CN105362314B - A kind of new application of twocolor sealavander herb ethyl acetate extract - Google Patents
A kind of new application of twocolor sealavander herb ethyl acetate extract Download PDFInfo
- Publication number
- CN105362314B CN105362314B CN201510899298.9A CN201510899298A CN105362314B CN 105362314 B CN105362314 B CN 105362314B CN 201510899298 A CN201510899298 A CN 201510899298A CN 105362314 B CN105362314 B CN 105362314B
- Authority
- CN
- China
- Prior art keywords
- cell
- liquid
- culture
- ethyl acetate
- twocolor sealavander
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of new applications of twocolor sealavander herb ethyl acetate extract, are especially treating osteosarcoma, especially U2-OS type osteosarcoma, colon cancer, particularly LOVO type colon cancer, the new application in terms of breast cancer, especially MCF-7 type breast cancer.Under same experimental conditions, the activity of twocolor sealavander herb ethyl acetate extract is better than 5 FU 5 fluorouracil, and having becomes the exploitation of replacement 5 FU 5 fluorouracil into the potentiality of new anticarcinogen.
Description
Technical field
The present invention relates to a kind of new applications of twocolor sealavander herb ethyl acetate extract, especially in treatment U2-OS type bone
New application in terms of sarcoma, LOVO colon cancer, MCF-7 breast cancer.
Background technique
Osteosarcoma (Osteosarcoma) is initiated by the malignant tumour of bone, and disease incidence is in primary bone malignant tumour
In occupy first place.75% is teenager in Patients with Osteosarcoma, and male patient is 1.5 times of female patient, and grade malignancy is very high,
Early stage Lung metastases are easy to happen, poor prognosis, previously one it is found that take amputation to treat more, and nonetheless, patient postoperative 5
Annual survival rate is also only 5-20%, this brings great moral injury for patient and its family, also all brings for society or even country
Heavy financial burden.Limb-sparing surgery, which is aided with high-dose chemotherapy gradually, at present becomes the preferred therapeutic scheme of osteosarcoma, but still has
20-40% patient dies of transfer.
Colon cancer (cancer of colon) is the most common malignant tumour of the developed countries such as West Europe, North America and China
One of nine big common cancers.Majority state or regional colon cancer in 30 years of past, including China
Disease incidence is in rising trend.In China, because of colon cancer died, male occupies the 5th of mortality of malignant tumors, and women occupies the 6th.
From the viewpoint of epidemiology, the morbidity of colon cancer and social environment, life style (especially eating habit, shortage muscular labor
It is dynamic), inherent cause it is related.Age, colorectal polypus history, ulcerative colitis and gallbladder removal be also colon cancer it is high-risk because
Element.
Malignant tumour in breast galandular epithelium tissue usually occurs for breast cancer (mammary cancer), is a kind of serious shadow
Ring one of the most common malignant tumour of the physically and mentally healthy even threat to life of women.According to statistics, it is various that disease incidence accounts for whole body
The 7-10% of malignant tumour is only second to uterine cancer in women, its morbidity it is often related with heredity and 40-60 years old between, menopause
Women's disease incidence before and after phase is higher, and breast cancer male is rare, and only the mammary gland patient of about 1-2% is male.It is global to die of cream every year
The case of gland cancer is about 41.1 ten thousand people, is first of the female cancer cause of the death.It is presented in the disease incidence of most countries, breast cancer
The trend of liter, China is the most fast country of breast cancer growth rate, and increased amplitude reaches 3-4% every year.
Twocolor sealavander herb (Limonium bicolor(Bunge) O.Kuntze) it is that Plumbaginaceae Statice gets over Nian Sheng
Herbaceous plant, alias dry branch plum, fly grass, burn plum wormwood artemisia at fly flower, and saline-alkali tolerant, arid, resistance is strong, is distributed mainly on China
Northwest, northeast and the coastal region in east China.Twocolor sealavander herb all herbal medicine, is common Chinese traditional herbs, sweet in flavor and neutral in nature, micro-puckery and
It is bitter, nontoxic, have the function of tonifying the blood and arrest bleeding, menstruation regulating QI invigorating, antibacterial anti-inflammatory, warming middle-JIAO and strengthening the spleen.Existing research shows twocolor sealavander herb
In containing ingredients, the Flavonoid substances therein such as flavones, polysaccharide, volatile oil, steroidal have preferable oxidation resistance, polysaccharide pair
Hela cell also has good inhibiting effect, and overground part extract has good inhibiting effect to glucuroide.
Although the effect of twocolor sealavander herb is studied scholar's discovery successively, in treatment osteosarcoma, especially U2-OS type
Osteosarcoma, colon cancer, particularly LOVO type colon cancer, the research in terms of breast cancer, especially MCF-7 type breast cancer do not appear in the newspapers yet
Road.
Summary of the invention
The present invention is intended to provide a kind of new application of twocolor sealavander herb ethyl acetate extract, especially in treatment bone and flesh
Tumor, especially U2-OS type osteosarcoma, colon cancer, particularly LOVO type colon cancer, breast cancer, especially MCF-7 type breast cancer side
The new application in face.
Twocolor sealavander herb raw material of the present invention is the herb of twocolor sealavander herb or the inflorescence of twocolor sealavander herb.
It is of the present invention to be extracted as cold soaking or heat reflux or ultrasound.
Its concentration of ethyl alcohol of the present invention is 50-100%.
Twocolor sealavander herb ethyl acetate extract of the present invention is to be mentioned using twocolor sealavander herb as raw material with ethyl alcohol
It takes, is filtered after extraction, after filtrate is concentrated into no alcohol taste, extracted with ethyl acetate, ethyl acetate extract is recycled, and is done
Dry, extract obtained is twocolor sealavander herb ethyl acetate extract.
PBS buffer solution preparation method used is as follows in the present invention: 8 gNaCl, 0.2 gKCl, 1.44 gNa2HPO4,0.20
gKH2PO4 adds ultrapure water to 1000 mL, adjusts PH7.2-7.4, (121 DEG C) sterilizing 20min of high pressure-temperature, and 4 DEG C of refrigerations are standby
With.
Pancreatin preparation method used is as follows in the present invention: weighing tryptose enzyme powder 1 g of 0.50 g and EDTA, is dissolved in 200
In mL PBS buffer solution.4 h of low temperature adjusts PH 7.2-7.4 after completely dissolution.0.22 μm of membrane filtration degerming is used
15 mL test tubes packing, -20 DEG C freeze it is spare.
RPMI1640 culture medium preparation method used is as follows in the present invention: by 1640 solid powder culture of RPMI Medium
6.75 g of base is dissolved in 500 mL ultrapure waters, and 32.5 mg penicillin, 50 mg streptomysins, 1 g NaHCO are added3, sufficiently dissolve；
0.22 μm of membrane filtration degerming, dispenses in superclean bench to 100mL bottles, every bottle of 85 mL, and 4 DEG C of refrigerations are spare.When use
Every bottle of addition 10 mL horse serums, 5 mL fetal calf serums, are configured to 15% culture medium.
DMEM culture medium preparation method used is as follows in the present invention: by Dulbecco's Modified Eagle Medium
6.75 g of solid powder culture medium is dissolved in 500 mL ultrapure waters, and 32.5 mg penicillin, 50 mg streptomysins, 1 g are added
NaHCO3, sufficiently dissolve；0.22μmMembrane filtration degerming, to dispensing in 100 mL bottles in superclean bench, every bottle of 90 mL, 4
DEG C refrigeration is spare.Every bottle of 10 mL fetal calf serums of addition, are configured to 10% culture medium when use.
F-12K culture medium preparation method used is as follows in the present invention: measuring the training of F-12K Nutrient Mixture liquid
500 mL of base is supported, 32.5 mg penicillin, 50 mg streptomysins are added, sufficiently dissolve；0.22μmMembrane filtration degerming, ultra-clean
To dispensing in 100 mL bottles in workbench, every bottle of 90 mL, 4 DEG C of refrigerations are spare.Every bottle of 10 mL fetal calf serums of addition, match when use
It is set to 10% culture medium.
MTT preparation method used is as follows in the present invention: weighing 20 mg MTT in 5 mL test tubes, it is slow that 4 mL PBS are added
Fliud flushing is made it completely dissolved using vortex instrument, 5 mg/mL MTT liquid；It is cold with -20 DEG C after 0.22 μm of millipore filter degerming
Freeze and saves.
Acid solution preparation method used is as follows in the present invention: weighing 120 g potassium bichromates, is added in 1000 mL ultrapure waters, adds
Heat auxiliary makes it completely dissolved；The lysate is slowly added in the 200 mL concentrated sulfuric acids, and is stirred continuously, until it is cooled down.
Step of the invention is as follows:
1, cell strain is recovered: distilled water is added in loosely capped vial, is put into 38 DEG C of thermostatic water baths, it will after its temperature is constant
The cell frozen in -198 DEG C of liquid nitrogen takes out, and is quickly put into loosely capped vial, shakes, makes to freeze liquid in pipe in 1-2 min
It dissolves.Cryopreservation tube is wiped with alcohol swab, is put into workbench, cell liquid is sucked in 15 mL test tubes, while 10 mL are added should
The fresh culture of cell strain, 1000 rpm are centrifuged 4 min.Liquid is discarded supernatant, liquid-transfering gun injects the piping and druming of 1 mL fresh culture
After dispersion, injection is had been loaded in the culture bottle of 7 mL fresh cultures, light spiral cover, in CO2Culture in incubator.
2, cell changes liquid: Tissue Culture Flask being tightened and is taken out after lid from incubator, the color of culture in glassware base is observed
And it is whether muddy, cell growth condition is observed under inverted microscope.Be put into ultra-clean work after culture bottle being wiped with alcohol swab
Platform removes lid, outwells culture medium.2 mL PBS are drawn with liquid-transfering gun to squeeze into culture bottle, after closeing the lid, are shaken several times,
PBS is outwelled, it is primary to repeat this operation.It is drawn in 8 mL fresh cultures injection culture bottle, is closed the lid, observation is thin with liquid-transfering gun
Born of the same parents' state.It is put into incubator after alcohol wipe body and bottleneck.
3, cell dissociation: Tissue Culture Flask is tightened and is taken out from incubator after lid, the color of culture in glassware base is observed
And it is whether muddy, cell growth condition is observed under inverted microscope.Be put into ultra-clean work after culture bottle being wiped with alcohol swab
Platform removes lid, outwells culture medium.2 mL PBS are drawn with sample injector to squeeze into culture bottle, after closeing the lid, are shaken several times
Afterwards, PBS is outwelled, it is primary to repeat this operation.1 mL pancreatin is drawn with liquid-transfering gun, is injected in culture bottle, is screwed lid, lay flat culture
Bottle makes pancreatin that can infiltrate culture face.It is observed under inverted microscope, cell becomes single circle, then needs to terminate disappearing for pancreatin
Change.Superclean bench is put into after alcohol swab wiping, pipette is drawn 1 mL fresh culture and squeezed into culture bottle, inhaled with liquid-transfering gun
The cell culture face of liquid piping and druming culture bottle in culture bottle is taken, liquid becomes cloudy state.Liquid after the digestion is moved into 10 mL
In test tube, lid is screwed, 1000 rpm are centrifuged 4 min, discard supernatant liquid.It is drawn in 2 mL PBS injecting tubes with liquid-transfering gun,
1000 rpm are centrifuged 4 min, discard supernatant liquid.
4, cell passes on: by cell digest liquid-transfering gun 2 mL fresh cultures of injection, and after blowing and beating dispersion, distinguishing
It draws the immigration of 1 mL liquid to have been loaded in the culture bottle of 7 mL fresh cultures, screws lid, be put into CO2It is cultivated in incubator.
5, cell count: by the good cell of above-mentioned digestion, injecting 4 mL fresh cultures with liquid-transfering gun, and blow and beat dispersion,
It is a little to draw cell suspension, is added dropwise at coverslip edge, is full of suspension between coverslip and tally.After standing a few minutes,
It is observed under inverted microscope, counts four big lattice total number of cells in cell plates.
It counts principle: being subject to cell ruling.It is not write down in note, the right side is disregarded on a meter left side.
Calculation formula: big lattice total number of cells/4 × 10 of cell number/mL=tetra-4It is a.
6, MTT measuring method: first by 96 orifice plates in superclean bench before experiment, ultraviolet light irradiation 3h.By it is above-mentioned carefully
The cell suspension that born of the same parents count, is added certain culture medium dilution, and adjustment cell concentration is 5000/100 μ L.It is inhaled with liquid-transfering gun
Take 200μThe cell suspension of L is in the hole of 96 orifice plates, wherein the surrounding of 96 orifice plates is not added, only plus intermediate 6 rows 10 column, totally 60
A hole.PBS buffer solution is added in the surrounding of 96 orifice plates, prevents edge effect.It closes the lid, and record date and time, alcohol spray
After plate, it is put into incubator and cultivates.
It is observed under inverted microscope after cultivating 12 h, after cell is adherent, alcohol sprays plate, is put into superclean bench, uses
200 μCulture medium in hole is sucked out L liquid-transfering gun, and the culture medium culture of the drug containing of various concentration, every hole 200 is addedμL.Lid
Upper cover is observed under inverted microscope, and alcohol sprays plate, is put into incubator.
Cultivate 24,48 h, every hole is added 10 in superclean bench after 60 or 72 hoursμL MTT, closes the lid, alcohol
It is put into incubator and cultivates after spray plate.Liquid in hole is sucked out with liquid-transfering gun after 4 h, every hole is added 150μL DMSO, shaking are uniform
Afterwards, it is put into microplate reader, is read under 570 nm.
7, calculation formula: growth of tumour cell inhibiting rate %=(OD control-OD sample)/OD compares × 100 %
8, experimental model is arranged: experimental group: trial drug is dissolved with DMSO, then keeps DMSO final with culture medium dilution
Concentration is 0.5%.Each monomeric compound sets three dosage groups, and each metering sample does the parallel laboratory test in 3 holes.Blank assay
Group: containing only culture medium, and there are the parallel laboratory tests in 3 holes in every piece of 96 orifice plates.Positive controls: contain a certain concentration positive drug
The culture medium of object 5 FU 5 fluorouracil (5-FU) has the parallel laboratory test in 3 holes in every piece of 96 orifice plates.Negative control group: contain 0.5%
The culture medium of DMSO, each sample do the parallel laboratory test in 3 holes.
9. cell strain and corresponding culture medium: LOVO colon cancer cell line, used medium are the F- containing 10% fetal calf serum
12K culture medium；U2-OS osteosarcoma cell line, used medium are to train containing the RPMI 1640 of 10% horse serum and 5% fetal calf serum
Support base；MCF-7 breast carcinoma cell strain, used medium are the DMEM culture medium containing 10% fetal calf serum.
10. in the present invention, the cell culture time is 12-72 hours.
Specific embodiment
Below with reference to embodiment, the present invention is further described below, but it is not represented as unique embodiment party of the invention
Formula.
Twocolor sealavander herb raw material of the present invention is the herb of twocolor sealavander herb or the inflorescence of twocolor sealavander herb.
It is of the present invention to be extracted as cold soaking or heat reflux or ultrasound.
The methanol or ethyl alcohol or acetone that Extraction solvent of the present invention is 50-100%.
Embodiment 1: twocolor sealavander herb ethyl acetate extract be using twocolor sealavander herb herb as raw material, with 80% methanol into
Row cold soaking extracts, and filters after extraction, after filtrate is concentrated into no alcohol taste, is extracted with ethyl acetate, and ethyl acetate extract carries out
Recycling, dry, extract obtained is twocolor sealavander herb ethyl acetate extract.U2-OS type osteosarcoma cell by recovery
Strain carries out cell as needed and changes liquid during the cultivation process, after cell adherent growth area reaches the 80% of cell bottle bottom,
Culture medium is outwelled, 2 mL PBS is drawn with sample injector and squeezes into culture bottle, after closeing the lid, after shaking several times, outwells PBS, weight
This multiple operation is primary.1 mL pancreatin is drawn with liquid-transfering gun, injects in culture bottle, screws lid, laying flat culture bottle makes the equal energy of pancreatin
The culture face of infiltration.It is observed under inverted microscope, cell becomes single circle, then needs to terminate the digestion of pancreatin.Alcohol cotton rub
Superclean bench is put into after wiping, pipette is drawn 1 mL fresh culture and squeezed into culture bottle, is drawn in culture bottle with liquid-transfering gun
Liquid blows and beats the cell culture face of culture bottle, and liquid becomes cloudy state.Liquid after the digestion is moved into 10 mL test tubes, rotation
Tight lid, 1000 rpm are centrifuged 4 min, discard supernatant liquid.With liquid-transfering gun draw 2 mL PBS injecting tubes in, 1000 rpm from
4 min of the heart discards supernatant liquid, carries out cell passage, the cell digested liquid-transfering gun is injected 2 mL fresh cultures, and blow
After playing dispersion, the immigration of 1 mL liquid is drawn respectively and is had been loaded in the culture bottle of 7 mL fresh cultures, lid is screwed, is put into CO2
It is cultivated in incubator.Cell after passing on, outwells culture medium, draws 2 mL PBS with sample injector and squeezes into culture bottle, covers
After lid, after shaking several times, PBS is outwelled, it is primary to repeat this operation.1 mL pancreatin is drawn with liquid-transfering gun, is injected in culture bottle, rotation
Tight lid, laying flat culture bottle makes pancreatin that can infiltrate culture face.It is observed under inverted microscope, cell becomes single circle, then
Need to terminate the digestion of pancreatin.Superclean bench is put into after alcohol swab wiping, pipette draws 1 mL fresh culture and squeezes into training
It supports in bottle, the cell culture face of liquid piping and druming culture bottle in culture bottle is drawn with liquid-transfering gun, liquid becomes cloudy state.It will be above-mentioned
The cell digested, is diluted with fresh culture, and blows and beats dispersion, and absorption cell suspension is a little, is added dropwise at coverslip edge, is made
Suspension is full of between coverslip and tally.It after standing a few minutes, observes, counts four big in cell plates under inverted microscope
Lattice total number of cells.Adjustment cell concentration is 5000/100 μ L.200 are drawn with liquid-transfering gunμThe cell suspension of L is in 96 orifice plates
Kong Zhong, wherein the surrounding of 96 orifice plates is not added, only plus 6 intermediate rows 10 are arranged, totally 60 holes.PBS buffering is added in the surrounding of 96 orifice plates
Liquid prevents edge effect.It closes the lid, and record date and time, after alcohol sprays plate, is put into incubator and cultivates.Cultivate 48h
It is observed under inverted microscope afterwards, after cell is adherent, alcohol sprays plate, is put into superclean bench, with 200μL liquid-transfering gun is by hole
Interior culture medium is sucked out, and is added 100μG ﹒ mL-1Drug containing culture medium culture, every hole 200μL.It closes the lid, in inversion
Microscopically observation, alcohol spray plate, are put into incubator.Every hole is added 10 in superclean bench after culture for 24 hoursμL MTT,
It closes the lid, is put into incubator and cultivates after alcohol spray plate.Liquid in hole is sucked out with liquid-transfering gun after 4 h, every hole is added 150μL
DMSO is put into microplate reader, reads under 570 nm after shaking uniformly.Calculation formula: growth of tumour cell inhibiting rate %=(OD
Control-OD sample)/OD × 100 % of control, experimental model setting: experimental group: trial drug is dissolved with DMSO, then with training
Supporting base dilution makes DMSO ultimate density 0.5%.Each monomeric compound sets three dosage groups, and each metering sample does 3 holes
Parallel laboratory test.Blank assay group: containing only culture medium, and there are the parallel laboratory tests in 3 holes in every piece of 96 orifice plates.Positive controls:
Contain 100μG ﹒ mL-1The culture medium of concentration positive drug 5 FU 5 fluorouracil (5-FU) has the parallel of 3 holes in every piece of 96 orifice plates
Experiment.Negative control group: the culture medium containing 0.5%DMSO, each sample do the parallel laboratory test in 3 holes.As the result is shown: negative
Control does not have any inhibiting effect, positive control 100 to U2-OS osteosarcoma cellμG ﹒ mL-1The 5 FU 5 fluorouracil of concentration is to U2-
The inhibiting rate of OS osteosarcoma cell is 66.7%, and twocolor sealavander herb ethyl acetate extract is 100μG ﹒ mL-1When to U2-OS bone and flesh
The inhibiting rate of oncocyte is 99.1%.
Embodiment 2: twocolor sealavander herb ethyl acetate extract be using twocolor sealavander herb inflorescence as raw material, with 70% ethyl alcohol into
Row circumfluence distillation filters after extraction, after filtrate is concentrated into no alcohol taste, is extracted with ethyl acetate, ethyl acetate extract into
Row recycling, dry, extract obtained is twocolor sealavander herb ethyl acetate extract.LOVO type colon cancer cell by recovery
Strain carries out cell as needed and changes liquid during the cultivation process, after cell adherent growth area reaches the 80% of cell bottle bottom,
Culture medium is outwelled, 2 mL PBS is drawn with sample injector and squeezes into culture bottle, after closeing the lid, after shaking several times, outwells PBS, weight
This multiple operation is primary.1 mL pancreatin is drawn with liquid-transfering gun, injects in culture bottle, screws lid, laying flat culture bottle makes the equal energy of pancreatin
The culture face of infiltration.It is observed under inverted microscope, cell becomes single circle, then needs to terminate the digestion of pancreatin.Alcohol cotton rub
Superclean bench is put into after wiping, pipette is drawn 1 mL fresh culture and squeezed into culture bottle, is drawn in culture bottle with liquid-transfering gun
Liquid blows and beats the cell culture face of culture bottle, and liquid becomes cloudy state.Liquid after the digestion is moved into 10 mL test tubes, rotation
Tight lid, 1000 rpm are centrifuged 4 min, discard supernatant liquid.With liquid-transfering gun draw 2 mL PBS injecting tubes in, 1000 rpm from
4 min of the heart discards supernatant liquid, carries out cell passage, the cell digested liquid-transfering gun is injected 2 mL fresh cultures, and blow
After playing dispersion, the immigration of 1 mL liquid is drawn respectively and is had been loaded in the culture bottle of 7 mL fresh cultures, lid is screwed, is put into CO2
It is cultivated in incubator.Cell after passing on, outwells culture medium, draws 2 mL PBS with sample injector and squeezes into culture bottle, covers
After lid, after shaking several times, PBS is outwelled, it is primary to repeat this operation.1 mL pancreatin is drawn with liquid-transfering gun, is injected in culture bottle, rotation
Tight lid, laying flat culture bottle makes pancreatin that can infiltrate culture face.It is observed under inverted microscope, cell becomes single circle, then
Need to terminate the digestion of pancreatin.Superclean bench is put into after alcohol swab wiping, pipette draws 1 mL fresh culture and squeezes into training
It supports in bottle, the cell culture face of liquid piping and druming culture bottle in culture bottle is drawn with liquid-transfering gun, liquid becomes cloudy state.It will be above-mentioned
The cell digested, is diluted with fresh culture, and blows and beats dispersion, and absorption cell suspension is a little, is added dropwise at coverslip edge, is made
Suspension is full of between coverslip and tally.It after standing a few minutes, observes, counts four big in cell plates under inverted microscope
Lattice total number of cells.Adjustment cell concentration is 5000/100 μ L.200 are drawn with liquid-transfering gunμThe cell suspension of L is in 96 orifice plates
Kong Zhong, wherein the surrounding of 96 orifice plates is not added, only plus 6 intermediate rows 10 are arranged, totally 60 holes.PBS buffering is added in the surrounding of 96 orifice plates
Liquid prevents edge effect.It closes the lid, and record date and time, after alcohol sprays plate, is put into incubator and cultivates.Cultivate 48h
It is observed under inverted microscope afterwards, after cell is adherent, alcohol sprays plate, is put into superclean bench, with 200μL liquid-transfering gun is by hole
Interior culture medium is sucked out, and is added 100μG ﹒ mL-1Drug containing culture medium culture, every hole 200μL.It closes the lid, in inversion
Microscopically observation, alcohol spray plate, are put into incubator.Every hole is added 10 in superclean bench after culture for 24 hoursμL MTT,
It closes the lid, is put into incubator and cultivates after alcohol spray plate.Liquid in hole is sucked out with liquid-transfering gun after 4 h, every hole is added 150μL
DMSO is put into microplate reader, reads under 570 nm after shaking uniformly.Calculation formula: growth of tumour cell inhibiting rate %=(OD
Control-OD sample)/OD × 100 % of control, experimental model setting: experimental group: trial drug is dissolved with DMSO, then with training
Supporting base dilution makes DMSO ultimate density 0.5%.Each monomeric compound sets three dosage groups, and each metering sample does 3 holes
Parallel laboratory test.Blank assay group: containing only culture medium, and there are the parallel laboratory tests in 3 holes in every piece of 96 orifice plates.Positive controls:
Contain 100μG ﹒ mL-1The culture medium of concentration positive drug 5 FU 5 fluorouracil (5-FU) has the parallel of 3 holes in every piece of 96 orifice plates
Experiment.Negative control group: the culture medium containing 0.5%DMSO, each sample do the parallel laboratory test in 3 holes.As the result is shown: negative
Control does not have any inhibiting effect, positive control 100 to LOVO colon cancer cellμG ﹒ mL-1The 5 FU 5 fluorouracil of concentration is to LOVO
The inhibiting rate of colon cancer cell is 72.3%, and twocolor sealavander herb ethyl acetate extract is 100μG ﹒ mL-1When to LOVO colon cancer
The inhibiting rate of cell is 87.9%.
Embodiment 3: twocolor sealavander herb ethyl acetate extract be using twocolor sealavander herb inflorescence as raw material, with 90% acetone into
Row ultrasonic extraction filters after extraction, after filtrate is concentrated into no alcohol taste, is extracted with ethyl acetate, and ethyl acetate extract carries out
Recycling, dry, extract obtained is twocolor sealavander herb ethyl acetate extract.MCF-7 breast carcinoma cell strain by recovery,
During the cultivation process, cell is carried out as needed and change liquid, after cell adherent growth area reaches the 80% of cell bottle bottom,
Fall culture medium, draw 2 mL PBS with sample injector and squeeze into culture bottle, after closeing the lid, after shaking several times, outwells PBS, repeat
This operation is primary.1 mL pancreatin is drawn with liquid-transfering gun, injects in culture bottle, screws lid, laying flat culture bottle soak pancreatin can
Moisten culture face.It is observed under inverted microscope, cell becomes single circle, then needs to terminate the digestion of pancreatin.Alcohol swab wiping
After be put into superclean bench, pipette is drawn 1 mL fresh culture and is squeezed into culture bottle, draws liquid in culture bottle with liquid-transfering gun
Body blows and beats the cell culture face of culture bottle, and liquid becomes cloudy state.Liquid after the digestion is moved into 10 mL test tubes, is screwed
Lid, 1000 rpm are centrifuged 4 min, discard supernatant liquid.It is drawn in 2 mL PBS injecting tubes with liquid-transfering gun, 1000 rpm centrifugation
4 min discard supernatant liquid, carry out cell passage, the cell digested liquid-transfering gun are injected 2 mL fresh cultures, and blow and beat
After dispersion, the immigration of 1 mL liquid is drawn respectively and is had been loaded in the culture bottle of 7 mL fresh cultures, lid is screwed, is put into CO2Training
It supports and is cultivated in case.Cell after passing on, outwells culture medium, draws 2 mL PBS with sample injector and squeezes into culture bottle, closes the lid
After son, after shaking several times, PBS is outwelled, it is primary to repeat this operation.1 mL pancreatin is drawn with liquid-transfering gun, injects in culture bottle, screws
Lid, laying flat culture bottle makes pancreatin that can infiltrate culture face.It is observed under inverted microscope, cell becomes single circle, then needs
Terminate the digestion of pancreatin.Superclean bench is put into after alcohol swab wiping, pipette draws 1 mL fresh culture and squeezes into culture
In bottle, the cell culture face of liquid piping and druming culture bottle in culture bottle is drawn with liquid-transfering gun, liquid becomes cloudy state.Disappear above-mentioned
The cell changed, is diluted with fresh culture, and blows and beats dispersion, and absorption cell suspension is a little, is added dropwise at coverslip edge, is made to hang
Liquid is full of between coverslip and tally.It after standing a few minutes, is observed under inverted microscope, counts four big lattice in cell plates
Total number of cells.Adjustment cell concentration is 5000/100 μ L.200 are drawn with liquid-transfering gunμThe cell suspension of L is in the hole of 96 orifice plates
In, wherein the surrounding of 96 orifice plates is not added, only plus 6 intermediate rows 10 are arranged, totally 60 holes.PBS buffer solution is added in the surrounding of 96 orifice plates,
Prevent edge effect.It closes the lid, and record date and time, after alcohol sprays plate, is put into incubator and cultivates.After cultivating 48h
It is observed under inverted microscope, after cell is adherent, alcohol sprays plate, is put into superclean bench, with 200μL liquid-transfering gun will be in hole
Culture medium be sucked out, be added 100μG ﹒ mL-1Drug containing culture medium culture, every hole 200μL.It closes the lid, it is aobvious in being inverted
Micro- microscopic observation, alcohol spray plate, are put into incubator.Every hole is added 10 in superclean bench after culture for 24 hoursμL MTT, lid
Upper cover is put into incubator and is cultivated after alcohol spray plate.Liquid in hole is sucked out with liquid-transfering gun after 4 h, every hole is added 150μL
DMSO is put into microplate reader, reads under 570 nm after shaking uniformly.Calculation formula: growth of tumour cell inhibiting rate %=(OD
Control-OD sample)/OD × 100 % of control, experimental model setting: experimental group: trial drug is dissolved with DMSO, then with training
Supporting base dilution makes DMSO ultimate density 0.5%.Each monomeric compound sets three dosage groups, and each metering sample does 3 holes
Parallel laboratory test.Blank assay group: containing only culture medium, and there are the parallel laboratory tests in 3 holes in every piece of 96 orifice plates.Positive controls:
Contain 100μG ﹒ mL-1The culture medium of concentration positive drug 5 FU 5 fluorouracil (5-FU) has the parallel of 3 holes in every piece of 96 orifice plates
Experiment.Negative control group: the culture medium containing 0.5%DMSO, each sample do the parallel laboratory test in 3 holes.As the result is shown: negative
Control does not have any inhibiting effect, positive control 100 to MCF-7 breast cancer cellμG ﹒ mL-1The 5 FU 5 fluorouracil pair of concentration
The inhibiting rate of MCF-7 breast cancer cell is 56.1%, and twocolor sealavander herb ethyl acetate extract is 100μG ﹒ mL-1When to MCF-7
The inhibiting rate of breast cancer cell is 80.8%.
Twocolor sealavander herb ethyl acetate extract is applied to treatment U2-OS type osteosarcoma, LOVO colon cancer, MCF- for the first time
In terms of 7 breast cancer, and result is good, and under identical drug concentration, the activity of twocolor sealavander herb ethyl acetate extract is better than
5 FU 5 fluorouracil, having becomes the exploitation of replacement 5 FU 5 fluorouracil into the potentiality of new drug.
Claims (1)
1. a kind of application of twocolor sealavander herb ethyl acetate extract in preparation treatment colon cancer drug；
The twocolor sealavander herb ethyl acetate extract is to be extracted using twocolor sealavander herb as raw material with ethyl alcohol, mistake after extraction
Filter, after filtrate is concentrated into no alcohol taste, is extracted with ethyl acetate, and ethyl acetate extract is recycled, dry, extract obtained
As twocolor sealavander herb ethyl acetate extract；
The twocolor sealavander herb raw material is the herb of twocolor sealavander herb or the inflorescence of twocolor sealavander herb；
Described is extracted as cold soaking or heat reflux or ultrasound；
Its concentration of the ethyl alcohol is 50-100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510899298.9A CN105362314B (en) | 2015-12-09 | 2015-12-09 | A kind of new application of twocolor sealavander herb ethyl acetate extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510899298.9A CN105362314B (en) | 2015-12-09 | 2015-12-09 | A kind of new application of twocolor sealavander herb ethyl acetate extract |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105362314A CN105362314A (en) | 2016-03-02 |
| CN105362314B true CN105362314B (en) | 2019-10-29 |
Family
ID=55365268
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510899298.9A Expired - Fee Related CN105362314B (en) | 2015-12-09 | 2015-12-09 | A kind of new application of twocolor sealavander herb ethyl acetate extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105362314B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106038624B (en) * | 2016-06-01 | 2019-11-29 | 鲁东大学 | Yantai herba limonii gmelinii anti-cancer effective component extract and its preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102920753A (en) * | 2012-11-05 | 2013-02-13 | 江苏省中国科学院植物研究所 | Dichromatism gmelin sealavender herb extractive and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080241282A1 (en) * | 2006-06-21 | 2008-10-02 | Rutgers, The State University | Sorghum Extract Compositions |
-
2015
- 2015-12-09 CN CN201510899298.9A patent/CN105362314B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102920753A (en) * | 2012-11-05 | 2013-02-13 | 江苏省中国科学院植物研究所 | Dichromatism gmelin sealavender herb extractive and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 二色补血草水溶性多糖、多酚类和发挥性成分的研究;张连茹;《武汉大学博士学位论文》;20041231;第62、74、78页，尤其是第3.2.3.1节、第3.3.3.1节及第3.5节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105362314A (en) | 2016-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108926584A (en) | The antimicrobial purposes of chimonanthea extract | |
| CN105362314B (en) | A kind of new application of twocolor sealavander herb ethyl acetate extract | |
| CN107320516B (en) | Application of bighead atractylodes rhizome in preparation of medicine for promoting proliferation of mesenchymal stem cells and medicine | |
| CN113143913A (en) | Application of eudesmane type sesquiterpene compound in preparation of anti-pancreatic cancer drugs | |
| CN105646516A (en) | Callicarpa nudiflora extract, diterpene compound and application of pharmaceutical composition to preparation of medicines for treating gliomas | |
| CN108452240B (en) | Anti-tumor traditional Chinese medicine composition and application thereof | |
| CN110478470A (en) | A kind of anti-gout composition of the small-molecular peptides containing sunflower disk and preparation method thereof | |
| CN105749154A (en) | Probiotic fermented traditional Chinese medicine compound composition for treating liver cancer and preparation and detection methods thereof | |
| CN103330781B (en) | There is the Chinese medicine composition of antitumor action and the preparation method of injection thereof | |
| CN107334793A (en) | The purposes of wintersweet platymiscium helicobacter pylori resistant | |
| CN107137457A (en) | Application of the flower bud of lily magnolia water extract as sole active agent in estrogenic is prepared | |
| CN101168008B (en) | Medicinal composition with tumor inhibition function and preparation method and application thereof | |
| CN105412059A (en) | New application of ethyl gallate to osteosarcoma treatment | |
| CN106890189A (en) | Application of the chonglou saponin in antineoplastic sensitizer is prepared | |
| CN107281235B (en) | The purposes of wintersweet platymiscium resisiting influenza virus | |
| CN105267201A (en) | Application of oridonin in preparing medicine for treating tumour | |
| US10004713B2 (en) | Uses of chlorogenic acid in the preparation of medicaments for treatment of oligodendroglioma | |
| CN105999245B (en) | Purposes of the pharmaceutical composition containing ulinastatin in preparation treatment gall-bladder cancer drug | |
| CN104825952A (en) | Traditional Chinese medicine for treating balanoposthitis | |
| CN104887690B (en) | Application of the chonglou saponin in antitumor drug is prepared | |
| CN108926583A (en) | The purposes of Zhejiang wintersweet anti-microbial infection | |
| CN109395015A (en) | Rheumatic pain killing Chinese medicine composition is preparing the purposes in anti-angiogenic drugs | |
| CN109758450B (en) | Antitumor compound, and preparation method and application thereof | |
| CN104083690B (en) | Application of Stahlianthus involucratus (King) Craib and its extract in preparation of drugs for treating and/or preventing breast cancer | |
| CN106860781A (en) | A kind of application of Rhizopus oryzae solid state fermentation extract, preparation method and its anticancer function |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191029 Termination date: 20211209 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |