CN104083690B - Application of Stahlianthus involucratus (King) Craib and its extract in preparation of drugs for treating and/or preventing breast cancer - Google Patents
Application of Stahlianthus involucratus (King) Craib and its extract in preparation of drugs for treating and/or preventing breast cancer Download PDFInfo
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Abstract
The invention relates to a new use of Stahlianthus involucratus (King) Craib, a Stahlianthus involucratus (King) Craib extract, a Stahlianthus involucratus (King) Craib fat-soluble extract and a Stahlianthus involucratus (King) Craib volatile oil composition, that is, a new use in the preparation of drugs for treating and/or preventing the breast cancer.
Description
Technical field
The present invention relates to the volatilization of Stahlianthus hainanensis (Hayata) T. L. Wu, Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat-soluble extract and Stahlianthus hainanensis (Hayata) T. L. Wu
The new application of oil, the i.e. new application in terms of preparing treatment and/or preventing breast cancer medicines.
Background technology
Breast cancer is the common cancer that women ranks the first, no matter domestic external, although the morbidity of breast cancer
Rate remains high, and the treatment means of breast cancer include operative treatment, radiotherapy, chemotherapy, endocrine therapy and molecule
Targeted therapy.Substantial amounts of research confirms that tumour is a class cell cycle disease, and one of feature of tumour is cell cycle exception,
The biosynthesis for making tumour cell infinite multiplication, any one phase of cell cycle is blocked, and can all make the company of cell cycle
Continuous property is interrupted, and the key link of oncotherapy is exactly to block the cell cycle of tumour cell, inducing cell apoptosis.
Apoptosis is betided in all of tumor tissues, with the generation of tumour, develop and shift closely related.Chemotherapeutic
Thing mainly reaches therapeutic effect by cancer cell specific induction of apoptosis, therefore, inducing apoptosis of tumour cell becomes research oncotherapy
Focus.
Apoptotic feature is that cell volume reduces, and is lost with the connection of neighbouring cell immediately, is disengaged from, and is lost
Microvillus, cytoplasmic condensation, reticulum dilatation in blister and and cell membrane fusion, mitochondria is without big change, nuclear chromatin condensation
In semilune, chromatin condensation forms apoptotic body close to nuclear membrane periphery, kernel cracking, and then cell membrane invagination, and cell withers
The process died does not cause lysosome to rupture, and leaks without cell inclusion, therefore does not cause inflammatory reaction and secondary damage, Ca2+/
Mg 2+Rely on endonuclease activity to increase, make DNA degradation, DNA electrophoresis show as scalariform, these changes at present have become judgement
Apoptotic important indicator.But the activation for not meaning endonuclease is necessary in apoptosis process, is being had
Not necessarily there is DNA electrophoresis in scalariform in a little Apoptosis.
Whether apoptotic generation needs the active of new RNA and protein to synthesize, depending on cell type and induction
The factor of apoptosis, perhaps some signal pathways need the expression of new gene, and some need not.Also have been reported that apoptotic cell does not have
DNA degradation, points out DNA degradation not essential in apoptosis process.Apoptosis be an extremely complex physiology and
Pathologic process, the inside and outside many factors of body can affect apoptotic generation, and substantially be affected by molecular genetics.It is different
Tumour cell it is different to the variable concentrations sensitiveness of different pharmaceutical or identical medicine, a kind of apoptosis of tumor cells can be activated
The material of effect, may be invalid to another kind of tumour cell.Such as taxol is primarily adapted for use in oophoroma and breast cancer, to lung
Cancer, colorectal cancer, melanoma, incidence cancer, lymthoma, brain tumor also have certain curative effect.And then effect is not to control other cancers
It is good.In a word, apoptotic molecular mechanism is unclear, awaits further investigation.
Stahlianthus hainanensis (Hayata) T. L. Wu is zingiberaceous plant ginger leaves pseudo-ginseng Stahlianthus involucratus (King ex Bak.)
The root-like stock and root tuber of Craib.The effect of tool heat-clearing, dampness removing, pain relieving, removing toxic substances, hemostasis, for stranguria with turbid discharge, have a stomachache, aphtha, hemorrhoid,
The diseases such as ulcer, traumatic injury, snake bite, traumatism and bleeding, are Guangxi Special Traditional Chinese Medicine material.
" China Zingiber medicinal plants study VI Radix Camptandrae Yunnanensis Analysis of The Essential Oil " (is published in《Chromatogram》1984
The phase of volume 1 the 1st), with GLC with GLC-mass spectrography each composition of Radix Camptandrae Yunnanensis volatile oil is shared and identified, identify
Go out Dihydrostahlianthusone, australene, camphene, nopinene, carene, limonene, Cineole, linalool, camphor, α-pepper alkene, trans-
15 kinds of compositions such as cloves alkene, aromadendrene, γ-muurolene, cadinene, Stahlianthusone.
Application No. CN200510098945.2 " a kind of externally applied drug of Stahlianthus hainanensis (Hayata) T. L. Wu ", discloses by Stahlianthus hainanensis (Hayata) T. L. Wu, diamond
Wind, prolongated spleenwort herb with root, yunnan rockvine stem and leaf, Chinese maple root, Prunus brachypoda Batal. Var.eglandulosa Cheng, caulis trachelospermi, a kind of externally applied drug of ethanol composition, to Fengshi Guanjie
Bitterly, bruise stasis caused pain, traumatic injury, traumatism and bleeding, arthralgia, muscles and bones sprain pain, arthralgia and myalgia, pain caused by ecchymoma, waist and knee pain
Bitterly, joint pain, wound stasis caused pain, soft tissue bruise determined curative effect, the course for the treatment of is shorter, better.
Additionally, patent application " a kind of damp-repellent pain-relieving medicinal liquor of Radix Vaccinii Fragilis ", the Publication No. of Publication No. CN1814229
A kind of patent application " manyspike chloranthus herb externally applied drug " of CN1742972, a kind of patent application " hill of Publication No. CN1814230
The active antalgesic of pea ", a kind of patent application " radix-Gynurae-Bodinieri collateral-flow-activating pain-relieving ointment for treating affection by dampness " of Publication No. CN1814228, publication number
Patent application " damp-clearing pain-relieving liquor and acupoint application treatment method " for CN101766729 refers to Stahlianthus hainanensis (Hayata) T. L. Wu or native pseudo-ginseng in bruise
Therapeutic effect in terms of damage or rheumatoid disease.
In existing open source literature, or the volatile oil component of Stahlianthus hainanensis (Hayata) T. L. Wu is mentioned, or mention Stahlianthus hainanensis (Hayata) T. L. Wu treatment bruise and damaged
The effect of the diseases such as wound, but not disclosed use Stahlianthus hainanensis (Hayata) T. L. Wu, Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat-soluble extract and ginger
Leaf notoginseng essential oil is used to treat tumour, the effect of various cancers, not yet has open Stahlianthus hainanensis (Hayata) T. L. Wu to can be used to treat breast cancer
Report, does not also report impact of the open Stahlianthus hainanensis (Hayata) T. L. Wu to breast cancer cell cycle, propagation or apoptosis.
The content of the invention
It is an object of the invention to provide Stahlianthus hainanensis (Hayata) T. L. Wu plant is preparing treatment and/or is preventing the application of breast cancer medicines；
It is an object of the invention to provide Stahlianthus hainanensis (Hayata) T. L. Wu extract is preparing treatment and/or is preventing the application of breast cancer medicines；
It is an object of the invention to provide Stahlianthus hainanensis (Hayata) T. L. Wu extractive of volatile oil is preparing treatment and/or is preventing breast cancer medicines
Using；
It is a further object of the present invention to provide the preparation method of Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil.
Stahlianthus hainanensis (Hayata) T. L. Wu is zingiberaceous plant ginger leaves pseudo-ginseng Stahlianthus involucratus (King ex Bak.)
The root-like stock of Craib, root tuber.Excavation after time of the year when autumn changes into winter blade is withered and yellow, removes impurity, cleans, and puts rapid iron in boiling water, dries.Receive
It is loaded in《Guangxi Chinese medicine standard》Nineteen ninety version page 8.
The another name of Stahlianthus hainanensis (Hayata) T. L. Wu has pseudo-ginseng ginger, Stahlianthus hainanensis (Hayata) T. L. Wu, native pseudo-ginseng, leaf of bamboo pseudo-ginseng, Stahlianthus hainanensis (Hayata) T. L. Wu, heart seven, red sand
Ginger, sharp pseudo-ginseng, drag top rifle (Zhuang), the interior son that disappears, do not beat to death.
According to inventor to Stahlianthus hainanensis (Hayata) T. L. Wu plant, Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat-soluble extract and ginger leaves
Notoginseng essential oil carries out clinical research and pharmacodynamic study, finds Stahlianthus hainanensis (Hayata) T. L. Wu plant, Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat
Soluble extract and Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil can be by suppressing Cells Proliferation of Human Breast Cancer, affecting breast cancer cell to wither
Die, and affect the change in breast cancer cell cycle, reach anti-human breast cancer cell activity, effect for the treatment of breast cancer.
On this basis, the present inventor is purposefully extracted and content by the active ingredient to Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil
After control so as to which the effect for treatment and/or pre- anti-cancer is more notable.Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is carried with other Stahlianthus hainanensis (Hayata) T. L. Wus
Take thing to compare, significant inhibitory action had more to breast cancer cell MCF-7, can effectively suppress the normal conversion of cell cycle,
Cell is set to pile up in the G1 phases, cell block, so as to prevent the mitosis of cell, is suppressed cell propagation in the G1 phases.
The security of patent clinical practice of the present invention is higher, a kind of good medicine of can yet be regarded as in the drug therapy of breast cancer, its
The new method of Chinese medicine preparation treatment breast cancer has been widened, it is especially suitable in the basic hospital that can not carry out PCI.
Therefore, applicant provides Stahlianthus hainanensis (Hayata) T. L. Wu plant, Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat-soluble extract, ginger leaves
Notoginseng essential oil is used to prepare the new application treated and/or prevent in terms of breast cancer medicines.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu extract is preparing treatment and/or is preventing the application of breast cancer medicines, the ginger
The preparation of leaf Notogineng Extract is comprised the following steps：The chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome is taken, extracting in water is decocted twice, is added for the first time
The decocting that medicinal material weight 2-6 times is measured is boiled, and 1-3 hours are boiled after boiling, adds the decocting that medicinal material weight 1-4 times is measured to boil 1-3 for the second time
Hour, filter, mix decoction liquor twice, 0.5-2 times of medicinal material gross weight is concentrated into, obtain final product.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu extract is preparing treatment and/or is preventing the application of breast cancer medicines, the ginger
Preparing for leaf Notogineng Extract is further comprising the steps of：The chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome is taken, adds alcohol reflux to extract 2-3 time, the
2-6 times of percent by volume is once added for the ethanol of 40-70%, 1-3 hours are extracted, volume basis are measured in second 1-4 times of addition
1-3 hours are extracted than the ethanol for 40-70%, collecting decoction is filtered, and reclaims ethanol, makes dry cream, is obtained final product.
The invention also discloses Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract is preparing treatment and/or is preventing breast cancer medicines
Using the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract is comprised the following steps：Take the chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome, plus oil
Ether refluxing extraction 2-3 time, adds every time the petroleum ether of medicinal material gross weight 3-5 times, and extraction time is 1-3 hours, merges extract,
Filter, reclaim petroleum ether, obtain Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract is preparing treatment and/or is preventing answering for breast cancer medicines
With the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract can also be comprised the following steps：The chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome is taken, plus
Ethyl acetate backflow is extracted 2-3 time, and the ethyl acetate of medicinal material gross weight 3-5 times is added every time, and extraction time is 1-3 hours, is closed
And extract, filter, ethyl acetate is reclaimed, obtain Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract is preparing treatment and/or is preventing answering for breast cancer medicines
With the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract can also be comprised the following steps：The chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome is taken, plus
Water-saturated n-butanol refluxing extraction 2-3 time, adds every time the water-saturated n-butanol of medicinal material gross weight 3-5 times, and extraction time is 1-3
Hour, merge extract, filter, water-saturated n-butanol is reclaimed, obtain Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract is preparing treatment and/or is preventing answering for breast cancer medicines
With the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract can also be comprised the following steps：The chopping of Stahlianthus hainanensis (Hayata) T. L. Wu rhizome is taken, plus
Alcohol reflux is extracted 2-3 time, and medicinal material gross weight 3-5 times, percent by volume are added every time for the ethanol of 30-50%, extraction time
For 1-3 hours, merge extract, filter, reclaim ethanol, obtain concentrate.
The concentrate adds 3 extractions of petroleum ether point, each petroleum ether consumption to be 3-5 times of the volume of concentrate, collect stone
Oily ether phase, concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The concentrate adds 3 extractions of ethyl acetate point, each ethyl acetate consumption to be 3-5 times of the volume of concentrate, receive
Collection petroleum ether phase, concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The concentrate adds 3 extractions of water-saturated n-butanol point, each water-saturated n-butanol consumption to be the volume of concentrate
3-5 times, water-saturated n-butanol phase is collected, concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
The invention discloses Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite is in preparation treatment and/or the application of prevention breast cancer medicines,
Contain active ingredient in the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil activity that the active ingredient is extracted by Stahlianthus hainanensis (Hayata) T. L. Wu
Composition is constituted, including monoterpenes compound, the oxide-based compound of monoterpene, and sesquiterpenoids, sequiterpene oxide
Class compound.
Preferably, described Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil active ingredient includes：The 3,6,7,8- tetrahydros of 20-30 weight portions-
3,3,6,6- tetramethyl cyclopenta [E] indenes -1 (2H) -one, the amphene of 8-20 weight portions；The 1,7,7- tri- of 6-15 weight portions
The ring of methyl-two [2.2.1] hept- 2- thatch ketone, the aromadendrene of 2-8 weight portions；The caryophyllene oxide of 1-6 weight portions.
It is highly preferred that described Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil active ingredient content includes：The 3,6,7,8- tetra- of 24-28 weight portions
- 3,3,6,6- tetramethyl cyclopenta [E] indenes -1 (2H) -one of hydrogenation, the amphene of 11-15 weight portions；The 1 of 8-12 weight portions,
Ring [2.2.1] the hept- 2- thatch ketone of 7,7- trimethyl-two, the aromadendrene of 3-6 weight portions；The caryophyllene oxide of 2-5 weight portions.
Applicant additionally provides a kind of method for preparing the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, and it comprises the steps：
Take Stahlianthus hainanensis (Hayata) T. L. Wu and be ground into meal, the distilled water immersion 0.5-3 hours of medicinal material gross weight 3-7 times are added, by water
Steam distillation extracts 3-6 hours, collects upper strata essential oil, obtains Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil.
Preferably, the preparation of Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is comprised the following steps：
Stahlianthus hainanensis (Hayata) T. L. Wu crushing, plus the distilled water immersion 1h of 5 times of medicinal material gross weight are taken, 4h is extracted by steam distillation, received
Collection upper strata essential oil obtains Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite.
Based on above-mentioned Stahlianthus hainanensis (Hayata) T. L. Wu extract, Stahlianthus hainanensis (Hayata) T. L. Wu fat-soluble extract, Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, the mesh of the present invention
A kind of new application for being to provide combination of the above thing, i.e., prepare treatment and/or prevent breast cancer medicines application.It can make
Into parenteral solution, powder ampoule agent for injection, freeze-dried powder injection, tablet, pill, powder, granule, mixture, syrup, capsule
Agent, dripping pill agent formulation, are preferably made parenteral solution, freeze-dried powder injection.The medicine of above-mentioned various formulations can be according to medicine
It is prepared by the conventional method in field.
The following is the crude drug source of Chinese medicine preparation of the present invention：
Stahlianthus hainanensis (Hayata) T. L. Wu：Zingiberaceous plant ginger leaves pseudo-ginseng Stahlianthus involucratus (King ex Bak.) Craib
Root-like stock and root tuber.
The invention discloses new application of the Stahlianthus hainanensis (Hayata) T. L. Wu in treatment breast cancer, Stahlianthus hainanensis (Hayata) T. L. Wu original plant and common process are carried
Thing is taken for treatment breast cancer has certain therapeutic effect, as a result visible experimental example 1 and experimental example 2, also, Stahlianthus hainanensis (Hayata) T. L. Wu toxicity
It is little, the strong side effect of such as chemotherapy etc will not be produced.
The extractive of volatile oil of Stahlianthus hainanensis (Hayata) T. L. Wu is with outstanding curative effect, the cell experiment as shown in experimental example, and its is dense
Degree just already has obvious activity in 120 μ g/ml, and the inhibiting rate of breast cancer cell is exceeded when 12 hours
It is higher than cis-platinum group that the volatile oil of control drug group, i.e. Stahlianthus hainanensis (Hayata) T. L. Wu suppresses the ability of human breast carcinoma activity, and its concentration is 480
During more than μ g/ml, its activity for killing human breast cancer cell even more can be more than 90%.
Description of the drawings
Fig. 1 is Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to breast carcinoma cell strain (MCF-7) proliferation inhibition rate line chart
Fig. 2 is impact of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to the MCF-7 cell cycles
Fig. 3 is Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to breast carcinoma cell strain (MCF-7) early apoptosis Annexin V-PI double-stainings
Fluorescence scatter diagram
Fig. 4 is Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to breast carcinoma cell strain (MCF-7) apoptosis rate bar chart
Specific embodiment
The present invention is further illustrated below by embodiment.It should be understood that embodiments of the invention are for illustrating
The present invention rather than limitation of the present invention.Essence of the invention belongs to the present invention to the simple modifications that the present invention is carried out
Claimed scope.Unless otherwise stated, the percentage in the present invention is percetage by weight (ethanol is percent by volume).
Embodiment 1：Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil
Take fresh Stahlianthus hainanensis (Hayata) T. L. Wu 150g and be ground into meal, plus the distilled water immersion 0.5h of medicinal material 450ml, steamed by water vapour
Extraction 3h is evaporated, upper strata essential oil is collected, Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil 1.7ml is obtained.
Gained Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is detected, component content result：3,6,7,8- tetrahydro -3,3,6,6- tetramethyls
Simultaneously [E] indenes -1 (2H) -one is 24% to butylcyclopentadiene, and amphene is 15%；Ring [2.2.1] the hept- 2- thatch ketone of 1,7,7- trimethyls-two
For 12%, aromadendrene is 3%；Caryophyllene oxide is 2%.
Embodiment 2：Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil
Take fresh Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 150g to crush, plus the distilled water immersion 1h of 750ml, extracted by steam distillation
4h, collects upper strata essential oil, obtains Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite 2.2ml.
Gained Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is detected, component content result：3,6,7,8- tetrahydro -3,3,6,6- tetramethyls
Simultaneously [E] indenes -1 (2H) -one is 28% to butylcyclopentadiene, and amphene is 11%；Ring [2.2.1] the hept- 2- thatch ketone of 1,7,7- trimethyls-two
For 8%, aromadendrene is 6%；Caryophyllene oxide is 5%.
Embodiment 3：Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil
Stahlianthus hainanensis (Hayata) T. L. Wu dry rhizome 150g crushing, plus 1050ml distilled water immersion 3h are taken, 6h are extracted by steam distillation,
Upper strata essential oil is collected, Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite 2.0ml is obtained.
Gained Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is detected, component content result：3,6,7,8- tetrahydro -3,3,6,6- tetramethyls
Simultaneously [E] indenes -1 (2H) -one is 30% to butylcyclopentadiene, and amphene is 20%；Ring [2.2.1] the hept- 2- thatch ketone of 1,7,7- trimethyls-two
For 15%, aromadendrene is 2%；Caryophyllene oxide is 1%.
Embodiment 4：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 5000g choppings, plus petroleum ether refluxing extraction 2 times are taken, 20000ml petroleum ethers are added for the first time,
Extract 2 hours, second addition 15000ml petroleum ether is extracted 2 hours, merges extract, is filtered, and reclaims petroleum ether, obtains ginger leaves
Pseudo-ginseng fat soluble component extract.
Embodiment 5：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 5000g choppings, plus petroleum ether refluxing extraction 3 times are taken, 25000ml petroleum ethers are added for the first time,
Extract 3 hours, second addition 20000ml petroleum ether is extracted 2 hours, it is 1 that third time adds 15000ml petroleum ether extraction times
Hour, merge extract, filter, petroleum ether is reclaimed, obtain Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 6：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 5000g choppings are taken, plus ethyl acetate backflow is extracted 3 times, and 25000ml acetic acid second is added for the first time
Ester, extracts 3 hours, and second addition 20000ml ethyl acetate is extracted 2 hours, and third time adds 15000ml ethyl acetate, carries
The time is taken for 1 hour, merges extract, filtered, reclaim ethyl acetate, obtain Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 7：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 5000g choppings, plus water-saturated n-butanol refluxing extraction 3 times are taken, 25000ml water is added for the first time
Saturation n-butanol, extracts 3 hours, and second addition 20000ml water-saturated n-butanol is extracted 2 hours, and third time adds 15000ml
Water-saturated n-butanol carried extraction time for 1 hour, merged extract, filtered, and reclaimed water-saturated n-butanol, obtained Stahlianthus hainanensis (Hayata) T. L. Wu liposoluble
Property component extract.
Embodiment 8：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu dry rhizome 5000g choppings are taken, adds alcohol reflux to extract 2 times, 25000ml volumes are added for the first time
Percentage is 40% ethanol, is extracted 3 hours, adds the ethanol that 15000ml percents by volume are 50% to extract 2 for the second time little
When, merge extract, filter, ethanol is reclaimed, concentrate adds 3 extractions of petroleum ether point, each petroleum ether consumption respectively to concentrate
5 times, 4 times, 3 times of liquid product, collect petroleum ether phase, and concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 9：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu dry rhizome 5000g choppings are taken, adds alcohol reflux to extract 3 times, 20000ml volumes are added for the first time
Percentage is 30% ethanol, is extracted 3 hours, adds the ethanol that 15000ml percents by volume are 40% to extract 2 for the second time little
When, third time adds the ethanol that 10000ml percents by volume are 50% to extract 1 hour, merges extract, filters, and reclaims ethanol,
Concentrate adds 3 extractions of ethyl acetate point, each ethyl acetate consumption to be respectively 5 times of the volume of concentrate, 4 times, 3 times, collect
Ethyl acetate phase, concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 10：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Stahlianthus hainanensis (Hayata) T. L. Wu dry rhizome 5000g choppings are taken, adds alcohol reflux to extract 3 times, 20000ml volumes are added for the first time
Percentage is 30% ethanol, is extracted 3 hours, adds the ethanol that 15000ml percents by volume are 40% to extract 2 for the second time little
When, third time adds the ethanol that 10000ml percents by volume are 50% to extract 1 hour, merges extract, filters, and reclaims ethanol,
Concentrate adds points of 3 times extractions of water-saturated n-butanol, each water-saturated n-butanol consumption be respectively 5 times of the volume of concentrate, 4 times, 3
Times, water-saturated n-butanol phase is collected, concentrate drying obtains Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 11：Stahlianthus hainanensis (Hayata) T. L. Wu water extract
Take fresh Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 1000g to shred, add 6000ml to decoct for the first time, 3h is kept after boiling, add for the second time
4000ml water boiling and extractions 1h.Merge decocting liquid twice, filter, be concentrated into 2000ml, obtain Stahlianthus hainanensis (Hayata) T. L. Wu water extract.
Embodiment 12：Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract
Fresh Stahlianthus hainanensis (Hayata) T. L. Wu rhizome 1000g choppings are taken, plus alcohol reflux is extracted 2 times, and 6 times of percents by volume are added for the first time
For 70% ethanol, extract 3 hours, second addition, 4 times of amount percents by volume are that 40% ethanol is extracted 1 hour, merge and decoct
Liquid, filters, and reclaims ethanol, makes dry cream, obtains final product Stahlianthus hainanensis (Hayata) T. L. Wu fat soluble component extract.
Embodiment 13：Stahlianthus hainanensis (Hayata) T. L. Wu volatilization fat injection
Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil in embodiment 1-3 is well mixed with glucose, appropriate cosolvent, appropriate solvent, is filtered
Cross, sterilize, prepare parenteral solution.
Embodiment 14：Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil freeze drying powder injection
Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil in embodiment 1-3 is well mixed with glucose, appropriate cosolvent, appropriate solvent, is gone out
Bacterium, in being sub-packed in the containers such as ampoule or cillin bottle, in sterile closed environment, freezes under low temperature, then by reducing ambient pressure,
The slow method for raising products temperature makes the solvent in product distil, and leaves the medicine of solid forms, obtains final product the lyophilized of the present invention
Powder pin.
Embodiment 15：Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil
Stahlianthus hainanensis (Hayata) T. L. Wu dry rhizome 150g crushing, plus 600ml distilled water immersion 2h are taken, 5h are extracted by steam distillation,
Upper strata essential oil is collected, Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite 1.9ml is obtained.
Gained Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is detected, component content result：3,6,7,8- tetrahydro -3,3,6,6- tetramethyls
Simultaneously [E] indenes -1 (2H) -one is 20% to butylcyclopentadiene, and amphene is 8%；Ring [2.2.1] the hept- 2- thatch ketone of 1,7,7- trimethyls-two
For 6%, aromadendrene is 8%；Caryophyllene oxide is 6%.
Effect experiment
The present invention of experimental example 1 is to Cells Proliferation of Human Breast Cancer inhibitory action
Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite, ligroin extraction in Example 2,5,6,7,11,12, ethyl acetate are carried
Thing, water-saturated n-butanol extract, water extract and ethanol extract are taken, is carried out said extracted thing with RPMI1640 nutrient solutions dilute
Release, 0.22 μm of millipore filter suction filtration is degerming, 4 DEG C save backup.
With the mtt assay detection present invention to Cells Proliferation of Human Breast Cancer inhibitory action.
1 sample and Experimental agents
1.1 breast cancer cell cultures
RPMI1640 of the human breast cancer cell (MCF-7) containing 10% hyclone and 100 μ g/ml Pen .- Streps
Culture medium, puts 37 DEG C, 5%CO2, cultivate and pass in constant-temperature enclosed incubator under the conditions of saturated humidity.0.02EDTA-
0.25% Trypsin Induced, passes on once per 2～3d.
1.2 Experimental agents
Experimental group：Embodiment 2,5,6,7,11,12.
Comparative example medicine：Cis-platinum, Dezhou Deyao Pharmaceutical Co., Ltd of manufacturer；Lot number：Chinese medicines quasi-word H37020524；
The μ g/ml of concentration 3.
2 mtt assay detect Stahlianthus hainanensis (Hayata) T. L. Wu extract to Cells Proliferation of Human Breast Cancer inhibitory action
The inhibiting rate of the embodiment of the present invention of table 1 and cis-platinum to Cells Proliferation of Human Breast Cancer
Experimental result shows：Embodiment 2 is significantly stronger than other embodiment group and suitable to Cells Proliferation of Human Breast Cancer inhibitory action
Platinum group；Each embodiment group compared with cis-platinum group, Cells Proliferation of Human Breast Cancer inhibitory action is had it is weak have strong, but embody to mammary gland
The propagation of cancer cell has inhibitory action.Conclusion can be obtained by more than：
1st, Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is substantially better than other extractions of Stahlianthus hainanensis (Hayata) T. L. Wu Jing to the inhibited proliferation of breast cancer cell
The extract that method is obtained, and it is substantially better than cis-platinum.
2nd, Stahlianthus hainanensis (Hayata) T. L. Wu ligroin extraction, ethyl acetate extract are better than ginger to the inhibited proliferation of breast cancer cell
Leaf pseudo-ginseng water saturation n-butanol extract, Stahlianthus hainanensis (Hayata) T. L. Wu water extract, Stahlianthus hainanensis (Hayata) T. L. Wu ethanol extract and cis-platinum.
3rd, Stahlianthus hainanensis (Hayata) T. L. Wu water extract, ethanol extract and water-saturated n-butanol extract press down to the propagation of breast cancer cell
Make of poor compared with cis-platinum group, but equally can show there is inhibitory action to Cells Proliferation of Human Breast Cancer.
The external anti-breast cancer cytoactive experiment of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil of experimental example 2
1 instrument and reagent
1.1 instrument volatile oil determination apparatuses meet the relevant standard of one annex XD determination of volatile oil method of Chinese Pharmacopoeia；
Galaxy170S type CO2 cell culture incubators (German Eppendorf companies), (Japanese BIO-RAD is public for MLDEL680 type ELIASAs
Department), Axiovert-40 type inverted phase contrast microscopes (Zeiss, Germany company), (Suzhou purification sets SW-CJ-2F type superclean benches
Standby Co., Ltd).
1.2 reagent hyclones, RPMI1640 culture mediums are purchased from hyclone companies of the U.S.；DMSO, tetramethyl azo
Azoles indigo plant (MTT) is purchased from Sigma companies；Other reagents such as ether are domestic pure analysis pure.
1.3 Experimental agents
Experimental group：Embodiment 1, Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil.
Comparative example medicine：Cis-platinum, Dezhou Deyao Pharmaceutical Co., Ltd of manufacturer；Lot number：Chinese medicines quasi-word H37020524；
The μ g/ml of concentration 3.
1.4 for examination breast cancer cell
Human breast cancer cell (MCF-7) is provided by Medical Colleges Of Guilin's scientific experiment center.
2 methods and result
2.1 method
2.1.1 medicine is prepared
The extractive of volatile oil (20mg/mL) of the gained of embodiment 1 is pressed into 1 with RPMI1640 nutrient solutions:10、1:13、1:20、
1:40 ratios are diluted, and 0.22 μm of millipore filter suction filtration is degerming, and 4 DEG C save backup.
2.1.2 experiment packet：
A：Blank control group
B：120 μ g/ml Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil groups
C：240 μ g/ml Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil groups
D：360 μ g/ml Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil groups
E：480 μ g/ml Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil groups
2.1.3 human breast cancer cell culture
RPMI1640 of the human breast cancer cell (MCF-7) containing 10% hyclone and 100 μ g/ml Pen .- Streps
Culture medium, puts 37 DEG C, 5%CO2, cultivate and pass in constant-temperature enclosed incubator under the conditions of saturated humidity.0.02EDTA-
0.25% Trypsin Induced, passes on once per 2～3d.
2.1.4 mtt assay detects medicine of the present invention to human breast cancer cell inhibited proliferation
The human breast cancer cell of exponential phase is pressed per 4000, hole, 96 well culture plates of inoculation, 37 DEG C, 5%CO2Culture
12h is cultivated in case, variable concentrations Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is separately added into cell by 24h, 36h, 48h, 60h after cell attachment
Strain has grown up in 96 porocyte culture plates of individual layer, and each concentration repeats 5 multiple holes, while setting up two control groups：One is
Blank control group (is not added with cell), and one is that (plus RPMI-1640, the concentration of Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is for 0) for cell controls group.
It is placed in 37 DEG C, 5%CO2Culture 24h in incubator.Culture terminates front 4h, and liquid in culture plate is abandoned in suction, and PBS is rinsed 3 times, kept away
MTT (5mg/ml) 20 μ l are added per hole under optical condition, 37 DEG C is placed in, 5%CO2Continue to cultivate 4h in incubator, after removing supernatant
DMSO150 μ l, horizontal shaker concussion 10min, elisa reading instrument colorimetric (wavelength 490nm) is added to survey absorbance per hole.Experiment repeats 3
It is secondary.The inhibitory action of experiment with computing medicine and control drug to breast cancer cell (MCF-7) as follows.Cell inhibitory rate
(%)=(1- medicine effect group absorbances/cell controls group absorbance) × 100%.
2.1.5 flow cytomery volatile oil is on breast cancer cell cycle and apoptotic impact
Human breast cancer cell (MCF-7) is divided into four groups：A groups (blank control group), B groups (0.5mg/ml), C group (1.0mg/
Ml), D groups (1.5mg/ml), Jing after processing, after all cells (adherent and suspension cell) of collection with PBS once, per component
100 μ l buffer are not added, piping and druming is uniform, then is separately added into 3 μ l PI and 3 μ Annexin-V, lucifuge is dyeed 1 hour, added
200 μ l buffer, 200 mesh filter screen filtration cells are collected in flow cytometer loading pipe.Using 488nm laser as exciting
Wavelength, 525nm as Detection wavelength, to 10,000 cell collection fluorescence intensity datas in each sample on flow cytometer.
2.1.6 cycle of flow cytomery breast cancer cell
Growth period different human breast cancer cells (MCF-7) of taking the logarithm are divided into A groups (blank control group), B groups (120 μ g/ml) C
Group (240 μ g/ml), D groups (360 μ g/ml) collect (the adherent and suspension of all cells Jing after different disposal in 15ml centrifuge tubes
Cell) after with PBS once, it is resuspended to be separately added into 1mL precooling PBS per group, and piping and druming is uniform.Centrifuge tube is positioned over into vortex shake
Swing on device, add 9ml70% ice-cold ethanols to fix in concussion, place -20 DEG C overnight.Washed with precooling PBS before dyeing, be centrifuged
(2000r/min, 4 DEG C) 5min, removes fixer, is digested with 500 μ L RNaseA, and 37 DEG C of water-bath 30min add 25 μ L iodine
Change the mixing of the third pyridine (propdium iodide, PI) dyeing liquor, room temperature lucifuge dyeing 30min, 200 mesh filter screen filtration cells are collected
In flow cytometer loading pipe.Flow cytometer carries out DNA content and cell cycle analysis, draws cell each phase of the cycles
Percentage.
2.1.7 statistical analysis
All experimental datas are with mean ± standard deviationRepresent, it is multigroup using SPSS17.0 typings and analyze data
Between data compare using one-way analysis of variance (One-Way ANOVA) check, P<0.05 expression difference has conspicuousness.
2.2 result
2.2.1 the present invention is to human breast cancer cell growth inhibition effect
The impact that Experimental agents are bred to human breast cancer cell (MCF-7) is analyzed using MTT experiment, as a result such as table 2 and Fig. 1
It is shown：
Table 2：Different time Breast cancer lines (MCF-7) proliferation inhibition rate (%,N=3)
Note：
◇：Embodiment group compares with cis-platinum group (positive controls), P values>0.05, i.e. embodiment group compare not with cis-platinum group
There is significant difference, the anti-breast cancer activity for illustrating embodiment group is essentially identical to cis-platinum group.
*：Embodiment group compares with cis-platinum group (positive controls), P values<0.05, i.e. embodiment group compare tool with cis-platinum group
There is significant difference, from its inhibiting rate analysis, embodiment group is higher than cis-platinum group inhibiting rate, illustrates the anti-mammary gland of embodiment group
Cancer activity is higher than cis-platinum group.
The experimental data of table 2 is carried out to understand after statistical analysis, after effect 24 hours, volatile oil of the present invention is to human breast carcinoma
The IC of cell (MCF-7)50For 246 μ g/ml.
Test result indicate that：The volatile oil of the present invention has significant inhibitory action to Breast cancer lines (MCF-7),
Its effect is substantially better than control drug cis-platinum group.And the increase of the prolongation and drug concentration with action time, its inhibitory action
Gradually strengthen, show significantly amount-effect and time-effect relationship.
2.2.2 impact of the flow cytomery Stahlianthus hainanensis (Hayata) T. L. Wu volatile-oil composite to the different human breast cancer cell cycles
Flow cytometry (FCM) result such as Fig. 2 and table 3 show：Different quality concentration Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil acts on human milk
After adenocarcinoma cell (MCF-7) 24h, compare with A groups (blank group), the change of B, C, D group (low middle dose group) cell quantity：
Fig. 2 and Biao 3 illustrate that G0/G1 phases cell quantity increases in B groups, C groups, and S phases, G2/M phases cell proportion are reduced, and D
Group (360 μ g/ml experimental groups) G1 phases cell proportion is reduced, it may be possible to because MCF-7 is arrested in the G1 phases by Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil,
Cause cell apoptosis in human breast cancer, and then cause what G1 phases cell proportion was reduced, point out Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to MCF-7 cells
With obvious G1 phases retardation.
Table 3：Impact (%) of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil to the MCF-7 cell cycles
2.2.3 the apoptosis of flow cytomery breast cancer cell
Jing after flow cytomery Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is acted on 24 hours, the apoptosis of human breast cancer cell (MCF-7)
Rate as shown in Figure 3, Figure 4, can be observed the increase with Stahlianthus hainanensis (Hayata) T. L. Wu volatilization oil concentration, and human breast cancer cell (MCF-7) withers
The ratio died gradually increases.
In the impact to MCF-7 apoptosis, each group compares P=0.00 with blank<0.01, with obvious statistics
Meaning.Apoptosis rate between tri- groups of B, C, D is without significant difference.
Test result indicate that：The basic, normal, high dosage group of Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil presents to MCF-7 human breast cancer cells
Significantly influence, has obvious G1 retardations to MCF-7 cells, can effectively suppress the normal conversion of cell cycle,
Cell is piled up in the G1 phases, cell block prevents the mitosis of cell in the G1 phases, be suppressed cell propagation, so as to reach
To anti-human breast cancer cell active function.
Claims (6)
1. Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is preparing treatment and/or is preventing the application of breast cancer medicines.
2. application as claimed in claim 1, it is characterised in that：Contain active ingredient in the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, the work
Sexual element is made up of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil active ingredient that Stahlianthus hainanensis (Hayata) T. L. Wu is extracted, including monoterpenes compound, monoterpene oxygen
Compound class compound, sesquiterpenoids, the oxide-based compound of sequiterpene.
3. application as claimed in claim 1, it is characterised in that：Contain active ingredient in the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, the work
Sexual element is made up of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil active ingredient that Stahlianthus hainanensis (Hayata) T. L. Wu is extracted, including the composition of following weight proportion：
The 3 of 20-30 weight portions, 6,7,8- tetrahydro -3,3,6,6- tetramethyl cyclopenta [E] indenes -1 (2H) -one, 8-20
The amphene of weight portion；The 1 of 6-15 weight portions, ring [2.2.1] the hept- 2- thatch ketone of 7,7- trimethyl-two, the aromadendrene of 2-8 weight portions；
The caryophyllene oxide of 1-6 weight portions.
4. application as claimed in claim 1, it is characterised in that：Contain active ingredient in the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil, the work
Sexual element is made up of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil active ingredient that Stahlianthus hainanensis (Hayata) T. L. Wu is extracted, including the composition of following weight proportion：
The 3 of 24-28 weight portions, 6,7,8- tetrahydro -3,3,6,6- tetramethyl cyclopenta [E] indenes -1 (2H) -one, 11-15
The amphene of weight portion；The 1 of 8-12 weight portions, ring [2.2.1] the hept- 2- thatch ketone of 7,7- trimethyl-two, the aromadendrene of 3-6 weight portions；
The caryophyllene oxide of 2-5 weight portions.
5. application as claimed in claim 1, it is characterised in that the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is comprised the following steps：
Take Stahlianthus hainanensis (Hayata) T. L. Wu and be ground into meal, the distilled water immersion 0.5-3 hours of medicinal material gross weight 3-7 times are added, by water vapour
3-6 hours are extracted in distillation, collect upper strata essential oil, are obtained final product.
6. application as claimed in claim 1, it is characterised in that the preparation of the Stahlianthus hainanensis (Hayata) T. L. Wu volatile oil is comprised the following steps：
Take Stahlianthus hainanensis (Hayata) T. L. Wu and be ground into meal, plus the distilled water immersion 1h of 5 times of medicinal material gross weight, 4h is extracted by steam distillation,
Upper strata essential oil is collected, is obtained final product.
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| CN201410174008.XA CN104083690B (en) | 2013-07-17 | 2014-04-28 | Application of Stahlianthus involucratus (King) Craib and its extract in preparation of drugs for treating and/or preventing breast cancer |
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| Title |
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| 主要栽培技术对"姜三七"产量与药效成分的影响;李丽淑等;《北方园艺》;20111231(第14期);168-170 * |
| 广西壮药姜三七对雌\孕激素负荷大鼠子宫肌瘤的影响;倪玲等;《时珍国医国药》;20130131;第24卷(第1期);128-130 * |
| 我国姜科药用植物研究Ⅵ姜三七挥发油化学成分分析;方洪钜等;《色谱》;19840130;第1卷(第01期);35-37 * |
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