CN106109416B - Anemarrhena saponin AIII nano liposomes and its preparation method and application - Google Patents
Anemarrhena saponin AIII nano liposomes and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to the formulation arts of medicine and Chinese pharmacology, disclose a kind of nano liposomes containing effective component of chinese medicine Anemarrhena saponin AIII, preparation method are as follows: (1) Anemarrhena saponin AIII solution is mixed with membrane material solution, remove solvent and moisture, obtain lipid membrane；(2) phosphate buffer aquation is added, obtains the thick suspension of Anemarrhena saponin AIII liposome；It is ultrasonically treated at (3) 40~60 DEG C or high-pressure homogeneous processing, obtains the nano liposomes of Anemarrhena saponin AIII.The Anemarrhena saponin AIII nano liposomes can be used for preparing the drug for inhibiting tumor cell proliferation, have a good application prospect.
Description
Technical field
The present invention relates to the formulation arts of medicine and Chinese pharmacology, more particularly, to effective component of chinese medicine Anemarrhena saponin AIII
Nano liposomes and preparation method thereof and the application in terms of preparing anti-tumor drug.
Background technique
Cancer (Cancer), also known as malignant tumour (Malignant neoplasm), for by control growth and proliferation of cell machine
Make it is not normal caused by disease.Tumour seriously threatens the major disease of human life as one kind, since its cure rate is low, recurrence
Rate is high, it has also become the principal element of human diseases death.It is reported that causing 8,200,000 people dead altogether in global range in 2012
It dies, wherein based on lung cancer, liver cancer, gastric cancer, colorectal cancer.The primary treatments of cancer have surgical operation, radiation at present
Treatment and chemotherapy etc., to treat disease or extending life and improve patients ' life quality.However these methods usually give patient with
Carry out huge pain, and generates biggish drug resistance and higher recurrence rate in therapeutic process.Chinese medicine and natural drug are to treatment
Cancer has certain curative effect, is gradually accepted extensively by domestic and international medical field.So, R&D of modern TCM skill can be utilized
Art, it is found that a kind of pair of tumor cell killing potential is strong, and the small drug of toxic side effect and treatment method have the clinical treatment of tumour
Far reaching significance.
Dry rhizome of the rhizoma anemarrhenae for liliaceae plant (Anemarrhena asphodeloides Bunge), bitter,
It is cold in nature, return lung, stomach, kidney channel.With clearing heat-fire, nourishing Yin and moistening dryness, relieving restlessness and other effects of quenching the thirst.The chemical component of rhizoma anemarrhenae mainly has steroid
Body saponin(e, double benzene pyrrones, flavonoids, lignanoids, polysaccharide, organic acid and microelement etc., wherein saponins are them
One of main ingredient accounts for about the 6% of Asphodeloides Bge Rhizome.Timosaponin is the primary pharmacological activity ingredient of rhizoma anemarrhenae, Anemarrhena saponin AIII
Content is very low in rhizoma ane marrhenae, and the higher timosaponin BII of content can be converted into 1-timosaponin A-1 by enzymatic hydrolysis in rhizoma anemarrhenae
Ⅲ.Modern pharmacology research shows increasing of the Anemarrhena saponin AIII to the tumour cell of lung cancer, liver cancer, intestinal cancer, colon cancer, cancer of pancreas
It grows with certain inhibiting effect.
Although according to cell assay in vitro as a result, Anemarrhena saponin AIII is able to suppress tumor cell proliferation；However, in reality
In the application of border, because the water solubility of Anemarrhena saponin AIII is poor, vivo biodistribution availability is lower, seriously affects the medicine of Anemarrhena saponin AIII
Effect plays.The present invention passes through the preparation of Anemarrhena saponin AIII nano liposomes, not only improves the water solubility of Anemarrhena saponin AIII, and
And improve its inhibiting effect to tumor cell proliferation.Anemarrhena saponin AIII liposome encapsulation is higher, and stability is preferable, whole
Body zoopery also indicates that Anemarrhena saponin AIII liposome has good inhibiting effect to tumour growth, is better than 1-timosaponin A-1
III, it can be used for the prevention and treatment of malignant tumour.
Summary of the invention
The present invention is intended to provide a kind of Anemarrhena saponin AIII nano liposomes.
Another object of the present invention is to provide the preparation method of this Anemarrhena saponin AIII nano liposomes.
The present invention also provides application of this Anemarrhena saponin AIII nano liposomes in terms of preparing anti-tumor drug.
Anemarrhena saponin AIII of the present invention, the enzyme of main saponin(e timosaponin BII in traditional Chinese medicine rhizoma anemarrhenae
Solve product, molecular formula C39H64O13, molecular weight 740.917660, structural formula is shown in formula I.
The technical scheme is that a kind of Anemarrhena saponin AIII nano liposomes, by groups such as Anemarrhena saponin AIII and phosphatide
At, in preparation prescription, Anemarrhena saponin AIII, phosphatide and distearoylphosphatidylethanolamine-polyethylene glycol (or distearyl
Phosphatidyl acetamide-methoxy poly (ethylene glycol)) molar ratio be 1:8~10:0~2.The 1-timosaponin A-1 prepared by this method
The drugloading rate of III liposome is higher than 5%, and encapsulation rate is higher than 75%.The stability of 1-timosaponin A-1 III nano liposomes under the prescription
Height is not easy to reveal.
The method for preparing Anemarrhena saponin AIII nano liposomes, comprising the following steps:
The membrane materials such as timosaponin, phosphatide are dissolved in organic solvent, the organic solvent is removed and forms adipose membrane, add
Buffer is hydrated, and the thick suspension of Anemarrhena saponin AIII liposome is obtained, and obtained thick suspension is carried out ultrasound or through high pressure
After the processing of homogeneous instrument, the Anemarrhena saponin AIII liposome is obtained.
Specifically, preparation method step are as follows:
(1) Anemarrhena saponin AIII solution is mixed with membrane material solution, removal organic solvent obtains lipid membrane；Membrane material includes
(A) phosphatide, (B) distearoylphosphatidylethanolamine-polyethylene glycol or the poly- second two of distearoylphosphatidyl acetamide-methoxyl group
Alcohol；
Anemarrhena saponin AIII, phosphatide and distearoylphosphatidylethanolamine-polyethylene glycol or distearoylphosphatidyl acetyl
Amine-methoxy poly (ethylene glycol) molar ratio is 1:8~10:0~2.
Phosphatide is selected from egg yolk lecithin (EPC), hydrogenated soy phosphatidyl choline (HSPC), Distearoyl Phosphatidylcholine
(DSPC), at least one of dimyristoyl phosphatidyl choline (DMPC), dipalmitoylphosphatidylcholine (DPPC).
The molecular weight of the distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is 2000~5000；
Distearoylphosphatidyl acetamide-methoxy poly (ethylene glycol) molecular weight is 1000~5000.The concentration of the membrane material solution is 20
~150mg/mL.
Preferably, Anemarrhena saponin AIII solution concentration is 0.5~4mg/mL；Anemarrhena saponin AIII solution methanol, ethyl alcohol or
Chloroform is prepared, more preferably methanol or ethyl alcohol.
Membrane material solution methanol, ethyl alcohol or chloroform are prepared；Its concentration is 15~50mg/mL.
Removing the method that organic solvent forms adipose membrane is one of drying method and decompression rotary evaporation.Using drying method
When, using nitrogen or inert gas, dried up under 15~40 DEG C (phase transition temperature of room temperature or phosphatide)；Using decompression rotary evaporation
When method, temperature is 40~60 DEG C, and vacuum degree is -0.09~-0.1MPa.
(2) phosphate buffer is added, aquation 30~120 minutes at 40~60 DEG C, it is thick to obtain Anemarrhena saponin AIII liposome
Suspension；The phosphate buffer solution concentration is 0.05~0.1mol/L, and pH value is 7.2~7.6；Preferably, hydration temperature
It is 45~50 DEG C.
Preferably, the amount ratio of membrane material and phosphate buffer is 10~70mg:1mL.
(3) the thick suspension of Anemarrhena saponin AIII liposome described in is ultrasonically treated at 40~60 DEG C or high-pressure homogeneous place
Reason, obtains the nano liposomes of Anemarrhena saponin AIII；
Preferably, supersonic frequency is 40~100Hz, 200~300W of power；Preferably, ultrasonic treatment condition is 45~50
Ultrasound 2~5 times, every time 3~5 minutes at DEG C.High-pressure homogeneous condition are as follows: recycled 2~6 times under the conditions of 10000~30000PSI；It is excellent
It is selected as, is recycled 2~4 times under the conditions of 10000~20000PSI.
It is in uniform milk-white coloured suspension by Anemarrhena saponin AIII nano liposomes appearance prepared by the above method, is averaged
Partial size (D90) is 25~80nm, -23~-35mV of Zeta potential range；Encapsulation rate is 75%~95%；PH value is 7.3~7.4,
Carrying drug ratio is 5wt%~10wt%.
Prepared Anemarrhena saponin AIII liposome solubility in physiological saline is preferably improved in aforementioned manners, molten
Xie Du reaches as high as 4mg/ml, improves 230 times or so relative to the solubility of Anemarrhena saponin AIII in water；And it is acquired
Anemarrhena saponin AIII liposome stability it is high, be not easy to reveal.
For Anemarrhena saponin AIII monomer, Anemarrhena saponin AIII liposome prepared by the present invention is thin for different tumours
The inhibiting effect of the proliferation of born of the same parents greatly enhances, IC50Reduce about one times or so.
Anemarrhena saponin AIII nano liposomes prepared by the present invention can be applied to the medicine that preparation inhibits tumor cell proliferation
Object；Tumour cell includes Humanmachine tumour A375,66C14, B16-F10 cell, breast cancer BT474 cell, people's non-small cell lung
Cancer cell A549, liver cancer HepG2, intestinal cancer HCT-15, gastric cancer HGC-27, breast cancer MCF-7, cancer of pancreas Panc-1 or glioma
U87；Being particularly useful for preparation inhibits Humanmachine tumour A375,66c14, B16-F10 cell and breast cancer BT474 cell to increase
The drug grown.Tumor killing effect on glioma U87 tumor bearing nude mice is up to 54.1%, and 1-timosaponin A-1 III liposome tumor suppression is made
With much stronger than 1-timosaponin A-1 III.
Anemarrhena saponin AIII nano liposomes Rats pharmacokinetics studies have shown that prepared by the present invention is quiet through tail
After arteries and veins administration, the AUC (0- ∞) of Anemarrhena saponin AIII improves 2.13 times with respect to proto-drug, and the internal residence time increases 3.7
Times, when illustrating that the intracorporal trap of drug can be improved in the preparation of Anemarrhena saponin AIII liposome, while drug effect can be extended
Between.
The present invention prepares Anemarrhena saponin AIII nano liposomes by simple method, and raw material is easy to get, and encapsulation rate is high；Gained
The Anemarrhena saponin AIII nano liposomes stability arrived is good, and water solubility is obviously improved, and is remarkably improved the internal of Anemarrhena saponin AIII
Trap extends drug treating time, and improves the inhibiting effect to tumor cell proliferation；Especially solves rhizoma anemarrhenae soap
Glycosides A III cannot lead to the problem of tumor killing effect in vivo.Anemarrhena saponin AIII is high in liposome internal stability, is not easy to reveal.
Therefore, this Anemarrhena saponin AIII nano liposomes can be used for preparing the drug for inhibiting tumor cell proliferation, have good application
Prospect.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of 1 Anemarrhena saponin AIII nano liposomes of embodiment.
Fig. 2~9 are the grain size distribution of Anemarrhena saponin AIII nano liposomes prepared by Examples 1 to 8.
Figure 10~17 are the Zeta potential figure of Anemarrhena saponin AIII nano liposomes prepared by Examples 1 to 8.
Figure 18 is Anemarrhena saponin AIII sample chromatogram figure.
Figure 19 is Anemarrhena saponin AIII liposomal samples chromatogram prepared by embodiment 1.
Specific embodiment
The present invention is further elaborated combined with specific embodiments below, but the present invention is not limited to following embodiments.Institute
State method is conventional method unless otherwise instructed.The raw material can be gotten from open business unless otherwise instructed.
Embodiment 1, Anemarrhena saponin AIII nano liposomes prepare (prescription 1)
0.8mg Anemarrhena saponin AIII is weighed to be dissolved in 400 μ L methanol；It weighs 8.35mg egg yolk lecithin (EPC) and is dissolved in 200 μ
In L chloroformic solution, ultrasonic dissolution.Merge above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until
One layer of uniform lipid membrane is formed on round bottom test tube wall.EPC and Anemarrhena saponin AIII molar ratio are 10:1.- 0.09MPa, 45~
50 DEG C are dried under reduced pressure and remove residual solvent and moisture, are added 200 μ L 0.01mol/L phosphate buffers (pH=7.4), 45~
Aquation 1 hour under 50 DEG C of water bath conditions, obtains the thick suspension of Anemarrhena saponin AIII liposome.By the thick suspension of Anemarrhena saponin AIII liposome
Ultrasound 3 times under conditions of supersonic frequency is 40-100Hz, power 200-300W, each ultrasonic time are 3-5min to get arriving
Anemarrhena saponin AIII nano liposomes.
Embodiment 2, Anemarrhena saponin AIII nano liposomes prepare (prescription 2)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 8.53mg Distearoyl Phosphatidylcholine
(DSPC) it is dissolved in 200 μ L chloroformic solutions, ultrasonic dissolution.Merge above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen removing
Methanol and chloroform, until forming one layer of uniform lipid membrane on round bottom test tube wall.DSPC is with Anemarrhena saponin AIII molar ratio
10:1.- 0.09MPa, 45~50 DEG C be dried under reduced pressure and remove residual solvent and moisture, it is slow that 200 μ L 0.01mol/L phosphate are added
Fliud flushing (pH=7.4) obtains the thick suspension of Anemarrhena saponin AIII liposome aquation 1 hour under 45-50 DEG C of water bath condition.By rhizoma anemarrhenae soap
The thick suspension of III liposome of glycosides A is 40-100Hz, power 200-300W in supersonic frequency, and ultrasound 3 times, each ultrasonic time is 3-
5min to get arrive Anemarrhena saponin AIII nano liposomes.
Embodiment 3, Anemarrhena saponin AIII nano liposomes prepare (prescription 3)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 7.51mg egg yolk lecithin (EPC), 3.02mg
Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) is dissolved in 500 μ L chloroformic solutions, and ultrasound is molten
Solution, merges above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until forming one on round bottom test tube wall
The uniform lipid membrane of layer.The molar ratio of EPC, DSPE-PEG2000 and Anemarrhena saponin AIII is 9:1:1.- 0.09MPa, 45~50
It DEG C is dried under reduced pressure and to remove residual solvent and moisture, be added 200 μ L 0.01mol/L phosphate buffers (pH=7.4), 45-50 DEG C
Aquation 1 hour under water bath condition obtains the thick suspension of Anemarrhena saponin AIII liposome.By the thick suspension of Anemarrhena saponin AIII liposome in super
Acoustic frequency is 40-100Hz, power 200-300W, ultrasound 3 times, and each ultrasonic time is 3-5min to get Anemarrhena saponin AIII is arrived
Nano liposomes.
Embodiment 4, Anemarrhena saponin AIII nano liposomes prepare (prescription 4)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 7.68mg Distearoyl Phosphatidylcholine
(DSPC), it is molten to be dissolved in 200 μ L chloroforms for 3.02mg distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000)
In liquid, ultrasonic dissolution merges above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until round bottom tries
One layer of uniform lipid membrane is formed on tube wall.The molar ratio of DSPC, DSPE-PEG2000 and Anemarrhena saponin AIII is 9:1:1.-
0.09MPa, it is dried under reduced pressure at 45~50 DEG C and removes residual solvent and moisture, 200 μ L 0.01mol/L phosphate buffers are added
(pH=7.4), aquation 1 hour under 45-50 DEG C of water bath condition, the thick suspension of Anemarrhena saponin AIII liposome is obtained.By 1-timosaponin A-1
The thick suspension of III liposome is 40-100Hz, power 200-300W in supersonic frequency, and ultrasound 3 times, each ultrasonic time is 3-5min,
Obtain Anemarrhena saponin AIII nano liposomes.
Embodiment 5, Anemarrhena saponin AIII nano liposomes prepare (prescription 5)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 7.51mg egg yolk lecithin (EPC), 5.40mg
Distearoylphosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG5000) is dissolved in 500 μ L chloroformic solutions, and ultrasound is molten
Solution, merges above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until forming one on round bottom test tube wall
The uniform lipid membrane of layer.The molar ratio of EPC, DSPE-PEG5000 and Anemarrhena saponin AIII is 9:1:1.- 0.09MPa, 45~50
It DEG C is dried under reduced pressure and to remove residual solvent and moisture, be added 200 μ L 0.01mol/L phosphate buffers (pH=7.4), 45-50 DEG C
Aquation 1 hour under water bath condition obtains the thick suspension of Anemarrhena saponin AIII liposome.By the thick suspension of Anemarrhena saponin AIII liposome in super
Acoustic frequency is 40-100Hz, power 200-300W, ultrasound 3 times, and each ultrasonic time is 3-5min to get Anemarrhena saponin AIII is arrived
Nano liposomes.
Embodiment 6, Anemarrhena saponin AIII nano liposomes prepare (prescription 6)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 7.67mg Distearoyl Phosphatidylcholine
(DSPC), it is molten to be dissolved in 200 μ L chloroforms for 5.40mg distearoylphosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG5000)
In liquid, ultrasonic dissolution merges above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until round bottom tries
One layer of uniform lipid membrane is formed on tube wall.The molar ratio of DSPC, DSPE-PEG5000 and Anemarrhena saponin AIII is 9:1:1.-
0.09MPa, 45~50 DEG C be dried under reduced pressure and remove residual solvent and moisture, 200 μ L 0.01mol/L phosphate buffer (pH are added
=7.4), aquation 1 hour under 45-50 DEG C of water bath condition, the thick suspension of Anemarrhena saponin AIII liposome is obtained.By Anemarrhena saponin AIII rouge
The thick suspension of plastid in supersonic frequency be 40-100Hz, power 200-300W, ultrasound 3 times, each ultrasonic time be 3-5min to get
To Anemarrhena saponin AIII nano liposomes.
Embodiment 7, Anemarrhena saponin AIII nano liposomes prepare (prescription 7)
It weighs 400mg Anemarrhena saponin AIII to be dissolved in 200ml methanol, weighs 3755mg egg yolk lecithin (EPC), 1510mg
Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) is dissolved in 250ml chloroformic solution, and ultrasound is molten
Solution, merges above-mentioned solution in a round bottom flask, is dried with nitrogen and removes methanol and chloroform, until forming one layer on round-bottomed flask wall
Even lipid membrane.The molar ratio of EPC, DSPE-PEG2000 and Anemarrhena saponin AIII is 9:1:1.- 0.09MPa, 45~50 DEG C subtract
Pressure dries and removes residual solvent and moisture, is added 100ml 0.01mol/L phosphate buffer (pH=7.4), 45~50 DEG C of water
Aquation 1 hour under the conditions of bath obtains the thick suspension of Anemarrhena saponin AIII liposome.By the thick suspension of Anemarrhena saponin AIII in high-pressure homogeneous instrument
Middle dispersion recycles 3 times under the conditions of 20000PSI to get to Anemarrhena saponin AIII nano liposomes.
Embodiment 8, Anemarrhena saponin AIII nano liposomes prepare (prescription 8)
It weighs 400mg Anemarrhena saponin AIII to be dissolved in 200ml methanol, weighs 3835mg Distearoyl Phosphatidylcholine
(DSPC), it is molten to be dissolved in 100ml chloroform for 1510mg distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000)
In liquid, ultrasonic dissolution merges above-mentioned solution in a round bottom flask, is dried with nitrogen and removes methanol and chloroform, until round-bottomed flask wall
One layer of uniform lipid membrane of upper formation.The molar ratio of DSPC, DSPE-PEG2000 and Anemarrhena saponin AIII is 9:1:1.-
0.09MPa, 45~50 DEG C be dried under reduced pressure and remove residual solvent and moisture, 100ml 0.01mol/L phosphate buffer (pH is added
=7.4), aquation 1 hour under 45-50 DEG C of water bath condition, the thick suspension of Anemarrhena saponin AIII liposome is obtained.Anemarrhena saponin AIII is thick
Suspension disperses in high-pressure homogeneous instrument, and 3 times are recycled under the conditions of 20000PSI to get Anemarrhena saponin AIII nano liposomes are arrived.
Reference examples 1, Anemarrhena saponin AIII nano liposomes prepare (prescription 9)
0.8mg Anemarrhena saponin AIII is weighed to be dissolved in 400 μ L methanol；It weighs 2.51mg egg yolk lecithin (EPC) and is dissolved in 60 μ L
In chloroformic solution, ultrasonic dissolution.Merge above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen and removes methanol and chloroform, until
One layer of uniform lipid membrane is formed on round bottom test tube wall.EPC and Anemarrhena saponin AIII molar ratio are 3:1.- 0.09MPa, 45~
50 DEG C are dried under reduced pressure and remove residual solvent and moisture, are added 200 μ L 0.01mol/L phosphate buffers (pH=7.4), 45~
Aquation 1 hour under 50 DEG C of water bath conditions, obtains the thick suspension of Anemarrhena saponin AIII liposome.By the thick suspension of Anemarrhena saponin AIII liposome
Ultrasound 3 times under conditions of supersonic frequency is 40-100Hz, power 200-300W, each ultrasonic time is 3-5min, acquired
Mixed liquor precipitating easy to form, uniform liposome suspension cannot be formed.
Reference examples 2, Anemarrhena saponin AIII nano liposomes prepare (prescription 10)
It weighs 0.8mg Anemarrhena saponin AIII to be dissolved in 400 μ L methanol, weighs 2.56mg Distearoyl Phosphatidylcholine
(DSPC) it is dissolved in 60 μ L chloroformic solutions, ultrasonic dissolution.Merge above-mentioned solution in round bottom tool plug test tube, is dried with nitrogen removing first
Pure and mild chloroform, until forming one layer of uniform lipid membrane on round bottom test tube wall.DSPC and Anemarrhena saponin AIII molar ratio are 3:
1.- 0.09MPa, 45~50 DEG C be dried under reduced pressure and remove residual solvent and moisture, 200 μ L 0.01mol/L phosphate buffers are added
(pH=7.4), aquation 1 hour under 45-50 DEG C of water bath condition, the thick suspension of Anemarrhena saponin AIII liposome is obtained.By 1-timosaponin A-1
The thick suspension of III liposome is 40-100Hz, power 200-300W in supersonic frequency, and ultrasound 3 times, each ultrasonic time is 3-5min,
Obtained mixed liquor precipitating easy to form, cannot form uniform liposome suspension.
The quality evaluation of embodiment 9, Anemarrhena saponin AIII nano liposomes
9.1 morphologic observation
Anemarrhena saponin AIII nano liposomes prepared by Example 1~8, with 0.01mol/L phosphate buffer (pH
=7.4) it is diluted to low concentration, drop with filter paper on copper mesh, blotting extra liquid, and acetic acid uranium dyeing 3min, dyeing is three times
The form of product is observed under transmission electron microscope afterwards.Anemarrhena saponin AIII nano liposomes prepared by Examples 1 to 8 it is rounded or
Oval microsphere, it is dispersion between particle, independent, and partial size < 100nm.The Anemarrhena saponin AIII nano liposomes of embodiment 1 are for example saturating
It penetrates shown in electron microscope Fig. 1.Anemarrhena saponin AIII nano liposomes prepared by reference examples 1,2 precipitate, not formed uniform rouge
Plastid suspension, partial size are greater than 1000nm.
9.2 solubility test
Using the dissolution in the solubility and liposome (prescription 1) in different saturated solutions of HPLC measurement Anemarrhena saponin AIII
Degree, seen from table 1, the solubility of Anemarrhena saponin AIII in water are 0.017mg/ml, and solubility is 4.00mg/ in liposome
Ml, solubility improve 230 times.
The quality evaluation result of 1 Anemarrhena saponin AIII liposome of table
9.3 partial sizes and Zeta potential measure
Appropriate Anemarrhena saponin AIII nano liposomes are taken, rouge is diluted to 0.01mol/L phosphate buffer (pH=7.4)
Plastid lipid concentration is 1mmol/L, measures its partial size and Zeta potential using Malvern zeta potential instrument.Using the above method point
Not Ce Ding liposome prepared by embodiment 1-8 partial size and current potential.Measure particle size range are as follows: 45.1-69.5nm (D90),
Zeta potential range are as follows: -24.8--31.8mV, grain size distribution is as shown in Fig. 2~9, Zeta potential figure such as Figure 10~17 institute
Show.Experimental result is shown in Table 2.
9.4pH value: Anemarrhena saponin AIII nano liposomes prepared by Example 1~8 detect pH value, and measurement result is
7.35±0.04。
The measurement of 9.5 encapsulation rates and carrying drug ratio: using the content of Anemarrhena saponin AIII in HPLC-ELSD measurement liposome.
9.5.1 chromatographic condition
1100 type high performance liquid chromatograph of Agilent (matches Alltech3300 evaporative light scattering detector；Chromatographic column is
AlltimaTM C18Chromatographic column (4.6mm × 250mm, 5 μm)；Mobile phase: methanol-water (85:15)；Flow velocity: 1mL/min；Sample introduction
Measure 20 μ L；Light scattering detector temperature is 60 DEG C, atomization gas flow rate 1.5L/min.Anemarrhena saponin AIII and liposomal samples
Chromatogram is shown in Figure 18,19.
9.5.2 the drafting of standard curve
Precision weighs 22.6mg Anemarrhena saponin AIII standard items, and 10mL is dissolved and be settled to methanol, obtains 2.26mg/ml,
It is diluted to the reference substance solution that concentration is 56.5,113,226,452,904,1130 μ g/mL respectively, is injected separately into liquid chromatogram
Instrument, sample volume is 20 μ L, and using the common logarithm of Anemarrhena saponin AIII peak area average value as ordinate, reference substance concentration is commonly used
Logarithm is that abscissa draws standard curve, calculates regression equation and obtains: y=1.3764x-1.6816, r=0.9995.Show rhizoma anemarrhenae
The common logarithm of saponin A III common logarithm and peak area of its concentration within the scope of 56.5~1130 μ g/mL is in good linear
Relationship.
9.5.3 in Anemarrhena saponin AIII nano liposomes Anemarrhena saponin AIII assay
20 μ L of Anemarrhena saponin AIII nano liposomes is accurately pipetted, 180 μ L methanol are added, is vortexed and mixes 5mins,
14000rpm/min is centrifuged 5mins, and supernatant sample introduction is taken to measure.
9.5.4 the measurement of encapsulation rate and carrying drug ratio
20 μ L Anemarrhena saponin AIII nano liposomes are taken, its mass concentration C1 is measured using HPLC.Separately take 50 μ L nano-lipids
Body is placed in 1000mL 0.01mol/L phosphate buffer (pH=in the bag filter that molecule interception is 8000-14000D
7.4) it dialyses for 24 hours in.Nano liposomes after taking out dialysis measure volume V2, and the accurate rhizoma anemarrhenae soap pipetted after 20 μ L dialysis
III nano liposomes of glycosides A, using the concentration C 2 of Anemarrhena saponin AIII in HPLC measurement bag inner lipid body.Calculate its encapsulation rate EE
(%)=C2*V2/C1*50 × 100%.
The measurement of carrying drug ratio: taking 200 μ L nano liposomes, its weighed quality is W1 after freeze-drying, and is measured according to the above method
The wherein weight W2 of Anemarrhena saponin AIII, carrying drug ratio DL (%)=W2/W1 × 100%.It calculates separately prepared by embodiment 1-8
Liposome encapsulation rate and carrying drug ratio, the results are shown in Table 2.
The quality evaluation result of 2 Anemarrhena saponin AIII nano liposomes of table
Conclusion: the Anemarrhena saponin AIII liposome encapsulation range of example 1-8 preparation are as follows: 78.3%~93.1%.It is preferred that locating
The Anemarrhena saponin AIII nano liposomes encapsulation rate just prepared is above rhizoma anemarrhenae prepared by reference examples 1 and 2 75% or more
III nano liposomes of saponin A.The nano liposomes carrying drug ratio of preparation is above 5%.
The experiment of 9.6 1-timosaponin A-1 III nano liposomes primary stabilities
The Anemarrhena saponin AIII nano liposomes (prescription 1) of preparation are placed 3 months under 4 DEG C of refrigerated conditions, with liposome
Mode of appearance, partial size and encapsulation rate variation be index investigate its stability.
3 Anemarrhena saponin AIII nano liposomes preliminarily stabilised of table is investigated
The result shows that after being placed 3 months under 4 DEG C of refrigerated conditions, Anemarrhena saponin AIII liposome mode of appearance, partial size and
Encapsulation rate variation is unobvious, shows that Anemarrhena saponin AIII liposome stability is good.
After the Anemarrhena saponin AIII nano liposomes of prescription 2-8 are placed 3 months under 4 DEG C of refrigerated conditions, mode of appearance, grain
Diameter and encapsulation rate are equally without significant change.
9.7 in vitro antitumor activity assays:
Humanmachine tumour A375,66c14, B16-F10 cell, breast cancer BT474 cell, people's non-small cell lung is respectively adopted
Cancer cell A549, liver cancer HepG2, intestinal cancer HCT-15, gastric cancer HGC-27, breast cancer MCF-7, cancer of pancreas Panc-1 are purchased from
Shanghai life science institute, the academy of sciences, state cell resource center, glioma U87 are limited purchased from Shanghai Austria terrestrial science and technology
Company.The cell strain cultivating system is DMEM culture solution (GIBCO) 90%, high-quality fetal calf serum 10%.5% carbon dioxide, temperature
37 DEG C of degree.0.25% trypsase, the passage of 0.02%EDTA digestive juice.
9.7.1 inoculation
The tumour cell in exponential phase of growth is taken respectively, is diluted to 1 with the DMEM culture solution containing 10% fetal calf serum
×106、4×105、1×105、1×105Cell/ml.50 μ L cell suspensions are added in the every hole of 96 orifice plates, are placed in 37 DEG C, 5%CO2
It is cultivated for 24 hours in incubator.
9.7.2 culture
96 orifice plates are placed in 37 DEG C, 5%CO2Continue culture in incubator for 24 hours.
9.7.3 dosing
Anemarrhena saponin AIII: being configured to stock solution with DMSO for Anemarrhena saponin AIII, is matched with DMEM culture solution (containing 10%FBS)
It makes each concentration working solution (3.8,7.7,15.4,20.5,30.754,41.0,54.7,109.4,164.0,218.7 μM), in every hole
Each liquid of 50 μ L or more is added, if 3 multiple holes.Each group final concentration is respectively 1.9,3.8,7.7,10.3,15.4,20.5,27.3,
54.7,82,109.4μM.Blank group and control group are set simultaneously, wherein blank group is to replace cell molten with isometric culture solution
Liquid, control group are to replace Anemarrhena saponin AIII solution to be added thereto with isometric PBS buffer solution.
Anemarrhena saponin AIII liposome: Anemarrhena saponin AIII liposome is each dense with DMEM culture solution (containing 10%FBS) preparation
It spends working solution (5.9,11.9,23.7,31.6,47.4,63.3,84.3,168.7,253.0,337.4 μM), 50 μ is added in every hole
Each liquid of L or more, if 3 multiple holes.Each group final concentration is respectively 3.0,5.9,11.9,15.8,23.7,31.6,42.2,84.3,
126.5,168.7μM.Blank group and control group are set simultaneously, wherein blank group is to replace cell molten with isometric culture solution
Liquid, control group are to replace Anemarrhena saponin AIII solution to be added thereto with isometric PBS buffer solution.
9.7.4 being incubated for
It is placed in 37 DEG C, 5%CO2Continue culture in incubator for 24 hours.
Plus MTT 9.7.5
MTT is made into 5mg/ml solution with phosphate buffer, the 5h before culture terminates, and 20 μ L are added in every hole, be placed in 37 DEG C,
5%C02Continue to be incubated in incubator.
9.7.6 measurement
Cell supernatant is discarded after culture, the 100 dissolving crystallized products of μ L DMSO are added in every hole.It is read with microplate reader
490nm wavelength OD value.Anemarrhena saponin AIII is recorded to the inhibiting rate of various cell Proliferations.
Inhibiting rate (%)=(A490,Control-A490,Sample)/(A490,Control-A490,BlankThe inhibiting rate of) × 100%
(%)=(A490,Control-A490,Sample)/(A490,Control-A490,Blank) × 100%
According to the Anemarrhena saponin AIII of various concentration and Anemarrhena saponin AIII liposome, (prepared by the method for Examples 1 to 4, use
III nano liposomes 1~4 of timosaponin TA indicate) to the inhibiting rate of various tumor cell proliferations, it is calculated using SPSS 18.0
IC50 is shown in Table 4,5.
The quality evaluation result of 4 Anemarrhena saponin AIII liposome of table
T is examined: * is compared with Anemarrhena saponin AIII group, P < 0.05；* is compared with Anemarrhena saponin AIII group, P < 0.01.
The quality evaluation result of 5 Anemarrhena saponin AIII liposome of table
The result shows that: timosaponin TAIII liposome can be improved drug to the inhibiting effect of tumor cell proliferation.
9.7 Anemarrhena saponin AIII and its liposome are to the subcutaneous tumor bearing nude mice tumor-inhibiting action of glioma U87
18 nude mices, weight 18-20g or so are raised in SPF environment, and nude mice foodstuff, drinking water, padding periodically pass through
Autoclaving sterilization, mouse cage, padding replace once a week.It is randomly divided into 4 groups after nude mice modeling: physiological saline group, 1-timosaponin A-1
III intraperitoneal injection treatment group (60mg/Kg), Anemarrhena saponin AIII liposome intraperitoneal injection treatment group (are calculated as with Anemarrhena saponin AIII
60mg/Kg), Normal group, every group 5.
When microscopically observation glioma U87 cell is in logarithmic growth phase, state is good, pollution-free, pancreatin digestion, system
For at cell suspension (concentration 2 × 107A/ml).Normal group does not do kind of a tumor operation.Other three groups to before on the right side of every nude mice
Oxter is inoculated with the U87 cell suspension of 0.1ml, and cell inoculation amount is 2 × 106A/ml.It is denoted as the 0th day on the day of tumor inoculation.One
It can touch tumour fritter after week, be divided into three groups: treatment group (60mg/Kg) is injected intraperitoneally in physiological saline group, Anemarrhena saponin AIII,
Treatment group (60mg/Kg) is injected intraperitoneally in Anemarrhena saponin AIII liposome, is tested with liposome prepared by example 8.Physiological saline
Group is each 9% physiological saline that 0.1ml is injected intraperitoneally；Anemarrhena saponin AIII intraperitoneal injection treatment group (60mg/Kg): rhizoma anemarrhenae is taken
III suspension of saponin A, intraperitoneal injection, dosage 60mg/Kg are administered once every other day；The intraperitoneal injection of Anemarrhena saponin AIII liposome
Treatment group (60mg/Kg): taking Anemarrhena saponin AIII liposome solutions, and intraperitoneal injection, dosage 60mg/Kg is administered once every other day；
Observation 18 days continuously records nude mice survival condition, changes of weight, tumor size, spirit during observation administration and after administration
The indexs such as state.After 18 days, all nude mices are put to death, tumor is taken to weigh, calculates knurl weight index, tumour inhibiting rate.Knurl weight index=tumour weight
Amount/nude mice weight × l00%, tumour inhibiting rate=l- experiment each group average knurl weight index/physiological saline group average knurl weight index ×
L00%, each group experimental result indicate that statistical comparison is examined using t with mean scholar SD, and P value is less than 0.05 for significant statistics
Learn difference.Experimental result such as table 6.
Anti-tumor activity experimental result in 6 Anemarrhena saponin AIII body of table
The result shows that although Anemarrhena saponin AIII cell experiment inhibits tumor cell proliferation to act on very strong, whole animal
The experimental results showed that Anemarrhena saponin AIII drug is not obvious the tumor killing effect of the subcutaneous tumor bearing nude mice of glioma, and rhizoma anemarrhenae soap
III liposome tumor killing effect of glycosides A is up to 54.1%, shows that drug 1-timosaponin A-1 itself can be enhanced in Anemarrhena saponin AIII liposome
III drug action.
Embodiment 10, Anemarrhena saponin AIII nano liposomes and Anemarrhena saponin AIII Rats pharmacokinetics Research on differences
SD rat 10,180-200g is divided into 2 groups immediately, divides Anemarrhena saponin AIII and its nano liposomes administration group, greatly
The disposable tail vein injection Anemarrhena saponin AIII of mouse or its liposome (prescription 4) 10mg/kg are (in terms of Anemarrhena saponin AIII actual content
Calculate), by 1mL/l00g tail vein injection.Be administered before eye socket take blood as blank sample, after administration in preset the time (2,5,
10,30min, 1,2,4,6,8,12,24,48,72h) eye socket blood sampling, take blood 0.2mL or so into test tube of hepari sample cell every time, from
The heart (5000r/min) 5min prepares blood plasma, handles sample by plasma sample preprocess method, measures blood plasma using HPLC-MS/MS
The concentration of middle Anemarrhena saponin AIII, DAS program carry out the dynamic analysis of Anemarrhena saponin AIII, and seek calculation pharmacokinetic parameter,
Compare Anemarrhena saponin AIII and its liposome in Pharmacokinetics in Rat difference, the results are shown in Table 7.
7 Anemarrhena saponin AIII nano liposomes of table and Anemarrhena saponin AIII Rats pharmacokinetics parametric results
The result shows that Anemarrhena saponin AIII liposome rat tail vein is administered, internal drug AUC (0- ∞) is 1-timosaponin A-1
2.13 times of III, the internal residence time increases 3.7 times, illustrates that drug body can be improved in the preparation of Anemarrhena saponin AIII liposome
Interior trap, while drug treating time can be extended, also further prove Anemarrhena saponin AIII lipidosome antineoplastic strong drug action
In Anemarrhena saponin AIII proto-drug.
Claims (9)
1. a kind of preparation method of Anemarrhena saponin AIII nano liposomes, which is characterized in that step includes:
(1) Anemarrhena saponin AIII solution is mixed with membrane material solution, removes solvent and moisture, obtains lipid membrane；Membrane material includes:
(A) phosphatide, (B) distearoylphosphatidylethanolamine-polyethylene glycol or the poly- second two of distearoylphosphatidyl acetamide-methoxyl group
Alcohol；Wherein, phosphatide is selected from egg yolk lecithin, hydrogenated soy phosphatidyl choline, Distearoyl Phosphatidylcholine, two myristoyl phosphatidyls
At least one of choline, dipalmitoylphosphatidylcholine, Anemarrhena saponin AIII, phosphatide and distearyl acyl group phosphatidyl ethanol
Amine-polyethylene glycol or distearoylphosphatidyl acetamide-methoxy poly (ethylene glycol) molar ratio are 1:8~10:0~2；
(2) phosphate buffer is added, aquation obtains the thick suspension of Anemarrhena saponin AIII liposome；
(3) the thick suspension of Anemarrhena saponin AIII liposome is ultrasonically treated or high-pressure homogeneous processing at 40~60 DEG C, obtains rhizoma anemarrhenae
The nano liposomes of saponin A III.
2. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that step (1) described rhizoma anemarrhenae
III solution concentration of saponin A is 0.5~4mg/mL, and Anemarrhena saponin AIII solution is methanol, ethyl alcohol, the chloroform or third of Anemarrhena saponin AIII
Ketone solution；Membrane material solution is methanol, ethyl alcohol, chloroform or the acetone soln of membrane material.
3. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that in step (1), two is hard
Fatty acyl group phosphatidyl-ethanolamine-polyethylene glycol molecular weight is 2000~5000；Distearoylphosphatidyl acetamide-methoxyl group is poly-
Molecular weight glycol is 1000~5000；The concentration of the membrane material solution is 20~150mg/mL.
4. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that in step (1), use
At least one of drying method or decompression rotary evaporation removal solvent and moisture；
When using drying method, using nitrogen or inert gas, dried up at 15~40 DEG C；
When using decompression rotary evaporation, temperature is 40~60 DEG C, and vacuum degree is -0.09~-0.1MPa.
5. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that in step (2), phosphoric acid
Salt buffer concentration is 0.05~0.1mol/L, and pH value is 7.2~7.6；Hydrating condition are as follows: 30~120 are handled at 45~50 DEG C
Minute.
6. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that in step (2), membrane material
Amount ratio with phosphate buffer is 10~70mg:1mL.
7. the preparation method of Anemarrhena saponin AIII nano liposomes described in claim 1, which is characterized in that described in step (3)
Ultrasonic treatment condition be 45~50 DEG C at ultrasound 2~5 times, 3~5 minutes every time, 40~100Hz of supersonic frequency, power 200~
300W；The high-pressure homogeneous condition are as follows: recycled 2~6 times under the conditions of 10000~30000PSI.
8. a kind of Anemarrhena saponin AIII nano liposomes, which is characterized in that pass through the described in any item method systems of claim 1~7
Standby, partial size D90 is 25~80nm, -23~-35mV of Zeta potential range；PH value be 7.3~7.4, carrying drug ratio be 5wt%~
10wt%, encapsulation rate 75%~95%.
9. application of the Anemarrhena saponin AIII nano liposomes described in claim 1 in terms of preparation inhibits tumor cell proliferation drug,
It is characterized in that, the tumour cell is Humanmachine tumour A375,66C14, B16-F10 cell, breast cancer BT474 cell, people
Non-small cell lung cancer cell A549, liver cancer HepG2, intestinal cancer HCT-15, gastric cancer HGC-27, breast cancer MCF-7, cancer of pancreas Panc-1
Or glioma U87.
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|---|---|---|---|---|
| CN1628790A (en) * | 2004-08-31 | 2005-06-22 | 中国人民解放军第二军医大学 | Injecta of anemarrhena extractive |
| WO2013149580A1 (en) * | 2012-04-03 | 2013-10-10 | Chiming Che | Timosaponin compounds |
| CN103540640A (en) * | 2012-07-09 | 2014-01-29 | 上海中医药大学 | Preparation method of timosaponin A III |
| CN103599122A (en) * | 2013-11-29 | 2014-02-26 | 沈阳药科大学 | Application of timosaponin AIII in anemarrhena to preparation of antitumor drugs |
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| CN1628790A (en) * | 2004-08-31 | 2005-06-22 | 中国人民解放军第二军医大学 | Injecta of anemarrhena extractive |
| WO2013149580A1 (en) * | 2012-04-03 | 2013-10-10 | Chiming Che | Timosaponin compounds |
| CN103540640A (en) * | 2012-07-09 | 2014-01-29 | 上海中医药大学 | Preparation method of timosaponin A III |
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