell in vitro permeability of the membrane,
Thus affecting the body absorption of medicine.
Additionally, with the ADG-NS of no TPGS contrast, the ADG-NS containing TPGS shows and significant higher penetrates through cell
Film, significantly increases (more than 87.1%) in Papp, by TPGS, P-gp inhibitor, can reduce the outer row function of P-gp.
Embodiment 12：The rat vivo biodistribution availability research of andrographolide nano suspension
Animal used as test：SPF level SD rat 24, male, body weight 200-220g.Animal origin：Nanjing University of Traditional Chinese Medicine is moved
Thing center.Animal quality certification number：SCXK (peaceful) 2013-0006.
Experimental technique：Rat is administered the configuration of liquid：Weigh andrographolide bulk drug, andrographolide physical mixed
Thing, dripping pills of andrographolide, the andrographolide nanosuspension frozen powder of embodiment 2 preparation, the Herba Andrographitis of embodiment 3 preparation
Lactone nanosuspension frozen powder (without TPGS) is appropriate, plus 0.5%CMC-Na solution becomes liquor strength to be punching
The liquid of lotus lactone 4mg/mL.
Reference substance solution configures：The configuration of andrographolide reference substance solution：Take andrographolide reference substance appropriate, accurate
Weighed, it is placed in the volumetric flask of 10mL, plus methyl alcohol dissolving, and it is settled to scale, shake up, be configured to reference substance storing solution (123.4
μ g/mL), put 4 DEG C of refrigerator cold-storages, treat that the used time is diluted to desired concn.
The configuration of Bilobalide inner mark solution：Take Bilobalide standard items appropriate, accurately weighed, it is placed in the volumetric flask of 10mL
In, plus methyl alcohol dissolving, and it is settled to scale, and shake up, be configured to inner mark solution (108.5 μ g/mL), put 4 DEG C of refrigerator cold-storages, stand-by
When be diluted to desired concn.
Rat packet and administration：24 male rats are randomly divided into 5 groups, every group 6, respectively andrographolide bulk drug
Group, andrographolide physical mixture group, dripping pills of andrographolide group, andrographolide nano suspension group, each after fasting 12h
Group gives the liquid prepared under 2.1 respectively with 10mL/kg gavage, and every group of dosage is all equivalent to andrographolide 40mg/
kg.Each group rat is 5 after gastric infusion, 10,20,30,45,60,120,240,360,480,720min eye socket take blood 0.2mL,
It is placed in the 1.5mL EP pipe containing 10 μ L liquaemins, 3000rpm is centrifuged 10min, takes blood plasma, be placed in freezing in -20 DEG C of refrigerators
Save backup.
Plasma sample processing method：According to document and preliminary experiment, the method for this experimental selection ethyl acetate extraction is locating
Regulating blood condition is starched, and concrete grammar is as follows：After blood plasma unfreezing, the accurate 100 μ L that draw are placed in EP pipe, are subsequently adding 10 μ L Bilobalides molten
Liquid (108.5ng/mL), the methyl alcohol (for calibration curve and quality control) of 10 μ L.Vortex 2min mixes, and adds 1mL acetic acid second
Ester, vortex 10min, 14000rpm are centrifuged 5min, take supernatant 800 μ L, put in reduced pressure concentration instrument and volatilize ethyl acetate.Use again
100 μ L mobile phases are redissolved, and vortex 5min, 14000rpm are centrifuged 5min, take supernatant 80 μ L, mass spectral analysis.
The foundation of the UPLC-MS/MS analysis method of andrographolide in plasma sample, carries out specificity, linear, minimum fixed
The Method validation such as amount limit, the degree of accuracy, precision, extraction recovery, matrix effect and stability
Chromatographic condition：Using HSS T3 chromatographic column (100mm × 2.1mm, i.d., 1.7 μm), column temperature is 40 DEG C, mobile phase
For water-methanol (30:70), flow velocity is 0.35mL/min, and sample size is 5 μ L, and run time is 2.5min.Mass Spectrometry Conditions
Electro-spray ionization (ESI), multiple-reaction monitoring ion scan pattern (MRM), using negative ion mode, ion gun temperature
150 DEG C of degree, 400 DEG C of desolventizing temperature, desolventizing flow velocity 800L h 1.Methodological study：To andrographolide in plasma sample
UPLC-MS/MS analysis method foundation, carry out specificity, linear, minimum quantitative limit, the degree of accuracy, precision, extract reclaim
The Method validation Drug-time curve such as rate, matrix effect and stability：After sample treatment, measure in each group Herba Andrographitis through LC-MS/MS
Ester blood concentration, draws Drug-time curve.
The calculating of pharmacokinetic parameters
By the blood concentration-time recording data with DAS 3.0 software, calculate each group pharmacokinetics ginseng through non-compartment model
Number, see table
Table 5- gavage gives andrographolide bulk drug, andrographolide nano suspension, andrographolide physical mixed
The related pharmacokinetic parameters (n=6) of thing and dripping pills of andrographolide
Compared with Herba Andrographitis bulk drug group,*p<0.05,**p<0.01；
Compared with andrographolide nano suspension group,#p<0.05,##p<0.01
Result shows：
(1) after andrographolide bulk drug gastric infusion, in rat body, Cmax is 77.74 ± 22.17 μ g/L, and AUC0-t is
5665.41 ± 1654.43 μ g/L min, andrographolide nano suspension (containing TPGS), andrographolide nano suspension are (no
Containing TPGS), andrographolide physical mixture group, dripping pills of andrographolide group Cmax be respectively 235.91 ± 53.73,200.41
± 18.91,80.25 ± 17.35,168.17 ± 47.28 μ g/L, AUC0-t are respectively 21960.14 ± 5392.04,14227.02
±306.24、9117.87±2126.20、10273.45±1699.48μg/L min.
(2) compared with andrographolide bulk drug andrographolide nano suspension (with or without TPGS), in Herba Andrographitis
Ester dripping pill group has higher blood plasma exposure, in CmaxAnd AUC0–tSupensoid agent is significantly increased, CmaxHigh by 116.3% respectively, 157.9%,
203.5%；AUC0–tHigh by 90.98% respectively, 164.48%, 308.3%.This shows, wears compared with andrographolide bulk drug
Heart lotus lactone nano suspension (with or without TPGS), dripping pills of andrographolide group are absorbed more in vivo.In addition with punching
Lotus lactone nano suspension (without TPGS) compares, and andrographolide nano suspension (containing TPGS) has significantly higher blood plasma
Exposure, its AUC0–tValue exceeds 54.3%.The AUC of andrographolide nano suspension (with or without TPGS)0–tWear and wear respectively
2.14,1.38 times of heart lotus lactone dripping pill.
Embodiment 13：Andrographolide nano suspension is studied to rat toes swelling antiinflammatory action
Animal used as test：SPF level SD rat, male, body weight 160 180g.Animal origin：In Nanjing University of Traditional Chinese Medicine animal
The heart.Animal quality certification number：SCXK (peaceful) 2014-0001.
Experimental technique：
Prepared by solution：Take a piece of aspirin effervescent tablet to be placed in the conical flask containing 26.67mL water, make 15mg/mL Ah
Department's woods aqueous solution.
Precision weighs andrographolide bulk drug 200mg and is placed in conical flask, accurate addition 20mL water, makes 10mg/mL
Andrographolide suspension.
Precision weighs dripping pills of andrographolide 796.52mg and is placed in conical flask, accurate addition 20mL water, makes 10mg/mL
Dripping pills of andrographolide suspension.
The andrographolide nanosuspension frozen powder 406.08mg that precision weighs embodiment 2 preparation is placed in conical flask,
Accurate addition 20mL water, makes 4mg/mL andrographolide nano suspension.
(1) andrographolide nano suspension Carrageenan causes the impact of rat toes swelling
Rat 60, is randomly divided into 6 groups, respectively control group, model group, positive drug group, andrographolide bulk drug group,
Dripping pills of andrographolide group, andrographolide nano suspension group, every group 10.Successive administration four days, control group, model group are given
Give running water, positive drug group gives the aspirin effervescent tablet aqueous solution (150mg/kg), andrographolide bulk drug group gives to wear
Heart lotus lactone suspension 40mg/kg, dripping pills of andrographolide group gives dripping pills of andrographolide suspension 40mg/kg, Herba Andrographitis
Lactone nano suspension group gives the andrographolide nano suspension of concentration 40mg/kg.Fasting 12h before last dose, can't help
Water., to the 4th day, with sufficient volume measuring apparatus measurement Rat Right metapedes volume after last dose, after 30 minutes, every mouse is right for gastric infusion
Afterwards toes hypodermic injection 1% carrageenan 0.1mL cause scorching, 0.5 after causing inflammation, 1,2,3,4, use sufficient volume determination again after 5h
Instrument measures Rat Right metapedes volume, and calculates swelling.
(2) impact to rat blood serum NO, SOD, IL-1 and TNF for the andrographolide nano suspension
Rat 60, is randomly divided into 6 groups, respectively control group, model group, positive drug group, andrographolide bulk drug group,
Dripping pills of andrographolide group, andrographolide nano suspension group, every group 10.Successive administration four days, control group, model group are given
Give running water, positive drug group gives the aspirin effervescent tablet aqueous solution (150mg/kg), andrographolide bulk drug group gives to wear
Heart lotus lactone suspension 40mg/kg, dripping pills of andrographolide group gives dripping pills of andrographolide suspension 40mg/kg, Herba Andrographitis
Lactone nano suspension group gives the andrographolide nano suspension of concentration 40mg/kg.Fasting 12h before last dose, can't help
Water., to the 4th day, last dose is after 30 minutes for gastric infusion, except blank group, toes hypodermic injection 1% carrageenan behind every mouse right side
0.1mL causes inflammation, takes blood 2mL after 0.5h, 3000rpm centrifugation 10min after blood clotting completely, separates serum, freezing is standby.
NO, SOD, IL-1 and TNF kit specification of Bioengineering Research Institute's offer is provided with reference to Nanjing, measures rat
Serum NO level, SOD, IL-1 and TNF content.
Statistical method：Experimental data is processed with SPSS 16 statistical software, carries out statistical analysis.Data with mean ±
Standard deviation represents.
Result shows：
Compare before and after (1) six experimental group Rat Right metapedes modeling, all occur different degrees of red, swollen.After Rat Right
Paw swelling.Compared with andrographolide bulk drug, in rat foot injection carrageenan 5h, andrographolide nano suspension
There is the rat toes swelling caused by preferable suppression carrageenan, optimal inhibition action time is 0.5h, and continues 8h, with mould
Type group compares foot swelling rate and reduces 52.45%.Compared with andrographolide bulk drug, dripping pills of andrographolide also has certain suppression
Rat toes swelling caused by carrageenan, but effect is then poorer compared with andrographolide nano suspension.
(2), after rat foot injection carrageenan 5h, model group rats serum NO, IL-1, TNF content increase, and SOD lives
Property reduce (p<0.01,p<0.05).Compare with model group, andrographolide nano suspension and dripping pills of andrographolide group can show
Write and reduce NO, IL-1, TNF content in rat blood serum, improve SOD activity (p<0.01,p<0.05).Show andrographolide nanometer
Supensoid agent and dripping pills of andrographolide have antiinflammatory action.Compare with andrographolide bulk drug, andrographolide nanometer suspension
Agent and dripping pills of andrographolide group also show and can substantially reduce NO, IL-1, TNF content in rat blood serum, improve SOD activity (p
<0.01,p<0.05).Compare with dripping pills of andrographolide group, andrographolide nano suspension reduces NO, IL- in rat blood serum
1st, TNF content, improves SOD active function more preferably, shows that andrographolide nano suspension antiinflammatory action is better than andrographolide
Dripping pill.
Summarize：
(1) andrographolide is P-gp substrate, and stabilizer T PGS used by nano suspension has P-gp inhibitory action, can
Outer row's effect to andrographolide for the effectively suppression P-gp, increases the absorption of medicine, thus the anti-inflammatory strengthening andrographolide is made
With.
(2) nano suspension is prepared by medium milling technology, nano suspension characterize average grain diameter be 215.6 ±
3.3nm, polydispersity coefficient is 0.165 ± 0.02, and Zeta potential is -36.9 ± 2.8mV.Short-term stability experiment shows that freezing is dry
ADG-NS after dry average grain diameter after a week does not have significant difference.The permeability test of Caco-2 cell shows:Herba Andrographitis
Lactone nano suspension has the potentiality improving ADG dissolution velocity, changes cell in vitro permeability of the membrane, thus affecting medicine
Body absorption.Dissolution in vitro test shows that ADG-NS dissolution rate is fast and dissolution rate is high, and pharmacodynamic experiment shows that ADG-NS resists
Scorching best results, foot swelling inhibition to greatest extent.Comprehensive above experiment is comprehensive to can be seen that nano suspension can strengthen ADG in vivo
Bioavilability and biological effect.
Claims (10)
1. a kind of andrographolide nano suspension, is made up of andrographolide, stabilizer it is characterised in that stabilizer is to have
The vitamin E polyethylene glycol succinic acid ester TPGS of P-gp inhibitory action and ionic stabilizer lauryl sodium sulfate SDS.
2. andrographolide nano suspension as claimed in claim 1 is it is characterised in that formulation is freeze-dried powder.
3. andrographolide nano suspension as claimed in claim 2 is it is characterised in that preparation method includes the step of following orders
Suddenly：(1) weigh andrographolide, add in the aqueous solution including TPGS and SDS；(2) aforesaid liquid is placed in refiner at a high speed
Shear to obtain suspension；(3) and then by above-mentioned suspension pour in medium grinder, grind, prepared andrographolide nanometer suspension
Agent；(4) gained nano suspension adds mannitol, is respectively charged in cillin bottle；(5) it is placed in -80 DEG C of refrigerator pre-freezes, then be positioned over
In freeze drier, prepare andrographolide nanosuspension frozen powder.
4. andrographolide nano suspension as claimed in claim 3 is it is characterised in that molten described in preparation method step (1)
It is 0.5g/100ml that the consumption of agent should make the concentration of andrographolide.
5. andrographolide nano suspension as claimed in claim 3 is it is characterised in that the described TPGS of preparation method step (1)
It is calculated as 0.62%, respectively 0.30%, 0.32% with stabilizer T PGS, SDS concentration in the aqueous solution of SDS with mass volume ratio.
6. andrographolide nano suspension as claimed in claim 3 is it is characterised in that the described medium of preparation method step (3)
In grinder, using 0.3-0.4mm zirconia bead 850g, rotating speed is 1056rpm, and milling time is 68min.
7. andrographolide nano suspension as claimed in claim 3 is it is characterised in that the described freezing of preparation method step (4)
Protection agent concentration is calculated as 1% mannitol with mass volume ratio.
8. andrographolide nano suspension as claimed in claim 3 is it is characterised in that the described pre-freeze of preparation method step (5)
Temperature is -80 DEG C, pre-freeze time 12h.
9. andrographolide nano suspension as claimed in claim 3 is it is characterised in that the described freezing of preparation method step (5)
Baking temperature is to maintain 10h when -30 DEG C, maintains 5h, 0 DEG C of maintenance 2h, 25 DEG C of maintenance 2h when -10 DEG C.
10. andrographolide nano suspension as claimed in claim 2 is it is characterised in that preparation method includes following orders
Step：(1) weigh 1g andrographolide, add and include 0.600gTPGS, in the 200mL aqueous solution of 0.640gSDS；(2) will be upper
State liquid and be placed in refiner 16000rpm high speed shear 5min；(3) and then by above-mentioned suspension pour in medium grinder, use
0.3-0.4mm zirconia bead 850g, grinds 68min, prepared andrographolide nano suspension under rotating speed 1056rpm；(4)
Gained nano suspension adds the mannitol being calculated as 1% with mass volume ratio, is respectively charged in cillin bottle, every bottle of 5mL；(5) put
In -80 DEG C of refrigerator pre-freeze 12h, then it is positioned in freeze drier, prepare andrographolide nanosuspension frozen powder.
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| CN108175752A (en) * | 2018-01-24 | 2018-06-19 | 广州中医药大学(广州中医药研究院) | Breviscapinun nano particle and preparation method thereof |
| CN108333024A (en) * | 2018-03-21 | 2018-07-27 | 杭州娃哈哈科技有限公司 | A kind of method for estimating stability of curcumin nanosuspension |
| CN109568264A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN109568266A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN109568265A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN111388466A (en) * | 2018-12-28 | 2020-07-10 | 澳门大学 | Pharmaceutical composition containing andrographolide and preparation thereof |
| CN115778907A (en) * | 2022-11-18 | 2023-03-14 | 江西中医药大学 | Andrographolide oral nanocrystal and preparation method and application thereof |
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| CN109568264A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN109568266A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN109568265A (en) * | 2017-09-28 | 2019-04-05 | 神威药业集团有限公司 | A kind of andrographolide nano suspension |
| CN109568264B (en) * | 2017-09-28 | 2021-09-28 | 神威药业集团有限公司 | Andrographolide nanosuspension |
| CN109568266B (en) * | 2017-09-28 | 2021-11-05 | 神威药业集团有限公司 | Andrographolide nanosuspension |
| CN108175752A (en) * | 2018-01-24 | 2018-06-19 | 广州中医药大学(广州中医药研究院) | Breviscapinun nano particle and preparation method thereof |
| CN108175752B (en) * | 2018-01-24 | 2021-01-15 | 广州中医药大学(广州中医药研究院) | Breviscapine nano-particles and preparation method thereof |
| CN108333024A (en) * | 2018-03-21 | 2018-07-27 | 杭州娃哈哈科技有限公司 | A kind of method for estimating stability of curcumin nanosuspension |
| CN111388466A (en) * | 2018-12-28 | 2020-07-10 | 澳门大学 | Pharmaceutical composition containing andrographolide and preparation thereof |
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