CN106942737A - Hippophate flavone and its application - Google Patents
Hippophate flavone and its application Download PDFInfo
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- CN106942737A CN106942737A CN201710146773.4A CN201710146773A CN106942737A CN 106942737 A CN106942737 A CN 106942737A CN 201710146773 A CN201710146773 A CN 201710146773A CN 106942737 A CN106942737 A CN 106942737A
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- hippophate flavone
- hippophate
- flavone
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- cell
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- 229930003944 flavone Natural products 0.000 title claims abstract description 69
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- 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)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of Hippophate flavone and its application.The Hippophate flavone, is extracted by fructus hippophae and obtained, and the extracting method comprises the following steps：(1) fructus hippophae obtains extract solution through organic solvent ultrasonic extraction；(2) extract solution, which concentrates and removes organic solvent, obtains crude extract；(3) crude extract obtains eluent through column chromatographic isolation and purification；(4) eluent obtains the Hippophate flavone through sour water solution.Hippophate flavone of the present invention is slightly carried by fructus hippophae through organic solvent, then through column chromatographic isolation and purification, then sour water solution acquisition.Different extracting methods has large effect to the content of each active ingredient in Hippophate flavone.Hippophate flavone of the present invention can be used the medicine or health food for preparing prevention or treatment prostate cancer it has been investigated that have certain treatment and prevention effect to prostate cancer.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to a kind of Hippophate flavone and its application.
Background technology
Prostate cancer refers to the epithelial malignancy for occurring in prostate.WHO in 2004《Urinary system and male genetic
Organ tumor pathology and science of heredity》Including on gland cancer (acinar adenocarcinoma), duct adenocarcinoma, urinary tract in middle prostate cancer histological type
Skin cancer, squamous cell carcinoma, adenosquamous carcinoma.Wherein adenocarcinoma of the prostate accounts for more than 95%, therefore, and usually said prostate cancer just refers to
Adenocarcinoma of the prostate.Prostate cancer be western countries' male's incidence of disease occupy first, fatal rate occupy deputy malignant tumour.
In recent years, the prostate-cancer incidence of China male was also raised year by year, and especially China just steps into aging society, following tens of
Probably there is prostate cancer onset peak in year.China's tumour registration area prostate-cancer incidence in 2012 is 9.92/10
Ten thousand, the 6th of the row male malignancy incidence of disease.Age of onset was in reduced levels before 55 years old, was gradually risen after 55 years old, sent out
Sick rate increases with advancing age, and the peak age is 70~80 years old.Familial inheritance type patients with prostate cancer age of onset is slightly
Early, the patient of age≤55 year old accounts for 43%.
Prostate cancer is due to the disguise of its physiological location, and early stage is often asymptomatic, tends to ignored, diagnosis discovery
Middle and advanced stage is in mostly afterwards, so as to lose the Best Times for the treatment of.
Chemical medicinal treatment occupies critical role in row gland cancer before the treatment.However, the chemotherapeutics of new production in recent years
Effect is bad always in terms of target-oriented drug and general toxicity, and this make it that active constituents of medicine is found from natural plants to be turned into
Develop the important means of antiprostate cancer.
Sea-buckthorn (Hippophae rhamnoides L.) also known as vinegar willow, black thorn, belong to Elaeangnaceae hippophae plant, extensively
NORTHWEST CHINA portion area is distributed in, is the traditional conventional crude drugs of China Tibetan medicine.At present, sea-buckthorn is allowed to make as medicine-food two-purpose crop
Used in industries such as food, medicines, it is with a wide range of applications.Hippophate flavone is the total of flavones in sea buckthorn fruit and Leaves of Hippophae L
Claim, different sources source and its flavone component of different activities position and content have a larger difference, but it is main all with quercetin aglycon,
Based on Isorhamnetin aglycon, Kaempferol and its glycoside.Flavonoid substances are one of topmost active component of sea-buckthorn, modern medicine
Prove, Hippophate flavone reducing blood lipid, it is hypoglycemic, improve myocardial hypoxia tolerance, it is anti-oxidant, antitumor in terms of have it is preferable
Physiological function.
Publication No. CN105982928A Chinese invention patent discloses fructus hippophae antineoplastic extract and combinations thereof
Medical usage and preparation method.A kind of Uygur medicine seabuckthorn fruit extract, Uygur medicine fructus hippophae is extracted through solvent supersonic, carried
Take after liquid concentration, use the silicagel column after silica gel post separation, loading, first with the dichloromethane that volume ratio is 100: 0-100: 10：First
Alcohol mixed solvent is eluted, after the dichloromethane with volume ratio 100: 12-100: 17：Methanol mixed solvent is eluted, and is with volume ratio
100: 12-100: 17 dichloromethane：The recovered solvent of eluent of methanol mixed solvent elution, it is the extract to dry.
Also disclose the Uygur medicine seabuckthorn fruit extract and prepare prevention and treatment breast cancer, lung cancer, stomach cancer, colon cancer, pancreas
Application in cancer, cervical carcinoma, glioma or liver cancer.
The pertinent literature that there is no Hippophate flavone at present is used for anti-prostate cancer is reported.
The content of the invention
The invention provides a kind of Hippophate flavone and its application, the Hippophate flavone is to be carried using the inventive method from sea-buckthorn
Take, there is certain curative effect to prostate cancer.
Hippophate flavone, is extracted by fructus hippophae and obtained, and the extracting method comprises the following steps：
(1) fructus hippophae obtains extract solution through organic solvent ultrasonic extraction；
(2) extract solution, which concentrates and removes organic solvent, obtains crude extract；
(3) crude extract obtains eluent through column chromatographic isolation and purification；
(4) eluent obtains the Hippophate flavone through sour water solution.
Step (1) is used for the active ingredient for tentatively extracting fructus hippophae, merges extract solution after can repeatedly extracting.Described sand
Organic solvent is 60%~80% ethanol solution in spine flavones, step (1).Described Hippophate flavone, temperature when ultrasonic is
50 DEG C~60 DEG C.Ultrasound assisted extraction, when ultrasonic, appropriate raising temperature is conducive to active ingredient to be dissolved into organic solvent.
Organic solvent in step (2) in removal step (1), can be by a variety of methods, such as vacuum distillation, or
Freeze-drying obtains powdered crude extract after concentration, is redissolved again with water when next step is isolated and purified.
In step (3), what column chromatography for separation was mainly removed is the materials such as carbohydrate, protein, tanning.The column chromatography is used
AB-8 macroreticular resins are used as filler.AB-8 type macroporous absorbent resins are styrene type low pole EVAs, and specific surface area is in 480-
520m2/ g or so, is adapted to the purifying of flavone compound；Other similar fillers also have D-101, HP-20 etc..Described sea-buckthorn
Flavones, macroporous absorbent resin uses 50% ethanol as eluting solvent when separating.During using AB-8 macroreticular resins as filler, on
After sample, first it is washed with water, then is washed with 10% ethanol, 30% ethanol gradient, washes away after impurity, 50% ethanol elution is finally used again, with
The component that 50% ethanol elution gets off is used for the sour water solution of next step.Certainly, concentration of alcohol when washing miscellaneous can suitably change, with
Clean impurity is defined, and the concentration of ethanol can also suitably change when eluting, and is advisable with that can elute object.
Hydrolyzate used in sour water solution is hydrochloric acid-methanol solution in described Hippophate flavone, step (4), dense by quality
Spend and 1: 3~5 mix acquisition by volume with methanol for 2%~6.5% hydrochloric acid.Sour water solution, can also use sulfuric acid or acetic acid
Deng other acid.Methanol is used as solvent, and other alcohol are also that can use with phase same-action, such as ethanol.Sour water solution is
Glycoside hydrolysis is turned into aglycon and glycosyl.
Before described Hippophate flavone, sour water solution, eluent is concentrated, removal solvent is freeze-dried.
Described Hippophate flavone, sour hydrolysis temperature is 60~80 DEG C, and hydrolysis time is 3~5h.
Described Hippophate flavone, by quality ratio, quercetin aglycon content are not less than 4.20%, Isorhamnetin Aglycones content
It is not less than 17.50%.
The medicine or health food of prevention or treatment prostate cancer are being prepared present invention also offers described Hippophate flavone
In application.
Hippophate flavone of the present invention is slightly carried by fructus hippophae through organic solvent, then through column chromatographic isolation and purification, then sour water solution is obtained
.Different extracting methods has large effect to the content of each active ingredient in Hippophate flavone.Hippophate flavone warp of the present invention
Research finds there is certain treatment and prevention effect to prostate cancer, can be used to prepare and prevents or treat prostate cancer
Medicine or health food.
Brief description of the drawings
Fig. 1 is mtt assay detection cytoactive result figure in embodiment 5, wherein, * is represented compared with control group, p<0.01,
With conspicuousness.
Fig. 2 monitors result figure of the Hippophate flavone to impact cell in real time for RTCA, wherein, curve a~h represents sea-buckthorn respectively
The μ g/mL of flavones concentration 0,0.78,1.56,3.13,6.25,12.5,25,50.
Result figure when Fig. 3 is Cell migration assay 24h, wherein, figure A is control group, and figure B Hippophate flavones concentration is 100 μ
G/mL, figure C Hippophate flavones concentration is 200 μ g/mL.
Fig. 4 is cell apoptosis assay result figure in embodiment 8, wherein, figure A is control group, and figure B is that Hippophate flavone concentration is
50μg/mL。
Fig. 5 is the statistical results chart of cell apoptosis assay in embodiment 8, wherein, " * ", which represents to have compared with control group, to be shown
Write sex differernce.
Fig. 6 is cell cycle experimental result picture in embodiment 9, wherein, figure A is control group, and figure B is that Hippophate flavone concentration is
50μg/mL。
Fig. 7 is the statistical results chart of cell cycle experiment in embodiment 9, wherein, " * ", which represents to have compared with control group, to be shown
Write sex differernce.
Fig. 8 is mtt assay detection cytoactive result figure in comparative example 1.
Embodiment
Experiment material and instrument：
Androgen Independent Prostate Cancer PC-3 cells (source：Shanghai Inst. of Life Science, CAS cell
Storehouse)；The ELIASAs of BIO-RAD 680 (BIO-RAD companies of the U.S.)；The real-time n cell functional analysis instrument (Ai Sen of RTCA S16
Biological (Hangzhou) Co., Ltd)；Tetrazolium bromide (MTT) (Beijing Lei Gen Bioisystech Co., Ltd)；6HT flow cytometries (the U.S.
BD companies)；Annexin V/PI cell apoptosis detection kits (Suo Laibao bio tech ltd)；DNA content (cell week
Phase) detection kit (Suo Laibao bio tech ltd)
Culture medium：Containing 10% hyclone and 1% streptomysin, (HyClone is public for the DMEM/F12 culture mediums of gibberellin
Department).
Embodiment 1
Dry fructus hippophae 100g is taken, 10 times of ethanol solutions of volume (1.0L) 70%, 55 DEG C of ultrasound assisted extraction 1h, weight is added
Multiple 3 times, filtering, merging filtrate, vacuum distillation removes ethanol, concentrating sample, it is freeze-dried after obtain fructus hippophae crude extract powder
Last 23.6g；Take above-mentioned fructus hippophae crude extract powder 10.0g, the 100mL that adds water to redissolve, take supernatant to cross AB-8 macroreticular resins, its
In, loading speed is 0.2BV/h, the evening of absorption one.Successively with 10%, 30%, 50% ethanol gradient elution, elution flow rate is
40mL/min, co-elute 1.5BV, retain 50% ethanol elution component, concentrate, freeze-drying；Take freeze-drying gained powder
1.0g, adds 600mL hydrolyzates, and hydrolyzate is mixed at 1: 4 by volume by mass concentration for 4% hydrochloric acid with methanol, and remaining is
Water, 70 DEG C of hydrolysis 4h, vacuum distillation removes methanol, and freeze-drying obtains Hippophate flavone sample.
Sampling is detected, with quercetin aglycon, Isorhamnetin aglycon standard items to reference substance, passes through high performance liquid chromatography
(HPLC) method is detected, wherein, quercetin aglycon content 5.20%, Isorhamnetin Aglycones content 19.02%.
Embodiment 2
Dry fructus hippophae 150g is taken, 10 times of ethanol solutions of volume (1.5L) 60%, 60 DEG C of ultrasound assisted extraction 1h, weight is added
Multiple 3 times, filtering, merging filtrate, vacuum distillation removes ethanol, concentrating sample, it is freeze-dried after obtain fructus hippophae crude extract powder
Last 40.18g；Take above-mentioned fructus hippophae crude extract powder 20.2g, the 200mL that adds water to redissolve, take supernatant to cross AB-8 macroreticular resins, its
In, loading speed is 0.2BV/h, the evening of absorption one.Successively with 10%, 30%, 50% ethanol gradient elution, elution flow rate is
40mL/min, co-elute 1.5BV, retain 50% ethanol elution component, concentrate, freeze-drying；Take freeze-drying gained powder
1.2g, adds 650mL hydrolyzates, and hydrolyzate is mixed to prepare at 1: 3 by volume by mass concentration for 6.5% hydrochloric acid with methanol,
Remaining is water, and 60 DEG C of hydrolysis 3h remove methanol, freeze-drying obtains Hippophate flavone sample.
Sampling is detected, with quercetin aglycon, Isorhamnetin aglycon standard items to reference substance, passes through high performance liquid chromatography
(HPLC) method is detected, wherein, quercetin aglycon content 4.98%, Isorhamnetin Aglycones content 20.87%.
Embodiment 3
Dry fructus hippophae 250g is taken, 10 times of ethanol solutions of volume (2.5L) 80%, 50 DEG C of ultrasound assisted extraction 1h, weight is added
Multiple 3 times, filtering, merging filtrate, vacuum distillation removes ethanol, concentrating sample, it is freeze-dried after obtain fructus hippophae crude extract powder
Last 60.2g；Above-mentioned fructus hippophae crude extract powder 30.0g is taken, 300mL distilled water is added and redissolves, take supernatant to cross AB-8 macropore trees
Fat, wherein, loading speed is 0.2BV/h, the evening of absorption one.Successively with 10%, 30%, 50% ethanol gradient elution, elution flow rate
For 40mL/min, co-elute 1.5BV, retain 50% ethanol elution component, concentrate, freeze-drying；The powder for taking freeze-drying to obtain
Last 2.0g, adds 1200mL hydrolyzates, and hydrolyzate is mixed to prepare at 1: 5 by volume by mass concentration for 2% hydrochloric acid with methanol,
Remaining is water, and 80 DEG C of hydrolysis 5h remove methanol, freeze-drying obtains Hippophate flavone sample.
Sampling is detected, with quercetin aglycon, Isorhamnetin aglycon standard items to reference substance, passes through high performance liquid chromatography
(HPLC) method is detected, wherein, quercetin aglycon content 5.21%, Isorhamnetin Aglycones content 19.47%.
Embodiment 4
Dry fructus hippophae 200g is taken, the ethanol solutions of 2.0L 70% are added, 60 DEG C of ultrasound assisted extraction 1h are repeated 3 times, mistake
Filter, merging filtrate, vacuum distillation remove ethanol, concentrating sample, it is freeze-dried after obtain sea-buckthorn pomace crude extract powder
45.8g；Above-mentioned sea-buckthorn pomace crude extract powder 15.0g is taken, 150mL distilled water is added and redissolves, take supernatant to cross AB-8 macropore trees
Fat, wherein, loading speed is 0.2BV/h, the evening of absorption one.Successively with 0,10%, 30%, 50% ethanol gradient elution, elution stream
Speed is 40mL/min, co-elute 1.5BV, retains 50% ethanol elution component, is concentrated, freeze-drying；Take above-mentioned dried powder
2.0g, adds 1.2L hydrolyzates, and hydrolyzate is mixed at 1: 4 by volume by mass concentration for 6.5% hydrochloric acid with methanol, and remaining is
Water, 60 DEG C of hydrolysis 3h, removes methanol, and freeze-drying obtains Hippophate flavone sample.
Using quercetin aglycon, Isorhamnetin aglycon standard items as reference substance, examined by high performance liquid chromatography (HPLC)
Survey, wherein quercetin aglycon content 4.20%, Isorhamnetin Aglycones content 17.50%.
Embodiment 5
The PC-3 cells newly recovered are taken in 37 DEG C, 5%CO2Cultivate, after cytotostatic is passed on 2 times, be used in incubator
Subsequent experimental.
Take the logarithm the PC-3 cells in growth period, after being digested with 0.25% pancreatin, count, adjustment cell concentration is 2 × 104
Individual/mL, 200 μ L cell suspending liquids of inoculation are in 96 well culture plates, in 37 DEG C, 5%CO2Incubator culture 24h makes cell attachment.More
Change nutrient solution, the μ L of nutrient solution 200 of Hippophate flavone sample (prepared by embodiment 1) are added per hole (sample 0.5%DMSO dissolves).
Blank group and sample sets are set, and every group of 6 multiple holes, concentration is respectively 50,100,150,200 μ g/mL, continue to cultivate 24h, 48h,
72h.4h before terminating reaction, the μ L of 5g/L MTT 20 are added per hole and continue to cultivate 4h.Nutrient solution in hole is abandoned in suction, and DMSO is added per hole
150 μ L, vibration 10min make after purple crystal is completely dissolved, in 570nm mensuration absorbances.
Versus cell vigor=(dosing group OD values-background OD values)/(blank control group OD values-background OD values)
Wherein, blank control group is 0.5%DMSO culture medium group；Background group is not add groups of cells.
Experimental data is represented with mean ± SD, using SPSS software analysis, and statistical check is examined with t and single factor test F is examined,
Insolation level p=0.01.
As a result as shown in figure 1, compared to control group, the Hippophate flavone of doses can substantially suppress the work of PC-3 cells
Power, and in dosage and time dependence.The IC of each sample treatment time section50Value is shown in Table 1.
Table 1
| Time | 24h | 48h | 72h |
| IC50(μg/mL) | 91.075 | 49.742 | 27.921 |
Embodiment 6
N cell analytical technology is by microelectronics cell to RTCA (Real Time Cellular Analysis) in real time
Sensor chip is incorporated into surface and migrates the micro- of plate with the bottom of the cell detection plate of growth or cellular infiltration suitable for cell attachment
Pore membrane, to build real-time, dynamic, the quantitatively tracking change such as cellular morphology and Proliferation, Differentiation cell impedance detection sensing system
System.The bottom of Tissue Culture Plate E-Plate16 in this experiment is integrated with micro- golden electronic sensor chip, and adherent growth is in micro- electricity
The cell state on pole surface, which changes, can cause the change of electrode impedance, and this change changes over related to the real-time status of cell
Property, thus can the measure based on electrical impedance represent cell state with cell index (Cell Index, CI), realize prison in real time
Survey.
Take the logarithm the PC-3 cells in growth period, be inoculated in the density in 5000/ hole in RTCA16 orifice plates, cultivate 24h, added
The μ L of sample solution 50 of various concentrations, continue to cultivate 72h, in real time the change of monitoring electrode impedance, and each concentration sets 3 repetitions.
As a result as shown in Fig. 2 compared to control group, the Hippophate flavone of doses can substantially suppress the propagation of PC-3 cells, and be in
Dosage and time dependence, influence of the Hippophate flavone to cell is monitored by RTCA in real time, finds the Hippophate flavone sample of low dosage
Product (6.25 μ g/mL) are the state that can significantly modify cell, cause the decline of cell index.
Embodiment 7
After the PC-3 cells of exponential phase digest through pancreatin, cell concentration is adjusted, appropriate (1 × 10 is added5It is individual) cell
Into 6 orifice plates, after cell attachment after the culture of a period of time.When cell length to when being paved with 6 orifice plate substantially, existed with 10 μ L pipette tips
The cell come off after cut, cut with 3 removals of PBS is carried out on 6 orifice plates.Various concentrations are added into 6 orifice plates respectively
Hippophate flavone sample (prepared by embodiment 1), while setting blank group, is put into incubator and continues to cultivate.Not enter in 12h, 24h, 36h
Capable migration situation of the observation of cell in scribe area of taking pictures.
As a result as shown in figure 3, compared to control group, 100 μ g/mL Hippophate flavone can substantially suppress the horizontal stroke of PC-3 cells
To migration, when concentration reaches 200 μ g/mL, PC-3 cells lose the ability of migration substantially.
Embodiment 8
Annexin V are a kind of cardiolipin binding proteins, can be turned in apoptosis process outside film phosphatidyl silk ammonia
Sour high-affinity specific bond, under the exciting of blue light, sends green fluorescence, so as to distinguish apoptotic cell and normal thin
Born of the same parents；Propidium iodide (PI) is a kind of nucleic acid dye, and it can not penetrate intact cell film, but to apoptosis late cell and dead cell
Damaged cell membrane can be penetrated, and make the red dye of nucleus.Annexin V are matched with PI and used, can be by apoptosis early stage, apoptosis
The cell differentiation in late period comes.
Take the logarithm the PC-3 cells in growth period, adjust cell concentration, be inoculated in the density in 20000/ hole in 6 orifice plates, trained
24h is supported, former culture medium is discarded, adds and contains various concentrations (25,50,100 μ g/mL) Hippophate flavone sample (prepared by embodiment 1)
Culture medium, continue cultivate 48h.Cell is collected according to the operation on Annexin V/PI cell apoptosis detection kit specifications
After dyeing, flow cytometry detection Apoptosis situation.
As a result as shown in figure 4, wherein Fig. 4 A are the control group of not dosing, Fig. 4 B are to add 50 μ g/mL Hippophate flavones
Experimental group.Left lower quadrant represents normal cell, and left upper quadrant represents mechanical damage cell, and it is thin that right lower quadrant represents early apoptosis
Born of the same parents, right upper quadrant represents non-viable apoptotic cell or non-viable non-apoptotic cell.
Statistics with Normal group as shown in figure 5, after processing 48h, compared, early apoptosis of cells rate in each experimental group
Although without significant change, late apoptic (necrosis) rate and total apoptosis rate all significantly raise (p<0.01), and in certain agent
Dependence is measured, illustrates that Hippophate flavone prepared by embodiment 1 has the effect for inducing PC-3 Apoptosis.
Embodiment 9
Cell cycle refers to that cycling cell terminates to mitosis next time to terminate to be undergone from a mitosis
Whole process, including G0/G1 phases, S phases, G2/M phases.Fluorescent dye propidium iodide PI can be combined with intracellular DNA, cell
Its DNA content of each period is different so as to cause the fluorescent dye combined different, and flow cytometer can be according to fluorescence intensity
Difference detects DNA content, so as to judge cell cycle distribution.
Take the logarithm the PC-3 cells in growth period, adjust cell concentration, be inoculated in the density in 20000/ hole in 6 orifice plates, trained
24h is supported, former culture medium is discarded, adds and contains various concentrations (25,50,100 μ g/mL) Hippophate flavone sample (prepared by embodiment 1)
Culture medium, continue cultivate 48h.Cell dye is collected according to the operation on DNA content (cell cycle) detection kit specification
After color, flow cytometry detection cell cycle stage.
As a result as shown in fig. 6, wherein Fig. 6 A are the control group of not dosing, Fig. 6 B are to add 50 μ g/mL Hippophate flavones
Experimental group, each peak represents different cell cycle phases.
Statistics with Normal group as shown in fig. 7, after Hippophate flavone sample effect PC-3 cells 48h, compared, sample
Cell G2/M phase ratios significantly raise (p in group<0.01), and in doses dependence.Therefore sample is mainly by by cell
Cycle Arrest in the G2/M phases, delay cell complete mitosis process, One-step delay of going forward side by side enter next cell cycle when
Between, the synthesis of influence material and DNA replication dna, so as to suppress PC-3 cells propagation.Influence DNA is synthesized and is delayed entering for cell cycle
OK, so as to suppress PC-3 cells propagation.
Comparative example 1
Hippophate flavone does not hydrolyze the preparation of sample, the preparation technology of Hippophate flavone：Dry fructus hippophae is taken, 10 times of volumes are added
70% ethanol solution, 55 DEG C of ultrasound assisted extraction 1h, is repeated 3 times, filtering, merging filtrate, concentration, it is freeze-dried after obtain sand
Spine fruit crude extract powder；Above-mentioned fructus hippophae crude extract powder is taken, add water redissolution, take supernatant to cross AB-8 macroreticular resins.Wherein, on
Sample speed is 0.2BV/h, the evening of absorption one.Successively with 10%, 30%, 50% ethanol gradient elution, elution flow rate is 40mL/min,
Co-elute 1.5BV, retains 50% ethanol elution component, concentrates, and freeze-drying obtains the non-hydrolyzation sample of Hippophate flavone.Wherein survey
Obtain quercetin aglycon content 0.1%, Isorhamnetin Aglycones content 0.08%.
Mtt assay detects the method be the same as Example 5 of cell viability.
Fig. 8 is shown in influence of the non-hydrolyzation sample of Hippophate flavone to PC-3 cell viabilities, compared to control group, 250 μ g/mL sand
Spine sample remains to promote the growth of PC-3 cells, in the absence of cel l proliferation is suppressed, and thus analysis is understood, the sand in the present invention
Spine flavones has to pass through the effect that hydrolysis competence exertion suppresses prostatic hyperplasia.
Claims (10)
1. Hippophate flavone, is extracted by fructus hippophae and obtained, it is characterised in that the extracting method comprises the following steps：
(1) fructus hippophae obtains extract solution through organic solvent ultrasonic extraction；
(2) extract solution, which concentrates and removes organic solvent, obtains crude extract；
(3) crude extract obtains eluent through column chromatographic isolation and purification；
(4) eluent obtains the Hippophate flavone through sour water solution.
2. Hippophate flavone as claimed in claim 1, it is characterised in that organic solvent is 60%~80% second in step (1)
Alcoholic solution.
3. Hippophate flavone as claimed in claim 1, it is characterised in that temperature when ultrasonic is 50 DEG C~60 DEG C.
4. Hippophate flavone as claimed in claim 1, it is characterised in that the column chromatography is using AB-8 macroreticular resins as filling out
Material.
5. Hippophate flavone as claimed in claim 1, it is characterised in that use 50% ethanol molten as eluting during column chromatography for separation
Agent.
6. Hippophate flavone as claimed in claim 1, it is characterised in that hydrolyzate used by sour water solution is hydrochloric acid-first in step (4)
Alcoholic solution, 1: 3~5 acquisition is mixed by mass concentration by volume for 2%~6.5% hydrochloric acid with methanol.
7. Hippophate flavone as claimed in claim 1, it is characterised in that before sour water solution, eluent is concentrated, removal is freeze-dried
Solvent.
8. Hippophate flavone as claimed in claim 1, it is characterised in that sour hydrolysis temperature is 60~80 DEG C, sour hydrolysis time is 3
~5h.
9. Hippophate flavone as claimed in claim 1, it is characterised in that by quality ratio, quercetin aglycon content is not less than
4.20%th, Isorhamnetin Aglycones content is not less than 17.50%.
10. the Hippophate flavone as described in claim 1~9 is any is preparing the medicine or health care food of prevention or treatment prostate cancer
Application in product.
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Application publication date: 20170714 |