CN107050053A - A kind of Contained Serum prepared by Ligusticum wallichii alcohol extract and its preparation method and application - Google Patents
A kind of Contained Serum prepared by Ligusticum wallichii alcohol extract and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of Contained Serum prepared by Ligusticum wallichii alcohol extract and its preparation method and application, and Contained Serum prepared by the Ligusticum wallichii alcohol extract is to carry out Ligusticum wallichii alcohol extract after gavage to rat, and blood sampling is obtained.Contained Serum prepared by the present invention can effectively suppress the propagation of the osteosarcoma cells of MG 63, promote its apoptosis, suppress its transfer, and its effect is substantially better than the medicine of Rhizoma Chuanxiong extract and existing treatment osteosarcoma；With the good result for suppressing osteosarcoma, medicine prepared by Contained Serum prepared by the Ligusticum wallichii alcohol extract also has significant effect when treating osteosarcoma；The preparation method of the present invention is safely controllable simultaneously, and is easy to operate, take care of, store and transport.
Description
Technical field
The invention belongs to field of biological medicine, and in particular to a kind of Contained Serum prepared by Ligusticum wallichii alcohol extract and its preparation
Method and the application in treatment osteosarcoma.
Background technology
Osteosarcoma is most common bone primary malignant neoplasm in B＆J system tumor.Osteosarcoma more than 85% is former
Hair property osteosarcoma, is often sent out in the Children and teenager phase, the incidence of disease about 6-10/100 ten thousand, men and women's incidence rate is about 1.5:1, its
In nearly 80-90% osteosarcoma disease send out in limb long tubular bone metaphysis.Osteosarcoma comes from a germinal layer tissue, its pathogenesis
It is due to the change of undifferentiated tumour cell developmental biology form, directly forms bone sample or immature osteoid tissue,
So far its genesis mechanism is not fully apparent from yet, and the research on osteosarcoma does not have very big progress yet.Therefore, searching can
Treating effective and Small side effects Chinese and western drugses of osteosarcoma turns into the pursuit direction of scholars' unremitting effort.With osteosarcoma
It is multiple, inhibitory action that many scholars grow in Chinese medical extract to osteosarcoma cell line in vitro culture and to cell week
Many explorations are made in terms of phase, Apoptosis, the influence of transfer ability, have been that the discovery and development of new plant type medicine are carried
Clue is supplied, also for the invention provides new thinking.
Chinese medicine is the rarity of Chinese nation's great culture legacy, and they play heavy to closing in the struggle of the mankind and disease
The role wanted, and it is seized of consequence in the medical development history in the world.With the continuous development of scientific technology, people couple
The understanding and requirement of Chinese medicine and Chinese medicine compound prescription are improved constantly, and the modernization development of Chinese medicine and Chinese medicine compound prescription is extremely urgent.In
Doctor thinks that the stagnant full course of disease through tumour of addiction is the main etiology and pathogenesis that malignant tumour occurs and developed.Cured according to middle
The principle of disease diagnosis and treatment is treated, using promoting blood circulationization addiction medicine treatment malignant tumour using relatively broad.More common work in clinic
Blood addiction medicine has Ligusticum wallichii, the red sage root, rheum officinale, safflower, Rhizoma Sparganii, river spine, returns tail etc., and the drug effect of wherein Ligusticum wallichii is splendid.Ligusticum wallichii is
The dry rhizome of Umbelliferae Jehol Ligusticum Rhizome platymiscium Ligusticum wallichii (Ligusticum Chuanxiong Hort), its main chemical composition is
Ligustrazine, forulic acid, ligustilide, ligustilide, chuanxingol, Chrysophanol, sedanoic acid, linoleic acid, loline etc..Its property is pungent
Fragrant row dissipates mildly bitter flavor, the gentle promoting blood circulation of the property of medicine, upper wardrobe top, under walk the sea of blood.Ligusticum wallichii is commonly used for blood-activating and qi-promoting, wind-expelling pain-stopping,
And treatment is had a headache, chest and hypochondrium, Amenorrhea is suffered from abdominal pain, rheumatalgia, the disease such as traumatic injury.In recent years, Ligusticum wallichii clinically extensive use
In illnesss such as treatment coronary heart disease, antimigraine, ischemia apoplexies, especially uniqueness is showed in the treatment of ischemia apoplexy
Advantage.There are some researches prove Ligusticum wallichii has good treatment to diseases such as lung cancer, prostate cancer, brain tumor, ulcerative colitis
Effect.And found through retrieval, ethanol and ether are to extract the conventional solvent of Ligusticum wallichii active ingredient, and ethanol to active ingredient because carrying
Take more complete and more commonly used, be extraction effect preferably solvent.Therefore the side for extracting Ligusticum wallichii active ingredient that the present invention takes
Method is ethanol extraction method.
The correlative study of Serum Pharmacology of Chinese Herbal Drugs has new progress on Chinese medicine or the studying of compound prescription of Chinese herbs in recent years, and it leads
If by simulating some processes of the digestion in vivo of Chinese medicine or Chinese medicine compound prescription, absorption, conversion, metabolism, then passing through collection
Take the blood of the animal of (or injection) Chinese medicine and separate serum, carry out a kind of new method of in vitro culture experimental study.In
The method and abbreviation that medicine is taken or injected are as follows：Be injected intravenously (iv), intraperitoneal injection (ip), oral (po), be subcutaneously injected (ih),
Gavage (ig) intramuscular injection (im), intravenous infusion (iv) etc..The proposition of this experimental method, can both exclude to a certain extent Chinese medicine or in
Medicine compound directly carries out the factor interference for being difficult to determine of experiment in vitro generation, and the confidence level of experimental result can also be made to carry significantly
It is high, it is often more important that medicine serum makes curative effect more notable.The research such as Zhang Minghui is found, is located after Ligusticum wallichii, bush gavage rat
The increasing of the high metastatic giant cell carcinoma of lung cell of PG people can be suppressed by managing when obtained Contained Serum acts on 24h under normal oxygen environment
Grow, and it is more beneficial in low-oxygen environment and promote PG cells propagation.The research such as Lu Chong is found, big through Sculellaria barbata alcohol extract gavage
The Contained Serum obtained after mouse has inhibitory action to HT-29 human colon cancer cells, and mechanism of action may be not only with suppressing cell life
Length is relevant, also relevant with retarding cell growth cycle and inducing cell apoptosis.The model research such as admire is found, after aspongopus gavage rat
Handle obtained Contained Serum and be incubated SW480 human colon cancer cells, can promote Apoptosis of Colon Cancer Cells, and influence apoptosis related because
Sub- P53, FADD expression, so as to reach antitumor action.The research discovery such as Feng Jundong, TMP (ligustrazine) Acidum Hydrochloricum Concentratums ip
The Contained Serum that injection rat post processing is obtained has certain inhibitory action to human liver cancer cell Hep G2 propagation.It is common at present
Osteosarcoma cell line have Saos-2, MG-63, M663, U2-OS, 143B, HOS, SOSP-9607, F5M2, F4, MNNG/HOS,
SW1353, UMR106 etc..But temporarily do not find that the Contained Serum prepared using Ligusticum wallichii is done correlation to MG-63 OS-732 cells and ground
The report studied carefully.
The content of the invention
Goal of the invention：The problem of existing for prior art, the present invention provides a kind of pastille prepared by Ligusticum wallichii alcohol extract
Serum；The Contained Serum can effectively suppress the propagation of osteosarcoma cell, promote its apoptosis, suppress its transfer.
The present invention also provides a kind of preparation method and applications of the Contained Serum prepared by Ligusticum wallichii alcohol extract.
Technical scheme：To achieve these goals, a kind of pastille blood prepared by Ligusticum wallichii alcohol extract as described in the present invention
Clearly, the Contained Serum is to carry out Ligusticum wallichii alcohol extract after gavage to rat, and blood sampling is obtained.
Wherein, the Ligusticum wallichii alcohol extract is that Ligusticum wallichii is ground into the powdered rear alcohol reflux that adds to extract, after freeze-drying
Pulverulent solids obtained by crushing.
The institute rat be cleaning grade SD male rats, rat be purchased from Yangzhou University's comparative medicine center, body weight be 250~
400g。
The preparation method of the Contained Serum of the present invention prepared by Ligusticum wallichii alcohol extract, comprises the following steps：
(1) weight ratio gavage Ligusticum wallichii alcohol extract (10-30) g/kg is pressed to rat；
(2) 1-3h is anaesthetized to rat after last gavage, is taken a blood sample under aseptic condition through abdominal aorta；
(3) blood is stood into 4-8h, centrifuges, take filtration sterilization after supernatant, inactivation, obtain Contained Serum, dispensed, freeze standby
With.
Preferably, step (1) described gavage can't help water for fasting before gavage, and it is daily 2-3 times, continue 3-5d.
Preferably, step (3) centrifugation is 3000-5000rpm, 4-6 DEG C of centrifugation 10-15min.
Preferably, step (3) inactivation is 56-60 DEG C of inactivation 25-30min.
Preferably, step (3) described filtration sterilization is degerming, preferably 0.22 μm of filter membrane by membrane filtration.
The Contained Serum of the present invention prepared by Ligusticum wallichii alcohol extract is applied in the medicine for preparing treatment osteosarcoma.
Further, the Contained Serum prepared by Ligusticum wallichii alcohol extract is preparing treatment MG-63 OS-732 cells
Medicine in apply.
Beneficial effect：Compared with prior art, the invention has the advantages that：
1st, the Contained Serum that is prepared by Ligusticum wallichii alcohol extract of the present invention according to Ligusticum wallichii it is promoting blood circulation and removing blood stasis, open Yu Hangqi, dispelling wind and relieving
Effect, can effectively suppress the propagation of osteosarcoma cell, promote its apoptosis, suppress its transfer.
2nd, the present invention is post-processed using MG-63 OS-732 cells as model using Ligusticum wallichii alcohol extract gavage rat
The Contained Serum culture MG-63 OS-732 cells arrived, for suppressing the propagation of osteosarcoma cell, promoting its apoptosis, suppression
Its effect shifted is substantially better than the medicine of Rhizoma Chuanxiong extract and existing treatment osteosarcoma；Wherein filled to rat by weight ratio
Contained Serum prepared by stomach Ligusticum wallichii alcohol extract (10-30) g/kg, takes the pastille that concentration is 15% (with DMEM culture volumes ratio)
Serum, which suppresses MG-63 OS-732 cells survival rate, can reach less than 26.69%, promote MG-63 OS-732 cells
Survival rate apoptosis rate reaches more than 40.92%；And Ligusticum wallichii alcohol extract and the Drug inhibition cell survival rate of existing treatment osteosarcoma
Best effect is only 72.34% and 42.15%, and the medicine of Ligusticum wallichii alcohol extract and existing treatment osteosarcoma promotes apoptosis rate best
Effect be 18.19% and 19.47%；The Contained Serum can effectively suppress moving for MG-63 OS-732 cells simultaneously
Shifting ability, so as to verify that it has the good result for suppressing osteosarcoma；It is prepared by Contained Serum prepared by Ligusticum wallichii alcohol extract of the present invention
Medicine treat osteosarcoma when also have significant effect.
3rd, preparation method of the invention is safely controllable, and is easy to operate, take care of, store and transport.
Brief description of the drawings
Fig. 1 is the Contained Serum and right for preparing embodiment 4 by weight ratio gavage Ligusticum wallichii alcohol extract 30g/kg to rat
MG-63 OS-732 cells are incubated according to group, Transwell cell migrations detect the schematic diagram of cell migration ability；
Fig. 2 is the Contained Serum and control group for preparing embodiment 5 by the clear 20g/kg of weight ratio gavage dysentery to rat
MG-63 OS-732 cells are incubated, Transwell cell migrations detect the schematic diagram of cell migration ability；
Fig. 3 is the Contained Serum and control group for preparing embodiment 6 by the clear 10g/kg of weight ratio gavage dysentery to rat
MG-63 OS-732 cells are incubated, Transwell cell migrations detect the schematic diagram of cell migration ability.
Embodiment
The invention will be further described with accompanying drawing with reference to embodiments.
Embodiment 1
The Contained Serum prepared by Ligusticum wallichii alcohol extract
(1) appropriate Ligusticum chuanxiong Hort is weighed first and is ground into powdered, the body that then 10 times of addition medicinal material weight is measured
The ethanol of fraction 80%, heating and refluxing extraction 2 times 30 minutes every time, merges after extract solution, by 200 mesh sieve net filtrations and
Distillation and concentration obtains lotion, then freeze-dried rear crush produces Ligusticum wallichii alcohol extract；Ligusticum wallichii is by commercially available；
(2) fasting before weight ratio gavage Ligusticum wallichii alcohol extract 30g/kg, gavage is pressed to 400g cleaning grade SD male rats can't help
Water, 3 times a day, continues 3d；
(3) 2h is anaesthetized to rat with yellow Jackets after last gavage, is adopted under aseptic condition through abdominal aorta
Blood；
(4) blood is stood into 8h, 5000rpm, 4 DEG C of centrifugation 10min, 0.22 μm is passed through after taking supernatant, 60 DEG C of inactivation 25min
Membrane filtration is degerming, obtains Contained Serum, packing, freezes standby in -80 DEG C of refrigerators.
Embodiment 2
The Contained Serum prepared by Ligusticum wallichii alcohol extract
(1) appropriate Ligusticum chuanxiong Hort is weighed first and is ground into powdered, the body that then 10 times of addition medicinal material weight is measured
The ethanol of fraction 80%, heating and refluxing extraction 2 times 30 minutes every time, merges after extract solution, by 200 mesh sieve net filtrations and
Distillation and concentration obtains lotion, then freeze-dried rear crush produces Ligusticum wallichii alcohol extract；
(2) fasting before weight ratio gavage Ligusticum wallichii alcohol extract 20g/kg, gavage is pressed to 300g cleaning grade SD male rats can't help
Water, 3 times a day, continues 3d；
(3) 2h is anaesthetized to rat with yellow Jackets after last gavage, is adopted under aseptic condition through abdominal aorta
Blood；
(4) blood is stood into 8h, 5000rpm, 4 DEG C of centrifugation 10min, 0.22 μm is passed through after taking supernatant, 60 DEG C of inactivation 25min
Membrane filtration is degerming, obtains Contained Serum, packing, freezes standby in -80 DEG C of refrigerators.
Embodiment 3
The Contained Serum prepared by Ligusticum wallichii alcohol extract
(1) appropriate Ligusticum chuanxiong Hort is weighed first and is ground into powdered, the body that then 10 times of addition medicinal material weight is measured
The ethanol of fraction 80%, heating and refluxing extraction 2 times 30 minutes every time, merges after extract solution, by 200 mesh sieve net filtrations and
Distillation and concentration obtains lotion, then freeze-dried rear crush produces Ligusticum wallichii alcohol extract；
(2) fasting before weight ratio gavage Ligusticum wallichii alcohol extract 10g/kg, gavage is pressed to 250g cleaning grade SD male rats can't help
Water, 2 times a day, continues 5d；
(3) 1h is anaesthetized to rat with yellow Jackets after last gavage, is adopted under aseptic condition through abdominal aorta
Blood；
(4) blood is stood into 4h, 3000rpm, 4 DEG C of centrifugation 15min, 0.22 μm is passed through after taking supernatant, 56 DEG C of inactivation 30min
Membrane filtration is degerming, obtains Contained Serum, packing, freezes standby in -80 DEG C of refrigerators.
Embodiment 4
Embodiment 1 is post-processed to obtained Contained Serum to rat by weight ratio gavage Ligusticum wallichii alcohol extract 30g/kg to be incubated
MG-63 OS-732 cells；
MG-63 OS-732 cells are thawed and add the DMEM containing (volume ratio) 10% hyclone (FBS) after recovering
Cell suspension is made in culture medium, and a point kind is placed in 5%CO2 (conventional environment of cell culture, CO2 volume fractions in blake bottle
For 5%), saturated humidity 95% is cultivated in 37 DEG C of incubators.1 nutrient solution is about changed during normal culture daily, when cell life
Long degrees of fusion carries out Secondary Culture when reaching 70%-80%；The old nutrient solution in blake bottle is discarded during Secondary Culture first, is added
37 DEG C of phosphate buffered saline solution (PBS) liquid wash bottles 2 times, add the trypsase that 2mL concentration is 0.25% in 25cm2 blake bottles
(i.e. 2.5g/L trypsin solution) is digested.Then carefully observed at any time under inverted microscope, when discovery cytoplasm
Retraction, space between cells terminate digestion when becoming big.Next trypsin solution is discarded, is added containing (volume ratio) 10% hyclone
DMEM culture mediums, softly blown and beaten with suction pipe makes attached cell suspend afterwards.And then the nutrient solution containing suspension cell is transferred to
In 10mL centrifuge tubes, 1500rpm centrifugation 5min abandon supernatant.Cell is resuspended in 5mL containing (volume ratio) 10% tire ox blood
In clear complete medium.Cell count terminates, and cell is dispensed into new sterile culture flask and cultivated by selection suitable concn.
Visible cell is adherent after last 6h, later secondary per liquid is changed within 2-3 days, after re-digesting passage after the Chang Full bottom of bottle 70%-80% of Xi Bao.
The well-grown in 4-6 generations is taken, the cell that degrees of fusion is about 60%-70% carries out cell experiment.In subconfluence
Cell, soft piping and druming is made into single cell suspension and is inoculated in 96 well culture plates, 24 well culture plates and Tranwell bed boards, carefully
With each packet MG-63 OS-732 cells are carried out with incubation 24h processing, specific packet situation is such as after born of the same parents' adherent growth 24h
Under：
Control group (sets 6 groups, group 1- groups 6) altogether：
1st, control group：With serum-free DMEM nutrient solution incubated cells 24h；
2nd, hyclone control group：Serum is only added in DMEM nutrient solutions, and (serum is standard hyclone, purchased from Hangzhoupro
State Chinese holly bio tech ltd), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
3rd, blank serum control group：Serum is only added in DMEM nutrient solutions, and (blood serum acquisition method is same as Example 1, no
It is same to be no gavage), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
4th, Ligusticum wallichii alcohol extract control group：(blood is mixed with Ligusticum wallichii alcohol extract with the serum that the rat without gavage obtains
Clear acquisition method is same as Example 1, and difference is no gavage, and Ligusticum wallichii alcohol extract and the weight ratio of the rat of embodiment 1 are
30g/kg), it is then added in DMEM nutrient solutions, serum-concentration (volume ratio) is 15%, with the DMEM nutrient solution incubated cells
24h；
5th, saline control group：Serum (the serum collection obtained to rat by weight ratio gavage physiological saline 30g/kg
Method is same as Example 1, and difference Ligusticum wallichii alcohol extract changes physiological saline into) it is added to addition, serum in DMEM nutrient solutions
Concentration (volume ratio) is 15%, with DMEM nutrient solution incubated cells 24h；
6th, existing drug control group：The serum obtained with existing treatment osteosarcoma drugs Cisplatin with the rat without gavage enters
(blood serum acquisition method is same as Example 1, and difference is no gavage, the weight of cis-platinum and the rat body weight of embodiment 1 for row mixing
Amount ratio is 30g/kg, and cis-platinum is bought in Qilu Pharmaceutical Factory), it is then added in DMEM nutrient solutions, serum-concentration (volume ratio) is
15%, with DMEM nutrient solution incubated cells 24h；
It is due to that cis-platinum injections are its conventional sides that cis-platinum gavage is not provided with control group and prepares Contained Serum control group
Method, unsuitable gavage is used, therefore cis-platinum injections control group and cis-platinum gavage control group are not provided with the present embodiment.
Treatment group (sets 2 groups, group 7 and group are 8) altogether：
With embodiment 1 DMEM trainings are added to rat Contained Serum as made from weight ratio gavage Ligusticum wallichii alcohol extract 30g/kg
In nutrient solution, Contained Serum concentration (volume ratio) is made for 15% and 20% two kind of DMEM nutrient solution, respectively with the He of concentration 15%
20% Contained Serum DMEM nutrient solution incubated cells 24h.
Embodiment 5
Embodiment 2 is post-processed to obtained Contained Serum to rat by weight ratio gavage Ligusticum wallichii alcohol extract 20g/kg to be incubated
MG-63 OS-732 cells, specific cell culture processes are same as Example 4.
Control group (sets 6 groups, group 1- groups 6) altogether：
1st, blank control group：With serum-free DMEM nutrient solution incubated cells 24h；
2nd, hyclone control group：Serum is only added in DMEM nutrient solutions, and (serum is standard hyclone, purchased from Hangzhoupro
State Chinese holly bio tech ltd), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
3rd, blank serum control group：Serum is only added in DMEM nutrient solutions, and (blood serum acquisition method is same as Example 2, no
It is same to be no gavage), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
4th, Ligusticum wallichii alcohol extract control group：(blood is mixed with Ligusticum wallichii alcohol extract with the serum that the rat without gavage obtains
Clear acquisition method is same as Example 2, and difference is no gavage, the weight ratio of Ligusticum wallichii alcohol extract and the rat body weight of embodiment 2
For 20g/kg), it is then added in DMEM nutrient solutions, serum-concentration (volume ratio) is 15%, is incubated with the DMEM nutrient solutions thin
Born of the same parents 24h；
5th, saline control group：Serum (the serum collection obtained to rat by weight ratio gavage physiological saline 20g/kg
Method is same as Example 2, and difference Ligusticum wallichii alcohol extract changes physiological saline into) it is added to addition, serum in DMEM nutrient solutions
Concentration (volume ratio) is 15%, with DMEM nutrient solution incubated cells 24h；
6th, existing drug control group：The serum obtained with existing treatment osteosarcoma drugs Cisplatin with the rat without gavage enters
(blood serum acquisition method is same as Example 2, and difference is no gavage, and cis-platinum and the rat body weight ratio of embodiment 1 are for row mixing
20g/kg), it is then added in DMEM nutrient solutions, serum-concentration (volume ratio) is 15%, with the DMEM nutrient solution incubated cells
24h；
Treatment group (sets 2 groups, group 7 and group are 8) altogether：
With embodiment 2 DMEM trainings are added to rat Contained Serum as made from weight ratio gavage Ligusticum wallichii alcohol extract 20g/kg
In nutrient solution, Contained Serum concentration (volume ratio) is made for 15% and 20% two kind of DMEM nutrient solution, respectively with the He of concentration 15%
20% Contained Serum DMEM nutrient solution incubated cells 24h.
Embodiment 6
Embodiment 3 is post-processed to obtained Contained Serum to rat by weight ratio gavage Ligusticum wallichii alcohol extract 10g/kg to be incubated
MG-63 OS-732 cells, specific cell culture processes are same as Example 4.
Control group (sets 6 groups, group 1- groups 6) altogether：
1st, blank control group：With serum-free DMEM nutrient solution incubated cells 24h；
2nd, hyclone control group：Serum is only added in DMEM nutrient solutions, and (serum is standard hyclone, purchased from Hangzhoupro
State Chinese holly bio tech ltd), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
3rd, blank serum control group：Serum is only added in DMEM nutrient solutions, and (blood serum acquisition method is same as Example 2, no
It is same to be no gavage), serum-concentration (volume ratio) is 15%, DMEM nutrient solution incubated cells 24h；
4th, Ligusticum wallichii alcohol extract control group：(blood is mixed with Ligusticum wallichii alcohol extract with the serum that the rat without gavage obtains
Clear acquisition method is same as Example 3, and difference is no gavage, the weight ratio of Ligusticum wallichii alcohol extract and the rat body weight of embodiment 3
For 10g/kg), it is then added in DMEM nutrient solutions, serum-concentration (volume ratio) is 15%, is incubated with the DMEM nutrient solutions thin
Born of the same parents 24h；
5th, saline control group：Serum (the serum collection obtained to rat by weight ratio gavage physiological saline 10g/kg
Method is same as Example 2, and difference Ligusticum wallichii alcohol extract changes physiological saline into) it is added to addition, serum in DMEM nutrient solutions
Concentration (volume ratio) is 15%, with DMEM nutrient solution incubated cells 24h；
6th, existing drug control group：(serum collection side is mixed with cis-platinum with the serum that the rat without gavage obtains
Method is same as Example 2, and difference is no gavage, and the rat body weight ratio of cis-platinum and embodiment 1 is 10g/kg), then add
Into DMEM nutrient solutions, serum-concentration (volume ratio) is 15%, with DMEM nutrient solution incubated cells 24h；
Treatment group (sets 2 groups, group 7 and group are 8) altogether：
With embodiment 3 DMEM trainings are added to rat Contained Serum as made from weight ratio gavage Ligusticum wallichii alcohol extract 10g/kg
In nutrient solution, Contained Serum concentration (volume ratio) is made for 15% and 20% two kind of DMEM nutrient solution, respectively with 15% and 20%
Contained Serum DMEM nutrient solution incubated cells 24h.
Test example 1MTT methods detect effect of the medicine to MG-63 OS-732 cells proliferation activities
MTT full name 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide,
Entitled 3- (4,5- dimethylthiazole -2) -2, the 5- diphenyltetrazolium bromide bromides of Chinese chemistry, are a kind of dyestuffs of yellow.In increasing
The amber dehydrogenase contained in the living cells cell mitochondrial for growing the phase, can make exogenous MTT be reduced into the needle-like of indissoluble
Formazan is crystallized, and in bluish violet, and is deposited in living cells, cell viability is directly proportional to Formazan forming amount, DMSO
Formazan crystallization dissolvings can be made, absorption value is determined at wavelength 570nm using ELIASA, reflect what cell was bred with reversed around here
Situation.In the range of certain cell number, MTT crystallizes the amount to be formed and is directly proportional to cell number.
The cell for 96 orifice plates that Example 4,5,6 is respectively prepared after packet is incubated, quantitatively adds 100 μ L to contain (body per hole respectively
Product ratio) 15% hyclone (FBS) complete medium；It is placed in 5%CO2, saturated humidity 95%, 37 DEG C of incubators and is cultivated
24~72h；4h adds the MTT in 10 μ L/ holes (concentration is 5mg/mL) before culture is terminated；Continue to cultivate after 4h, careful inhale abandons supernatant
Add 100 μ L/ holes dimethyl sulfoxide (DMSO) (DMSO) terminating reactions after nutrient solution, in shaking 10min or so on oscillator, fill crystal
Divide dissolving；ELIASA 570nm detects OD values, and experiment is at least in triplicate.Result is recorded, survival rate is calculated.
Cell survival rate (%)=treatment group mean OD value/control group mean OD value × 100%.
As a result as shown in 1 to 3.
Mtt assay detects MG-63 OS-732 cells survival rates during the 30g/kg of table 1 Ligusticum wallichii alcohol extract gavage rat
* represent notable with control group comparing difference
Mtt assay detects MG-63 OS-732 cells survival rates during the 20g/kg of table 2 Ligusticum wallichii alcohol extract gavage rat
* represent notable with control group comparing difference
Mtt assay detects MG-63 OS-732 cells cell survivals during the 10g/kg of table 3 Ligusticum wallichii alcohol extract gavage rat
Rate
* represent notable with control group comparing difference
From table 1-3, to rat by weight ratio containing of preparing of gavage Ligusticum wallichii alcohol extract 30,20,10g/kg respectively
Medicine serum, when concentration (volume ratio) is 15% and 20% Contained Serum, MG-63 OS-732 cells survival rates can
To reach 26.69% and less than 26.45%, with the cell survival rate of Ligusticum wallichii alcohol extract control group and existing drug control group
Cell survival rate, which is compared, has obvious advantage, all suppresses MG-63 OS-732 cells survival benefits, explanation with good
Contained Serum prepared by the embodiment of the present invention has the effect of the survival of good suppression MG-63 OS-732 cells.
As shown in Table 2, to rat Contained Serum effect as made from weight ratio gavage 20g/kg preferably, concentration is 15% He
The cell survival rate of 20% Contained Serum group is 24.69%, 24.58% respectively；Cell survival rate is during 20% Contained Serum group
24.58%, and 15% Contained Serum group no difference of science of statistics, therefore, by pressing weight ratio gavage Ligusticum wallichii alcohol extract to rat
Contained Serum made from 20g/kg, the Contained Serum of concentration 15% can just obtain good suppression MG-63 people into bone and flesh
Oncocyte survival benefit.
The Apoptosis by Flow Cytometry of test example 2
Phosphatide phthalein serine (PS) is only distributed in the inner side of cell membrane lipid bilayer, and in Apoptosis early stage, cell membrane
In phosphatidylserine turned on one's side laterally in adipose membrane.Under the constraint and encirclement of sheath fluid, the cell of sample to be tested lines up list
Row are sprayed by flow chamber nozzle at high speeds, and form cell fluid column.Fluid column is when by detection zone, under the irradiation of incoming laser beam
Side scattered light (SSC) and forward scattering light (FSC) can be produced, they reflect cell granulations degree and size respectively, according to these
Characteristic is by cell classification.Sample after one or more of special fluorescence labelings, can under the exciting of laser beam generation specific fluorescent,
The specific fluorescent by optical system detection and can be analyzed, and thus obtain corresponding various cell characteristics.AnnexinV is a kind of point
35~36kDa of son amount Ca2+Dependence cardiolipin binding protein, has high affinity, therefore can pass through cell with phosphatidylserine
The phosphatidylserine of outside exposure is combined with the after birth of apoptosis early stage cell.AnnexinV is subjected to FITC marks, to mark
AnnexinV utilizes flow cytometer to can detect the generation of Apoptosis as fluorescence probe.
The cell for 24 orifice plates that Example 4,5,6 is respectively prepared after packet is incubated, discards every hole after culture supernatant and adds respectively
Enter the trypsase (i.e. 2.5g/L trypsin solution) that 0.5mL concentration is 0.25%, observed under inverted microscope, treat thin
Born of the same parents start to be rounded, plus a small amount of serum-containing medium, are moved to after terminating digestion, piping and druming suspension cell in centrifuge tube, under 1000rpm from
Heart 10min, abandons supernatant, is washed with the PBS of precooling 2 times, and cell is resuspended with 250 μ L combination buffers.Adjust its concentration for 1 ×
106/ mL, takes 100 μ L cell suspensions in 5mL streaming pipes, and the PI for adding the 5 μ L AnnexinVFITC and μ g/mL of 10 μ L 20 is molten
Room temperature lucifuge is incubated 15min after liquid, mixing, and 400 μ L PBS, flow cytometer analysis are added in reaction tube.Experimental result
Through computer software ModFit LT3.0 analysis software cell cycle cell numbers, every group of experiment is at least repeated 3 times；As a result see
Shown in table 4 to 6.
The change of MG-63 OS-732 cells apoptosis rates during the 30g/kg of table 4 Ligusticum wallichii alcohol extract gavage rat
* represent notable with control group comparing difference
The change of MG-63 OS-732 cells apoptosis rates during the 20g/kg of table 5 Ligusticum wallichii alcohol extract gavage rat
* represent notable with control group comparing difference
The change of MG-63 OS-732 cells apoptosis rates during the 10g/kg of table 6 Ligusticum wallichii alcohol extract gavage rat
* represent notable with control group comparing difference
From table 4 to 6, to rat by weight ratio containing of preparing of gavage Ligusticum wallichii alcohol extract 30,20,10g/kg respectively
Medicine serum, when the Contained Serum of concentration (volume ratio) 15% and 20%, MG-63 OS-732 cells apoptosis rate can be with
40.92% and more than 39.16% are reached, it is thin with the apoptosis rate of Ligusticum wallichii alcohol extract control group and existing drug control group
Born of the same parents' apoptosis rate, which is compared, has obvious advantage, can greatly increase the apoptosis rate of MG-63 OS-732 cells, illustrates this
Contained Serum prepared by inventive embodiments has the effect of the apoptosis of good promotion MG-63 OS-732 cells.
As shown in Table 5, to rat Contained Serum effect as made from weight ratio gavage Ligusticum wallichii alcohol extract 20g/kg preferably, it is dense
The apoptosis rate spent for 15% and 20% Contained Serum group is 43.57%, 43.38% respectively；It is thin during 20% Contained Serum group
Born of the same parents' apoptosis rate and 15% Contained Serum group no difference of science of statistics, therefore, by pressing weight ratio gavage Ligusticum wallichii alcohol extract to rat
Contained Serum made from 20g/kg, using its concentration for 15% Contained Serum can just obtain it is good promote MG-63 people into
Apoptosis in osteosarcoma cells effect.
Test example 3Transwell Matrigels detection Ligusticum wallichii alcohol extract prepares shadow of the Contained Serum to cell invasion ability
Ring.
Cell migration assay is that Transwell cells are put into culture plate, small indoor referred to as upper chamber, under claiming in culture plate
Room, levels nutrient solution is separated by with polycarbonate membrane, by the tumour cell kind of research in upper interior, lower room add containing FBS or certain
The nutrient solution of some specific chemotactic factor (CF)s, because polycarbonate membrane has a permeability, tumour cell can to nutritional ingredient it is high under
Room is migrated, and counts the transfer ability for entering the cell quantity of lower room with regard to energy reacting cells.
The Transwell bed board cells that each packet of the process of Example 4,5,6 is incubated respectively, the final concentration of cell is big
Small about 5 × 105/mL；The lower indoor μ L of addition 500 of the 24 orifice plates DMEM containing (volume ratio) 20%FBS,
The small indoor μ L of addition 100 of Transwell cell suspension, cellar culture 24h；After the completion of cell culture, Transwell is gently carried small
Room, topples over the culture medium of interior, washs cell 2 times with appropriate PBS, adds 1mL 4% paraformaldehyde per hole afterwards
(4% paraformaldehyde is melted into 100mLPBS for 4g paraformaldehydes and is made；The preparation of PBS：NaCl8g, KCl 0.2g,
Na2HPO4 1.44g, KH2PO4 0.24, adds 800mL deionized waters, is sufficiently stirred for, and pH value is adjusted to 7.4, supplement with HCl
Deionized water is settled to 1000mL, and high pressure steam sterilization is stored in 4 DEG C.) fixed cell 15min, PBS clean 3 times again, every time
5min, reuse 0.1% crystal violet stained cells 30min, washed with PBS 3 times, each 5min is gently rushed using ultra-pure water
The cell of moistening chamber surface is washed, the cell for not migrating film is finally gently wiped with moistening cotton swab, blower fan is air-dried；It is inverted
Transwell cells, microscope is observed and taken pictures.Cells are observed in randomly selecting 5 visuals field under microscope, and counting is moved
Move cell number.
Fig. 1 is that embodiment 1 is incubated MG-63 to rat by the weight ratio gavage Ligusticum wallichii alcohol extract 30g/kg Contained Serums prepared
OS-732 cells cell, Transwell cell migrations detection cell migration ability, specific packet reference embodiment 4, wherein
1 blank control group；2 hyclone control groups；3 blank serum control groups；4 Ligusticum wallichii alcohol extract control groups；5 saline controls
Group；The 6 existing Contained Serum groups of the concentration of drug control group 7 (volume ratio) 15%；The Contained Serum group of 8 concentration (volume ratio) 20%.Figure
Middle * is P<0.05, significant difference.
Fig. 2 is that embodiment 2 is incubated MG-63 to rat by the weight ratio gavage Ligusticum wallichii alcohol extract 20g/kg Contained Serums prepared
OS-732 cells cell, Transwell cell migrations detection cell migration ability, specific packet reference embodiment 5, wherein
1 blank control group；2 hyclone control groups；3 blank serum control groups；4 Ligusticum wallichii alcohol extract control groups；5 saline controls
Group；6 existing drug control groups；The Contained Serum group of 7 concentration (volume ratio) 15%；The Contained Serum group of 8 concentration (volume ratio) 20%.Figure
Middle * is P<0.05, significant difference.
Fig. 3 is that embodiment 3 is incubated MG-63 to rat by the weight ratio gavage Ligusticum wallichii alcohol extract 10g/kg Contained Serums prepared
OS-732 cells cell, Transwell cell migrations detection cell migration ability, specific packet reference embodiment 6, wherein
1 blank control group；2 hyclone control groups；3 blank serum control groups；4 Ligusticum wallichii alcohol extract control groups；5 saline controls
Group；6 existing drug control groups；The Contained Serum group of 7 concentration (volume ratio) 15%；The Contained Serum group of 8 concentration (volume ratio) 20%.Figure
Middle * is P<0.05, significant difference.
From Fig. 1 to 3 as can be seen that distinguishing to rat by weight ratio after gavage Ligusticum wallichii alcohol extract 10,20,30g/kg through abdomen master
After arterial blood extracting centrifugation, it is configured to Contained Serum of the DMEM nutrient solution volumes than 15%, 20% to MG-63 people into bone and flesh
Oncocyte is incubated, and as a result shows the MG-63 OS-732 cells of Contained Serum incubation relative to the Ligusticum wallichii alcohol extracting of control group 4
Thing control group, 6 existing drug control group transfer abilities are significantly reduced, illustrate the embodiment of the present invention prepare Contained Serum have it is good
The effect of good suppression MG-63 OS-732 cells migration.
Claims (10)
1. a kind of Contained Serum prepared by Ligusticum wallichii alcohol extract, it is characterised in that the Contained Serum is by Ligusticum wallichii alcohol extract pair
Rat is carried out after gavage, and blood sampling is obtained.
2. Contained Serum according to claim 1, it is characterised in that the Ligusticum wallichii alcohol extract is that Ligusticum wallichii is ground into powder
Add alcohol reflux after shape to extract, the pulverulent solids after freeze-drying obtained by crushing.
3. Contained Serum according to claim 1, it is characterised in that the rat is cleaning grade SD male rats.
4. a kind of preparation method of the Contained Serum as claimed in claim 1 prepared by Ligusticum wallichii alcohol extract, it is characterised in that bag
Include following steps：
(1) weight ratio gavage Ligusticum wallichii alcohol extract (10-30) g/kg is pressed to rat；
(2) 1-3h is anaesthetized to rat after last gavage, is taken a blood sample under aseptic condition through abdominal aorta；
(3) blood is stood into 4-8h, centrifuges, take filtration sterilization after supernatant, inactivation, obtain Contained Serum, dispensed, freeze standby.
5. Contained Serum according to claim 1, it is characterised in that step (1) described gavage be can't help for fasting before gavage
Water, it is daily 2-3 times, continue 3-5d.
6. Contained Serum according to claim 1, it is characterised in that step (3) centrifugation is 3000-5000rpm, 4-
6 DEG C of centrifugation 10-15min.
7. Contained Serum according to claim 1, it is characterised in that step (3) inactivation is 56-60 DEG C of inactivation 25-
30min。
8. Contained Serum according to claim 1, it is characterised in that step (3) described filtration sterilization is to pass through filter membrane mistake
Filter out bacterium.
9. a kind of Contained Serum as claimed in claim 1 prepared by Ligusticum wallichii alcohol extract is in the medicine for preparing treatment osteosarcoma
Using.
10. application according to claim 9, it is characterised in that the Contained Serum prepared by Ligusticum wallichii alcohol extract is in system
Applied in the medicine of standby treatment MG-63 OS-732 cells.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109295160A (en) * | 2018-10-24 | 2019-02-01 | 常州大学 | Dictamnus dasycarpus Turcz is as dipeptide peptidase i inhibitor and preparation method and purposes |
| CN114910573A (en) * | 2021-02-10 | 2022-08-16 | 陕西中医药大学附属医院 | Preparation and identification method of traditional Chinese medicine-containing serum for in vitro cell culture |
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2017
- 2017-01-25 CN CN201710055451.9A patent/CN107050053A/en active Pending
Non-Patent Citations (3)
| Title |
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| 王勇等: "川芎嗪对人骨肉瘤MG-63细胞生长的抑制作用观察及机制探讨", 《山东医药》 * |
| 赵丽恋等: "川芎醇提工艺研究", 《中成药》 * |
| 雷志丹等: "川芎血清药物化学的初步研究", 《中国实验方剂学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295160A (en) * | 2018-10-24 | 2019-02-01 | 常州大学 | Dictamnus dasycarpus Turcz is as dipeptide peptidase i inhibitor and preparation method and purposes |
| CN114910573A (en) * | 2021-02-10 | 2022-08-16 | 陕西中医药大学附属医院 | Preparation and identification method of traditional Chinese medicine-containing serum for in vitro cell culture |
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