CN104667102B - The Preparation method and use of galangal aerial parts volatile oil - Google Patents
The Preparation method and use of galangal aerial parts volatile oil Download PDFInfo
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Abstract
The present invention is suitable for pharmaceutical technology field, provides a kind of Preparation method and use of galangal aerial parts volatile oil.The galangal aerial parts volatile oil contains following components：1H imidazole, 4,5 dihydros 2 (benzyl), 3 cyclohexene, 1 methanol, α, α, 4 trimethyls, bicyclic [3.1.1] 2 heptene, 2,6 dimethyl 6 (4 methyl), naphthalene, 1,2,3,4,4a, 5,6,7 methyl of 8a octahydros, aromadendrene, eucalyptol, acetic acid fennel seeds ester, benzenpropanal, Bronyl acetate, benzylacetone, bicyclic 2 ketone of [2.2.1] heptane is weight percent >=50% of above compound component in terms of 100% by the gross weight of galangal aerial parts volatile oil.
Description
Technical field
The invention belongs to pharmaceutical technology field more particularly to the Preparation method and uses of galangal aerial parts volatile oil.
Background technology
Tumour is to threaten the first big killer of human life and health.Currently, the treatment for cancer, mostly uses greatly chemistry
The method of drug therapy, although chemotherapy can kill or inhibit the growth of tumour cell to a certain extent, however,
Existing antineoplastic also have while killing tumor cell, to normal cell it is larger lethal, toxicity it is larger and
Drug resistance is easy tod produce, tumour is still to seriously threaten one of the disease of people's life and quality of life.Seek new, efficiently low
The antitumor drug of poison becomes the important topic studied now.Natural drug is proposed from nature animals and plants or microorganism
The active medicine separated has many advantages, such as that toxic side effect is small, is not likely to produce drug resistance, is safe and effective, therefore, in crude drug
The ingredient that treatment tumour is found in object is expected to carry out new hope for the treatment zone of tumour.
Galangal system zingiberaceous plant galangal (Alpinia officinarum Hance), also known as galingal, version over the years
《Chinese Pharmacopoeia》Record.For galangal main product in provinces such as Guangdong, Guangxi, Yunnan, Hainan, Zhanjiang is China galangal road
Ground is most strong and the producing region most concentrated.Southern medicine galangal can be traced back to as Zhanjiang specialty, the galangal scale plantation of Xuwen County
16 beginnings of the century were then established in 1998 as southern medicine galangal Development Base, and from creating so far, cultivated area is stablized always
40000 mu or so, main integrated distribution is in big pool Hua Linyi bands.It is main using galangal under ground portion as medicine-food two-purpose at present
Material, and galangal aerial part is mainly handled in a manner of waste.For galangal aerial part research report compared with
It is few, and relevant report there have been no to the research of galangal aerial parts volatile oil.
Invention content
The purpose of the present invention is to provide a kind of galangal aerial parts volatile oils, it is intended to solve existing galangal overground part
The problem of being allocated as offal treatment and ingredient and its pharmacological research do not carried out to galangal aerial parts volatile oil.
Another object of the present invention is to provide a kind of preparation methods of galangal aerial parts volatile oil.
Another object of the present invention is to provide a kind of galangal aerial parts volatile oils as pharmaceutical preparation or medicine group
Point, the application in anti-curing cancers.
It is still another object of the present invention to provide a kind of galangal aerial parts volatile oils as pharmaceutical preparation or medicine group
Point, the application in preventing osteoporosis.
The invention is realized in this way a kind of galangal aerial parts volatile oil, the volatile oil contain following compounds
Component：1H- imidazole, 4,5- dihydro -2- (benzyl), 3- cyclohexene -1- methanol, α, α, 4- trimethyls, bicyclic [3.1.1] 2-
Heptene, 2,6- dimethyl -6- (4- methyl), naphthalene, 1,2,3,4,4a, 5,6,8a- octahydro -7- methyl, aromadendrene, eucalyptol, second
Sour fennel seeds ester, benzenpropanal, Bronyl acetate, benzylacetone, bicyclic [2.2.1] heptane -2- ketone are waved with galangal aerial part
The gross weight of hair oil is 100% meter, weight percent >=50% of above compound component.
Correspondingly, a kind of preparation method of galangal aerial parts volatile oil, includes the following steps：
Galangal aerial part is taken, after crushing processing, galangal aerial part 10-15 times of water measured of weight is added,
Steam distillation is carried out, distillate is obtained；
The distillate is detached with oil water separator, obtains galangal aerial parts volatile oil.
And a kind of galangal aerial parts volatile oil is as pharmaceutical preparation or drug component, answering in anti-curing cancers
With.
A kind of galangal aerial parts volatile oil is as pharmaceutical preparation or drug component, answering in preventing osteoporosis
With.
Galangal aerial parts volatile oil provided by the invention is extracted using discarded galangal aerial part as raw material
It obtains, inhibiting effect can be generated to tumour, meanwhile, also there is good function of resisting osteoporosis.The galangal overground part
Divide volatile oil in addition to active anticancer, also there is good function of resisting osteoporosis.Made with galangal aerial parts volatile oil
For antitumor and osteosporosis resistant medicament, there is fine curative effect, and toxic side effect is few, is not likely to produce drug resistance.
The preparation method of galangal aerial parts volatile oil provided by the invention directly utilizes discarded galangal overground part
It is allocated as raw material, turning waste into wealth；Preparation method is simple for this, easy to operate, it can be achieved that large-scale production.
Description of the drawings
Fig. 1 is various concentration galangal of embodiment of the present invention aerial parts volatile oil (0-2.5 μ L/mL) to 6 kinds of cancer cells
The inhibiting effect figure of (A-549, NCI-H460, NCI-H23, MCF-7, HL-60 and Hela) proliferation；
Fig. 2 is various concentration galangal aerial parts volatile oil of the embodiment of the present invention (0-10mL/kg) to human lung carcinoma cell
The growth inhibition effect of A549 transplanted tumor in nude miceResult figure；
Fig. 3 is that various concentration of the embodiment of the present invention (0-0.3125 μ L/mL) galangal aerial parts volatile oil acts on for 24 hours
Afterwards, the influence schematic diagram to A549 Apoptosis；
Fig. 4 is the embodiment of the present invention by DAPI nuclear targetings, observes the various concentration (0-0.3125 μ L/mL) height
Galingal aerial parts volatile oil acts on for 24 hours the influence schematic diagram to A549 nucleus afterwards；
Fig. 5 is that the embodiment of the present invention dyes detection various concentration (0-0.3125 μ L/mL) galangal overground part by JC-1
Divide after volatilizing oil processing A549 cells for 24 hours, the influence schematic diagram to mitochondrial membrane potential；
Fig. 6 is various concentration of the embodiment of the present invention (0-0.15625 μ L/mL) galangal aerial parts volatile oil to tire mouse head
The influence schematic diagram of lid bone osteoblastic proliferation；
Fig. 7 is various concentration of the embodiment of the present invention (0-0.15625 μ L/mL) galangal aerial parts volatile oil to newborn big
The influence schematic diagram of mouse osteoblast alkaline phosphatase activities.
Fig. 8 is that various concentration of the embodiment of the present invention (0-0.15625 μ L/mL) galangal aerial parts volatile oil acts on 20 days
Afterwards, influence situation schematic diagram neonate rat osteoblast bone tubercle formed.
Specific implementation mode
In order to make technical problems, technical solutions and advantageous effects to be solved by the present invention be more clearly understood, below in conjunction with
Accompanying drawings and embodiments, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
An embodiment of the present invention provides a kind of galangal aerial parts volatile oil, the volatile oil contains following compound groups
Point：1H- imidazole, 4,5- dihydro -2- (benzyl), 3- cyclohexene -1- methanol, α, α, 4- trimethyls, bicyclic [3.1.1] 2- heptan
Alkene, 2,6- dimethyl -6- (4- methyl), naphthalene, 1,2,3,4,4a, 5,6,8a- octahydro -7- methyl, aromadendrene, eucalyptol, acetic acid
Fennel seeds ester, benzenpropanal, Bronyl acetate, benzylacetone, bicyclic [2.2.1] heptane -2- ketone are volatilized with galangal aerial part
The gross weight of oil is 100% meter, weight percent >=50% of above compound component.
In the embodiment of the present invention, the galangal aerial parts volatile oil is extracted from galangal aerial part
Volatile oil component, the galangal aerial part refer to the stem and leaf part on galangal ground or more.The galangal aerial part
In volatile oil, the composition and content of each ingredient can with extracting method and its technological parameter, the change difference of human factor,
In the embodiment of the present invention, the galangal aerial parts volatile oil mainly contains following compounds, and preferred content is as described below：
It is in terms of 100% by the gross weight of galangal aerial parts volatile oil, the weight percent of above compound component >=
50%.
As further preferred embodiments, in the galangal aerial parts volatile oil, also contain following compound components,
β-carypohyllene, α-humulene, 1H- cyclopropyl [e] decahydronaphthalene azulenes -1,1,7- trimethyls, α-humulene, α-farnesene,
Decahydronaphthalenes -4a- methyl-1s-methylene -7- methyl, naphthalene, 1,2,3,5,6,8a- hexahydro -4,7- dimethyl, naphthalene, 1,2- dihydro -
1,1,6- trimethyl, caryophyllene oxide, azulenes, 1,2,3,3a, 4,5,6,7- octahydros-Isosorbide-5-Nitrae-dimethyl, decahydronaphthalenes-
4a- methyl-1s-methylene -7- (1- Methylethyls), α-santalol, bicyclic [3.1.1] hept-2-ene", 2,6- dimethyl -6- (4-
Methyl), 3,6- bis- (4- tolyls) -1,2- dialins, each component preferred content is as described below：
Be in terms of 100% by the gross weight of galangal aerial parts volatile oil, the weight percent of each compound component it
With >=22%.
In the embodiment of the present invention, each compound component in the galangal aerial parts volatile oil, using gas chromatography mass spectrometry
(GC-MS) it is measured, and is obtained by standard items comparison and its computer search identification.
Galangal aerial parts volatile oil provided in an embodiment of the present invention has preferable antitumor and anti-osteoporosis
Effect, and its high-efficiency low-toxicity are used as the pharmaceutical preparation or component for the treatment of tumour or osteoporosis.
Galangal aerial parts volatile oil described in the embodiment of the present invention can be obtained by following preparation methods, certainly,
The method that above-mentioned galangal aerial parts volatile oil can be prepared by other obtains.
Correspondingly, an embodiment of the present invention provides a kind of preparation method of galangal aerial parts volatile oil, including it is as follows
Step：
S01. galangal aerial part is taken, after crushing processing, is added what 10-15 times of galangal aerial part weight was measured
Water carries out steam distillation, obtains distillate；
S02. the distillate is detached with oil water separator, obtains galangal aerial parts volatile oil.
Specifically, in above-mentioned S01 steps, galangal aerial part of the embodiment of the present invention is taken, is dried after being cleaned.In order to
So that following step in it is more abundant to the extraction of the galangal aerial parts volatile oil, by the galangal aerial part into
Row pulverization process, the pulverization process can be used to crush in pulverizer and realize.As a preferred embodiment, the height after pulverization process
Galingal aerial part carries out sieving processing, obtains the galangal aerial part of the mesh of grain size≤40.The galangal of the particle size
Upper part, facilitates following step to extract it, improves the yield of galangal aerial parts volatile oil.
Typically for the extraction of volatile oil component in natural drug, steam distillation can be used, solvent extraction method, surpass
A variety of methods such as critical extraction.In the embodiment of the present invention, the extraction for galangal aerial parts volatile oil, using vapor
Distillation not only have the characteristics that it is easy to operate, of low cost, pollution-free, be suitable for mass produce, can also fully extract height
Galingal aerial parts volatile oil reduces the loss of volatile oil component.It, can be right before steam distillation in order to improve extraction efficiency
The galangal aerial part of above-mentioned flour carries out immersion treatment, and soaking time was advisable with 2-48 hours.
It tests and finds by inventor, as a preferred embodiment, the temperature of the steam distillation is 80-120 DEG C, distillation
Time is 2-6 hours.The vapo(u)rizing temperature and distillation time can preferably improve extraction efficiency, ensure volatile oil in the galangal
The abundant extraction of ingredient, meanwhile, prevent temperature is excessively high from causing volatile oil component excessively to volatilize to cause damages.
In above-mentioned S02 steps, the distillate is carried out by water-oil separating using oil water separator, after removing water phase, is obtained
The galangal aerial parts volatile oil.As a preferred embodiment, it volatilizees to preferably remove above-mentioned galangal aerial part
Moisture in oil, can be dried the galangal aerial parts volatile oil processing, and the drying process preferably uses drying
Agent is realized.As specific embodiment, the drier includes but not limited to anhydrous sodium sulfate, other with described in the embodiment of the present invention
The not aitiogenic drier of galangal aerial parts volatile oil, is used equally for the embodiment of the present invention.
As further preferred embodiments, the quality of the galangal aerial parts volatile oil obtained in order to prevent not by
Destruction-such as ingredient volatilization decomposes in light, and can will be protected from light processing after galangal aerial parts volatile oil sealing, and be placed in
It is stored in refrigerator, the temperature of refrigerator is advisable with≤4 DEG C.
The preparation method of galangal aerial parts volatile oil provided in an embodiment of the present invention, it is not only simple and practicable, it is easy behaviour
Make, and the galangal aerial parts volatile oil yield up to it is more than 1.5% (v/w dry weights), it can be achieved that large-scale production, and
Discarded galangal aerial part can be directly utilized, volatile oil is therefrom extracted, turns waste into wealth.
The embodiment of the present invention additionally provides a kind of conduct pharmaceutical preparation or the drug of the galangal aerial parts volatile oil
Component, the application in anti-curing cancers.As a preferred embodiment, the galangal aerial parts volatile oil as pharmaceutical preparation or
Drug component is in anti-curing cancers in application, further including pharmaceutically acceptable carrier.
It is tested by internal and external test, test cell line and subcellsular level, to galangal described in the embodiment of the present invention
Upper part volatile oil carries out the Effect study of cancer cell multiplication, cancer cell early apoptosis, nucleus and mitochondria, the results showed that institute
It states galangal aerial parts volatile oil and shows very strong inhibitory activity to cancer cell multiplication in vivo and in vitro；Meanwhile the height
Galingal aerial parts volatile oil to the test result of cancer cell early apoptosis, nucleus and mitochondria with showing the galangal
Upper part volatile oil is by caused by mitochondria pathway cancer cell specific induction of apoptosis to the inhibiting effect of cancer cell multiplication.The present invention
Galangal aerial parts volatile oil described in embodiment can be used for preventing the pharmaceutical preparation or drug component of Cancerous disease.
And the embodiment of the present invention additionally provides a kind of galangal aerial parts volatile oil as pharmaceutical preparation or medicine
Object component, the application in preventing osteoporosis.As a preferred embodiment, the galangal aerial parts volatile oil is as drug
Preparation or drug component are further including pharmaceutically acceptable carrier when being applied in preventing osteoporosis.The galangal is on the ground
Part volatile oil can promote differentiation and the mineralising of osteoblast, have the function of promoting the formation of osteoblast bone tubercle, can be used for
Prevention to bone loss disorders.
In the embodiment of the present invention, as pharmaceutical preparation or the galangal overground part of the anti-curing cancers of drug component or osteoporosis
Divide volatile oil, the galangal aerial parts volatile oil described in the embodiment of the present invention can be made, or press preparation side of the embodiment of the present invention
The galangal aerial parts volatile oil that method obtains.
The galangal aerial parts volatile oil provided in an embodiment of the present invention, in addition to having good antitumor action
Outside, also there is good function of resisting osteoporosis.In addition, the anti-curing cancers of the galangal aerial parts volatile oil or osteoporosis
When, have the advantages that efficient, low toxicity, is expected to as antitumor and osteosporosis resistant medicament new in the market.
It is illustrated with reference to specific embodiment.
Embodiment one
A kind of preparation method of galangal aerial parts volatile oil, includes the following steps：
S11. dry galangal aerial part is taken, after crushing processing with pulverizer, crosses 40 mesh sieve.Weigh 1000-
Galangal aerial part 10-15 times of water measured of weight is added in the above-mentioned galangal aerial parts of 1500g, carries out steam distillation, obtains
To distillate；Wherein, the temperature of the steam distillation is 80-120 DEG C, and distillation time is 2-6 hours；
S12. it detaches the distillate with oil water separator and uses anhydrous slufuric acid after removing the water phase in the distillate
Processing is dried in sodium, obtains galangal aerial parts volatile oil, is kept in dark place in 4 DEG C of refrigerator interior sealings；Wherein, described anhydrous
The adding proportion of sodium sulphate is：The galangal aerial parts volatile oil volume：Anhydrous sodium sulfate weight=(20-30) ml：(5-
10)g。
The galangal aerial parts volatile oil yield up to 1.5% (v/w dry weights) more than.
Embodiment two
The galangal aerial parts volatile oil that above-described embodiment one obtains, using Japanese SHIMADZU GCMS-QP2010 types
Gas chromatography-mass spectrum (GC-MS) combined instrument analyzes galangal aerial parts volatile oil obtained, and test condition is such as
Under：
GC conditions：HP-Innowax capillary columns (30m × 0.25mm × 0.25 μm)；240 DEG C of injector temperature；It is diverted into
Sample, split ratio 20:1；1 μ L of volatile oil sample size；Carrier gas (99.999% high-pure helium) constant linear velocity 40cm/sec；Purge stream
Measure 3mL/min.Temperature program：50 DEG C of initial temperature keeps 2min；Again with 10 DEG C/min temperature programmings to 280 DEG C, keep
10min。
MS conditions：Electron bombardment ionization source (Electron Impact, EI), electron energy 70eV, 200 DEG C of ion source temperature,
220 DEG C of interface temperature.When choosing SCAN patterns, mass scan range is 30-450 (m/z), solvent delay 2.5min.
Similar to search is carried out in NIST databases according to retention time, while to each compound mass spectrum, each component is carried out
Qualitative analysis, relative amount are calculated using area normalization method.It reflects altogether from the galangal aerial parts volatile oil
53 compounds are defined, retention index, title and the weight percentage of 53 each compound are shown in Table 1,53 eachization
The sum of the weight for closing object accounts for 93.46% (wt) of the galangal aerial parts volatile oil total content.Wherein main compound has
1H- imidazole, 4,5- dihydro -2- (benzyl), 3- cyclohexene -1- methanol, α, α, 4- trimethyls, bicyclic [3.1.1] 2- heptene,
2,6- dimethyl -6- (4- methyl), naphthalene, 1,2,3,4,4a, 5,6,8a- octahydro -7- methyl, aromadendrene, eucalyptol, acetic acid fennel fruit
Fragrant ester, benzenpropanal, Bronyl acetate, benzylacetone, bicyclic [2.2.1] heptane -2- ketone etc..
1 galangal aerial parts volatile oil main chemical compositions of table
Three galangal aerial parts volatile oil antitumaous effect of embodiment
1.MTT cytotoxic activities are tested
Take 6 kinds a concentration of 5 × 104Cancer cell A-549, NCI-H460, NCI-H23, MCF-7, HL-60 of a/mL and
Hela is respectively placed in 96 well culture plates, using the RPMI1640 culture medium cell suspensions containing 10% fetal calf serum, in 37 DEG C of items
Under part, 5%CO2It is cultivated for 24 hours in incubator.Experimental group be separately added into ultimate density be respectively 2.5 μ L/mL, 1.25 μ L/mL,
The galangal aerial parts volatile oil of 0.625 μ L/mL, 0.3125 μ L/mL and 0.15625 μ L/mL, control group are then added in equal volume
Acetone, every group of 5 holes set 37 DEG C, after 5%CO2 incubators culture 48 hours, discard supernatant liquid, are added that 200 μ L are fresh to be matched per hole
The serum-free medium of the MTT containing 50mg/mL of system continues to cultivate 4h under the conditions of 37 DEG C.Liquid is discarded supernatant, 100 μ L are added
DMSO, after shaking mixing, with microplate reader using 450nm as the OD values of reference wavelength, measurement wavelength 550nm.Drug is calculated as follows
Inhibiting rate is obtained to growth of tumour cell, experiment 3 times is repeated, is averaged.
Growth of tumour cell inhibiting rate %=(1- experimental groups mean OD value/control group mean OD value) × 100%
Cytotoxic activity experimental result is shown, compared with Normal group, galangal overground part described in the embodiment of the present invention
Divide the tumor cell proliferation rate that volatile oil acts on 48 hours on a declining curve, and as the galangal aerial part volatilizees
The raising of oily farmland, tumor cell proliferation rate decline obviously, and experimental result is as shown in Fig. 1.
2. galangal aerial parts volatile oil is to the growth inhibition effect of human lung cancer cell A549's transplanted tumor in nude mice
BALB/C/nu strain Female nude mices, 6 week old are raised and are tested in SPF grades of barrier systems.The present invention is real
It applies example and establishes animal tumor model using A549 cells, using culture cell allotransplantation method, carry out internal efficacy of medicine observing.Greatly
After measuring subculture typeⅡ pneumocyte and collecting cell, diluting cells suspension to a concentration of 2 × 107/ mL is inoculated with 0.1mL
It is subcutaneous in the right armpit of nude mouse.The 8th day after inoculation, there is apparent tumor tissue block at nude inoculation position, is considered as and models successfully, tumor formation
Rate basically reaches 100%.Wait for tumour growth to about 100mm3When, experiment nude mouse is randomly divided into five groups, solvent glycerol control
Group, galangal aerial parts volatile oil administration group (low dosage 1mL/kg, middle dosage 5mL/kg, high dose 10mL/kg), cis-platinum sun
Property control group, every group 10, each group be injected intraperitoneally medication observe 20 days, the next day be administered.All nude mices normally drink during administration
Food, drinking-water observe its quality of life and measure record weight and tumour growth volume change situation.Last dose for 24 hours after with neck
Vertebra dislocation method puts to death animal and terminates to test, and claims nude mice weight, knurl weight respectively, measures tumorous size, and calculate its tumour inhibiting rate.Using
SPSS15.0 software statistics analyze experimental data and carry out therapeutic evaluation, as a result see shown in attached drawing 2.The experimental results showed that galangal
Volatile oil administration group can obviously inhibit the growth of tumour, reduce animal tumor mass, hence it is evident that improve tumour inhibiting rate.High dose group 10mL/kg,
The weight of knurl is significantly lower than positive controls cis-platinum group.Changes of weight situation after observation medication, cis-platinum medication group nude mice are thin
Small, weight is decreased obviously, and illustrates the toxic side effect for having certain, and galangal aerial parts volatile oil medication group and control group phase
Than without apparent difference, it is seen that its toxic side effect is relatively low.Generally speaking, galangal volatile oil 10mL/kg, 5mL/kg,
When 1mL/kg dosage, there is certain inhibiting effect to human lung cancer cell A549's transplanted tumor in nude mice, and is in dose-effect relationship.
3. galangal aerial parts volatile oil influences to test on apoptosis of tumor cells
The galangal aerial parts volatile oil is implemented using the present invention, using Annexin V-FITC/PI methods, to people
The influence of body Increase Apoptosis of Lung Cancer Cells is evaluated.
Wherein Annexin V-FITC/PI methods are as follows：
(1) by a concentration of 2 × 105The cell inoculation in a/hole contains 1640 culture mediums of 10% fetal calf serum in 6 orifice plates
After overnight incubation, it is changed to containing different galangal aerial parts volatile oil concentration (0 μ L/mL, 0.078125 μ L/mL, 0.15625 μ L/
ML, 0.3125 μ L/mL) DMEM culture for 24 hours；
(2) through without EDTA pancreatin digestion after, merges with the cell in cell culture supernatant, centrifugation 1500 × g, 4
DEG C centrifugation 5min collect；
(3) resuspension of 190 μ L Annexin V-FITC combination liquid is added in cell；
(4) add 5 μ L mixings of Annexin V-FITC；
(5) room temperature is protected from light 10min, and 1000g centrifuges 5min, abandons supernatant, and 190 μ L Annexin V-FITC knots are added
Liquid is closed to be resuspended；
(6) 10 μ L propidium iodide stain liquid are added, gently mixing, ice bath avoid light place；
(7) it in 1h, is detected on flow cytometer.
Experimental result is as shown in Fig. 3, wherein 1 is non-viable non-apoptotic cell, and 2 be non-viable apoptotic cell, and 3 be normal cell, and 4 are
Viable apoptotic cell.Experimental result is shown：The early apoptosis rate of control group human lung cancer cell A549 is 4.13%, and the present embodiment is high
The early apoptosis of each concentration group of galingal aerial parts volatile oil (0.078125 μ L/mL, 0.15625 μ L/mL, 0.3125 μ L/mL)
Rate is respectively 21.8%, 32.6% and 40.7%, the significant difference (p compared with Normal group<0.01), and in apparent
Dose-effect relationship.
4. galangal aerial parts volatile oil influences to test on neoplastic cell nuclei
The galangal aerial parts volatile oil galangal aerial parts volatile oil is implemented using the present invention, using DAPI
Decoration method evaluates the influence of Human Lung Cancer nucleus.
Wherein DAPI decoration methods are as follows：
(1) by a concentration of 1 × 105The cell inoculation in/hole in 12 porocyte culture plates for 24 hours after, culture medium is changed to and is contained
The culture solution of 10% serum and various concentration drug continues to cultivate 48h；
(2) cell is rinsed with PBS buffer solution and is then slowly added to twice in the absolute ethyl alcohol of 1mL-20 DEG C of precooling,
The shaken well when being added；
1h or more is fixed under the conditions of (3) -20 DEG C, abandons supernatant.4 DEG C of precooling PBS 1.5mL rinsings are primary, abandon supernatant；
(4) DAPI dyeing liquor 0.5mL are added per hole, are placed at room temperature for 5min, absorbs DAPI dyeing liquors, is washed 2 times with PBS,
In fluorescence microscopy microscopic observation.
Experimental result is as shown in Fig. 4, as a result shows：The morphological change of nuclei dyeing chromaticness point in apoptosis process
For three phases：The nucleus of I phase is corrugated or is in crease sample, and enrichment stage occurs in part chromatin；The dyeing of II a phase nucleus
Matter high degree of coagulation, marginalisation；The nucleus of II b phases is cracked into fragment, generates apoptotic body.It, can be according to caryoplasm after DAPI contaminates core
Form judge the process of apoptosis.Show this product galangal aerial parts volatile oil in 0.15625 μ L/mL effects of concentration
Under, DAPI dyeing, it is more compared with negative control group that enrichment stage cell is presented in A549 lung carcinoma cells.Show lung cell A549 in this hair
It is bright implement the galangal aerial parts volatile oil under the action of, cause Apoptosis.
5. galangal aerial parts volatile oil influences to test on tumour cell mitochondrial membrane potential
Using galangal aerial parts volatile oil described in the embodiment of the present invention, using mitochondrial membrane potential detection kit
(JC-1), the influence of Human Lung Cancer cell A549 mitochondrial membrane potentials is evaluated.
Wherein mitochondrial membrane potential detection kit (JC-1) method is as follows：
(1) by a concentration of 1 × 105The cell inoculation in/hole in 12 porocyte culture plates for 24 hours after, culture medium is changed to and is contained
The culture solution of 10% serum and various concentration drug continues to cultivate 48h；
(2) culture solution is absorbed, wash that cell is primary with PBS, addition cell culture fluids of the 1mL containing serum；
(3) 1mL JC-1 are added and dye working solution, mix well.It is incubated 20 minutes for 37 DEG C in cell incubator；
(4) in incubation period, the ratio of 4mL distilled water is added according to every 1mL JC-1 dye solutions (5 ×), prepares suitable
The JC-1 dye solutions (1 ×) of amount, and it is positioned over ice bath；
After (5) 37 DEG C are incubated, supernatant is absorbed, is washed 2 times with JC-1 dye solutions (1 ×)；
(6) 2mL cell culture fluids are added, serum and phenol red can be contained in culture solution；
(7) fluorescence microscopy microscopic observation.
After dyeing detection various concentration galangal aerial parts volatile oil processing A549 cells for 24 hours by JC-1, to line grain
The influence of body film potential, experimental result are as shown in Fig. 5, wherein B2 represents the percentage composition of low potential cell.Mitochondrial membrane electricity
The decline of position is the hallmark events of Apoptosis early stage.The result shows that tumour cell A549 mitochondrial membrane potentials influence in fact
It tests and shows that the ratio for increasing low potential cell with the galangal aerial parts volatile oil concentration constantly rises.This product Gao Liang
Ginger aerial parts volatile oil can induce lung cell A549 and generate Apoptosis.
Example IV galangal aerial parts volatile oil function of resisting osteoporosis is studied
1, the preparation and culture of neonate rat cranium osteoblast
Newborn SD rat (within for 24 hours) is taken, it is sterile to take cranium, it shreds, uses after rejecting the connective tissue and blood vessel sticked
After 0.25% trypsase predigestion, adds and digested containing the digestive juice of 0.2% Type I collagen enzyme and 0.1% hyaluronidase, digestion 6
Cell that is secondary, taking last 4 digestion obtained, 3000 × g centrifuge 10min, abandon supernatant, sedimentation cell washing centrifugation is primary, most
It is resuspended afterwards with 10% fetal calf serum DMEM culture solutions, inoculation after blowing and beating uniformly is placed in 37 DEG C, 5%CO2Incubator culture, for 24 hours
Liquid is changed, is changed the liquid once later every 2-3d, is passed on.
2, galangal aerial parts volatile oil acts on neonate rat osteoblastic proliferation
By above-mentioned neonate rat cranium osteoblast with 2 × 104/cm2Density be inoculated with into 96 orifice plates, the first hole is sky
White control group.Cell inoculation for 24 hours after, the galangal aerial parts volatile oil of various concentration is added, final concentration is made to be respectively：
0.01953125 μ L/mL, 0.0390625 μ L/mL, 0.078125 μ L/mL and 0.15625 μ L/mL.After dosing for 24 hours, 48h and
72h is detected, and 4h adds the MTT (5mg/mL) of 20 μ L before experiment terminates, and is continued at 37 DEG C, 5%CO2Under the conditions of be incubated 4h, inhale
Add 150 μ L of dimethyl sulfoxide (DMSO) after taking culture solution, shake after being completely dissolved to crystallization, its absorbance is detected using microplate reader, detects
Wavelength 570nm, reference wavelength 630nm.
Osteoblastic proliferation plays an important role in determining new bone formation.
Experimental result is as shown in Fig. 6, and experimental result is shown, the galangal aerial parts volatile oil is in high concentration
(0.078125 μ L/mL, 0.15625 μ L/mL) has neonate rat cranium osteoblastic proliferation the inhibiting effect of very little, low dense
It spends little to the proliferative effect of neonate rat cranium osteoblast when (0.01953125 μ L/mL, 0.0390625 μ L/mL).
3, galangal aerial parts volatile oil is to neonate rat osteoblast alkaline phosphatase (ALP) active effect
Cell is with 2 × 104/cm2Density be inoculated in 48 orifice plates.Cell inoculation for 24 hours after, the product of various concentration is added
Galangal aerial parts volatile oil makes final concentration be respectively：0.0390625 μ L/mL, 0.078125 μ L/mL and 0.15625 μ L/
mL.Cell ALP contents are measured in drug effect within the 5th, 7,9 day.Remove culture solution when measurement, PBS is rinsed, and cell pyrolysis liquid is added
200 μ L, freeze thawing 30min, then ultrasound cracking 20min, be added diethanol amine containing 25mmol/L, 1mmol/L magnesium chlorides with
Reaction solution 400 μ L, the 37 DEG C of placement 0.5h of 6mmol/L PNPP, then use the 100 μ L of NaOH of 0.1mol/L to terminate reaction,
Microplate reader measures OD values under 405nm wavelength.Sample OD values read enzymatic activity value (U/L) on ALP standard curves.
ALP is the specific parameters for reflecting osteoblast differentiation early activity, and ALP is in one in osteoblast differentiation early stage
The process of rising, in the process its expression number can reflect the differentiation degree and functional status of osteoblast.
Experimental result is as shown in Fig. 7, the results showed that, neonate rat osteoblast volatilizees in the galangal aerial part
Oil concentration is under the stimulation of 0.0390625 μ L/mL, 0.078125 μ L/mL and 0.15625 μ L/mL, and alkaline phosphatase activities are notable
It increases.
4, the effect that product galangal aerial parts volatile oil forms neonate rat osteoblast bone tubercle
Neonate rat osteoblast maturation mineralising is induced using vitamin C and p- sodium glycero-phosphates, in product galangal
After upper part volatile oil is handled 18 days, after exhausting each hole culture supernatant of 12 orifice plates, culture plate is rinsed 2 times with PBS, 95% ethyl alcohol
Fixed 10min, is rinsed 3 times again with PBS, and 0.1% alizarin red 1mL is added in each hole, and 30min, distilled water punching are placed at 37 DEG C
It washes, dry.Under the microscope in light, 100 μm of standard is more than with red dye, sharpness of border, minor axis, to each hole point under 40 times of light microscopics
It does not count, each hole is repeated three times.
It is characterized by the formation of bone tubercle and quantity in the mineralization of extracellular matrix phase.Bone tubercle is differentiation and maturation skeletonization
Cell forms the tubercle spline structure of multiple-layer stacked, while bone tubercle is to weigh osteoblast differentiation late period active index.
Experimental result is as shown in Fig. 8, the results showed that, the galangal aerial parts volatile oil is a concentration of
After 0.0390625 μ L/mL, 0.078125 μ L/mL and 0.15625 μ L/mL are acted on 20 days, neonate rat osteoblast can be promoted
Bone tubercle is formed.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Claims (8)
1. a kind of galangal aerial parts volatile oil, which is characterized in that the volatile oil contains following compound components：1H- is different
Pyrazoles, 4,5- dihydro -2- (benzyl), 3- cyclohexene -1- methanol, α, α, 4- trimethyls, bicyclic [3.1.1] 2- heptene, 2,6-
Dimethyl -6- (4- methyl), naphthalene, 1,2,3,4,4a, 5,6,8a- octahydro -7- methyl, aromadendrene, eucalyptol, acetic acid fennel seeds
Ester, benzenpropanal, Bronyl acetate, benzylacetone, bicyclic [2.2.1] heptane -2- ketone, with the total of galangal aerial parts volatile oil
Weight is 100% meter, weight percent >=50% of above compound component；
The preparation method of the galangal aerial parts volatile oil, includes the following steps：
Galangal aerial part is taken, after crushing processing, galangal aerial part 10-15 times of water measured of weight is added, carries out
Steam distillation obtains distillate, wherein the temperature of the steam distillation is 80-120 DEG C, and distillation time is 2-6 hours；
The distillate is detached with oil water separator, obtains galangal aerial parts volatile oil.
2. a kind of galangal aerial parts volatile oil as described in claim 1, which is characterized in that the volatile oil also contains following
Compound component：β-carypohyllene, α-humulene, 1H- cyclopropyl [e] decahydronaphthalene azulenes -1,1,7- trimethyls, α-Acacia
Alkene, decahydronaphthalenes -4a- methyl-1s-methylene -7- methyl, naphthalene, 1,2,3,5,6,8a- hexahydro -4,7- dimethyl, naphthalene, 1,2- bis-
Hydrogen -1,1,6- trimethyls, caryophyllene oxide, azulenes, 1,2,3,3a, 4,5,6,7- octahydros-Isosorbide-5-Nitrae-dimethyl is decahydronaphthalene
Naphthalene -4a- methyl-1s-methylene -7- (1- Methylethyls), α-santalol, bicyclic [3.1.1] hept-2-ene", 2,6- dimethyl -6-
(4- methyl), 3,6- bis- (4- tolyls) -1,2- dialins, by the gross weight of galangal aerial parts volatile oil be 100% in terms of,
The sum of weight percent of each compound component >=22%.
3. galangal aerial parts volatile oil as described in claim 1, it is characterised in that：Galangal after pulverization process is on the ground
Part carries out sieving processing, obtains the galangal aerial part of the mesh of grain size≤40.
4. galangal aerial parts volatile oil as described in claim 1, it is characterised in that：Further include to the galangal overground part
Divide volatile oil that processing is dried, described be dried is realized using drier.
5. a kind of any galangal aerial parts volatile oils of claim 1-4 are being made as pharmaceutical preparation or drug component
Application in standby prevention and control of cancer drug.
6. the application of galangal aerial parts volatile oil as claimed in claim 5, which is characterized in that the galangal overground part
Point volatile oil is as pharmaceutical preparation or drug component in application, further including pharmaceutically acceptable in preparing prevention and control of cancer drug
Carrier.
7. a kind of any galangal aerial parts volatile oils of claim 1-4 are being prepared as pharmaceutical preparation or drug component
Application in osteoporosis protective agents.
8. the application of galangal aerial parts volatile oil described in a kind of claim 7, which is characterized in that the galangal overground part
It further includes that can pharmaceutically connect when being applied in preparing osteoporosis protective agents to divide volatile oil as pharmaceutical preparation or drug component
The carrier received.
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| 高良姜挥发油对不同肿瘤细胞株增殖的影响;李宁等;《山东医药》;20121231;第52卷(第11期);第19-21页 * |
| 高良姜提取物高效液相色谱分析方法的优化;邓亦峰等;《时珍国医国药》;20121231;第23卷(第5期);第1282-1283 * |
| 高良姜根茎叶挥发油化学成分的比较;罗辉等;《时珍国药研究》;19971231;第8卷(第4期);第319-320页 * |
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