CN105777518B - Double rhizoma nardostachyos ketenes A compounds and preparation method and application - Google Patents
Double rhizoma nardostachyos ketenes A compounds and preparation method and application Download PDFInfo
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Abstract
The present invention provides double rhizoma nardostachyos ketenes A (Dinardokanshone A,DNK A) compound and preparation method and application,The compound is (2'R,4aR,4'aR,5S,8'S,8'aS) 1 hydroxyl 4a,4',5,1 alkene of 8'a tetramethyl 8'(propane, 2 base) 1',2',4a,4'a,5,5',6,6',7,7',8',Ten dihydros [2 of 8'a,The double naphthalenes of 2'] 3 (4H) ketone,It is to isolate and purify and obtain from the rhizome of Valerianaceae rhizoma nardostachyos platymiscium rhizoma nardostachyos (Nardostachys chinensis Batal.),It is strongly active with 5 hydroxytryptamine transporters,It can be used for depression preparing,Anxiety disorder,Schizophrenia,Obsession,Applied in terms of the medicine of the diseases such as nervus retrogression.
Description
Technical field
The present invention relates to traditional Chinese medicine research field, especially double rhizoma nardostachyos ketenes A compounds and preparation method and application.
Background technology
Serotonin transporter (SERT) is a kind of transmembrane transporter for having high affinity to 5-HT, containing about 630
Amino acid residue, its encoding gene (SLC6A4) are about respectively outside 14 of 35kb by span in No. 7 and No. 11 chromosomes
Aobvious son composition.SERT albumen includes 12~13 transmembrane regions, and N-terminal and C-terminal are located in kytoplasm, has cAMP dependences at N-terminal
Protein kinase binding site, has one to be located at extracellular annulus between the 3rd and the 4th transmembrane region, is the sugar of N- connections
Base site.
SERT belongs to Na+/Cl-Dependent form transport protein, is predominantly located at 5-HT serotonergic neurons.SERT is from nerve synapse gap
In reuptake 5-HT and enter presynaptic neuron, directly affect synaptic cleft 5-HT concentration, change postsynaptic receptor mediation letter
Number amount and acting duration.It moreover has been found that SERT intestinal epithelial cell, blood platelet, placenta tissue, marrow, kidney,
Lung, the heart, adrenal gland, liver, parathyroid gland, thyroid gland, pancreas etc. are also distributed, and prompt SERT to participate in different physiological roles.
SERT is the important molecule for transporting 5-HT, with many Physiological Psychology functions such as mood, appetite, sleep, memory, study
Correlation, SERT and 5-HT expression changes can cause anxiety, depression, obsession, neurosis, or even schizophrenia, and and medicine
It is additive closely related；In addition, being distributed a large amount of SERT on intestinal epithelial cell film, played in gastrointestinal function disease
Key player, such as clinically slow Constipation, irritable bowel syndrome, functional distension functional gastrointestinal disease, use
Antidepressants, anxiolytic drugs for SERT achieve curative effect.
SERT is the important target spot of clinical medicine research and development.Classical antidepressants based on SERT target spots belong to selectivity more
SERT inhibitor (SSRI), such as Prozac (Fluoxetine)；Serotonin reuptake transporter accelerating agent (SSRE) then rare report,
So far reported that SSRE has Tianeptine (tianeptine), alias Tatinol (stablon), its chemical constitution category tricyclic antidepressants resists
Depressant drug, clinic are mainly used for antidepression and antianxiety.Experimental studies have found that Tianeptine can promote 5-HT reuptakes, in nerve
In terms of structure plasticity, Tianeptine to stress/the hippocampal neuron dendron atrophy of cortisone induction has prevention effect, can
Hippocampus precursor propagation, the decline of hippocampus volume and the N- acetyl aspartates concentration for resisting stress induction decline, and prevent apricot
Benevolence core dendron hyperplasia.In terms of nerve excitability, Tianeptine, which can overcome, stress block hippocampal long-term humidification,
Reverse stress be to inhibitory action of hippocampus-preceding cortex cynapse etc..In terms of neuroprotection, Tianeptine can reduce hippocampus and temporo
The Apoptosis of leaves layer.In terms of memory function, Tianeptine to stress reduced space memory impairment there is blocking effect, increase
Add memory to retain, contribute to notice to concentrate (the McEwen BS, Olie such as behavior, the illeffects of antagonism alcohol
JP.2005.Neurobiology of mood,anxiety,and emotions as revealed by studies of a
unique antidepressant: tianeptine.Molecular Psychiatry 10,525–537).In addition, thiophene how
General spit of fland can raise maincenter 5-HT metabolins 5-hydroxyindoleacetic acid, after deduction may be due to presynaptic membrane reuptake -5-HT increases,
5-HT catabolism accordingly increases related in nerve；Tianeptine may act on hypothalamus-pituitary-adrenal axis, make hypothalamus
Cortin releasing factor and anterior pituitary adrenal cortical hormone concentration decline.
Tianeptine is effective to major depressive disorder, is better than Prozac to depression long-term efficacy, and safe, bad anti-
Should lack, be suitable for depression in old age, anxiolytic effect better than Prozac (sprout recklessly, Li Zhen .2007. Tianeptines pharmacological researches and
Clinical advances Guangdong medicine 28 (7)：1192-1193.) Tianeptine includes the action character of human body：It is disorderly to mental state
There is certain effect, between sedating antidepressants and excitability antidepressants；To Somatic discomfort, especially for anxiety and
The disorderly related upset,gastro-intestinal of mental state has obvious effect；The personality and conduct disorder occurred to alcoholism patient during abstinence from alcohol
There is certain effect；Moreover, Tianeptine to following aspect without ill-effect：Sleep and vigilance；Cardiovascular system；Cholinergic system
(nonreactive cholinergic symptoms)；Drug habit.Research has prompted serotonin reuptake transporter accelerating agent (SSRE) in clinical practice above
The characteristics of middle and advantage.
Rhizoma nardostachyos (Nardostachys chinensis Batal.) is Valerianaceae rhizoma nardostachyos platymiscium, has regulating qi-flowing for relieving pain, opens
It is strongly fragrant to be amusing；The effect of external application clearing damp is subsided a swelling, rhizoma nardostachyos it has been reported that bioactivity act on nervous system including (1), such as anti-suppression
Strongly fragrant, calm and anticonvulsion, anti-Parkinson and reminiscence；(2) cardiovascular system is acted on, such as blood pressure lowering, anti-arrhythmia, is resisted
Myocardial ischemia, anti-cardiovascular injury；(3) respiratory system is acted on, such as strengthens hypoxia-bearing capability；(4) it is antibacterial；(5) anti-liver injury
Deng.The chemical composition of rhizoma nardostachyos includes sequiterpene, triterpene, iridoid, cumarin, phenolic acid, flavones etc., sequiterpene be its mainly into
Point, the wherein research such as nardosinone, aristolene report is more, and other active ingredients are particularly a small amount of or micro constitutent rarely has report
Road, bioactivity and related mechanism need to be further discovered that.
The content of the invention
The technical problems to be solved by the invention are to provide double rhizoma nardostachyos ketenes A compounds.
Another technical problem to be solved by this invention is the preparation method for providing above-mentioned double rhizoma nardostachyos ketenes A compounds.
Another technical problem to be solved by this invention is the application for providing above-mentioned double rhizoma nardostachyos ketenes A compounds.
In order to solve the above technical problems, the technical scheme is that：
A kind of double rhizoma nardostachyos ketenes A compounds (Dinardokanshone A, DNK-A), chemical system be named as (2'R,
4aR, 4'aR, 5S, 8'S, 8'aS) -1- hydroxyls -4a, 4', 5,8'a- tetramethyl -8'- (propane -1- alkene -2- bases) -1', 2', 4a,
Ten dihydro of 4'a, 5,5', 6,6', 7,7', 8', 8'a--[the double naphthalenes of 2,2'-] -3 (4H) -one【(2'R,4aR,4'aR,5S,8'S,8'
aS)-1-hydroxy-4a,4',5,8'a-tetramethyl-8'-(prop-1-en-2-yl)-1',2',4a,4'a,5,5',
6,6',7,7',8',8'a-dodecahydro-[2,2'-binaphthalen]-3(4H)-one】, there is following structural formula (I)
Formula：
Above-mentioned double rhizoma nardostachyos ketenes A compounds are formed by connecting by 1 sequiterpene and 1 drop sequiterpene by carbon carbon, on parent nucleus
9'- olefinic carbons hydroxyl can be substituted base acylation, phenolic ether, acid amides chemical conversion ester, phenolic ether or nitrogenous compound.
Preferably, above-mentioned double rhizoma nardostachyos ketenes A compounds, its physical chemistry and Spectroscopic Properties are：White, needle-shaped crystals (dichloro
Methane), it is single spot in various development systems, 10% sulfuric acid ethanol shows purple dot；UV(MeOH)λmax:202nm；
CD (MeOH, c=2.0 × 10-3)λ(Δε):306(+2.65)、267(-3.40)、238(-0.02)、213(-1.22)nm；Nuclear-magnetism
Data are：1H-NMR(CDCl3,600MHz):δH1.81,1.27 (each 1H, m, 2H-1), 1.49~1.66 (2H,
Overlapped, 2H-2), 1.46,1.17 (each 1H, m, 2H-3), 1.97 (1H, m, H-4), 1.60 (1H, dd, J=7.2,
12.6Hz, Ha-6), 1.11 (1H, dd, J=12.0,12.6Hz, Hb-6), 3.93 (1H, m, H-7), 5.43 (1H, s, H-8),
2.05(1H,m,H-10)、4.74,4.73(each 1H,br s,2H-12)、1.74(6H,s,3H-13,3H-15)、0.91(3H,
S, 3H-14), 6.46 (1H, t, J=3.6Hz, H-1'), 2.26 (1H, m, H-2'a), 1.49~1.66 (3H, overlapped,
H-2'b, 2H-3'), 1.63 (1H, m, H-4'), 2.58,2.16 (each 1H, d, J=15.6Hz, 2H-6'), 1.04 (3H, s,
3H-11'), 0.93 (3H, d, J=6.6Hz, 3H-12'), 6.92 (1H, s ,-OH)；13C-NMR data are shown in embodiment portion
Divide table 1.
The preparation method of above-mentioned double rhizoma nardostachyos ketenes A compounds, comprises the following steps that：
(1) rhizoma nardostachyos rhizome per 20Kg with 70% alcohol steep 3 times, every time 48 it is small when, merging extracting solution, is concentrated under reduced pressure, obtains
Crude extract medicinal extract 3Kg；Then put on 70% ethanol heat and state the dregs of a decoction 3 times, every time 2 it is small when, merge extracting solution, be concentrated under reduced pressure, obtain
Crude extract medicinal extract 400g；Merge crude extract twice and obtain total medicinal extract 3.4Kg；
(2) the gained total medicinal extract of crude extract, after moisture dissipates, respectively with isometric petroleum ether, ethyl acetate and n-butanol
Extract successively, obtain petroleum ether part 320g, ethyl acetate extract 1Kg, n-butanol portion 600g, water layer 1.2Kg；
(3) petroleum ether part 320g (is mixed into sample silica gel 100-200 mesh 400g, column silica gel 200-300 mesh through silica gel column chromatography
3.3Kg, petroleum ether：Ethyl acetate=100:0~100:50 solvent system gradient elutions), obtain 22 fraction Fr.1~22；
(4) fraction Fr.5 (petroleum ethers：Ethyl acetate=100:2 solvents elute fraction), through silica gel column chromatography (oil repeatedly
Ether：Ethyl acetate=100:0~100:2 gradient elutions), isolated this pair of rhizoma nardostachyos ketenes A compounds.
Above-mentioned double rhizoma nardostachyos ketenes A compounds answering in terms of promotion serotonin transporter (SERT) active medicine is prepared
With.
Preferably, above-mentioned double rhizoma nardostachyos ketenes A classes compounds are preparing depression, anxiety disorder, schizophrenia, are forcing
Application in terms of the medicines of disease such as disease, nerve degenerative diseases, drug addiction and digestive system function disorder.
Pharmaceutical composition with above-mentioned double rhizoma nardostachyos ketenes A class compounds, comprising treatment and/or prevention effective dose it is double sweet
Loose ketenes A compounds and optional pharmaceutically acceptable excipient.
Above-mentioned pharmaceutically acceptable excipient can be any conventional excipient, particular excipient in field of pharmaceutical preparations
Selection by depending on the administering mode or disease type and state for treating particular patient, the conjunction for specific administration pattern
The preparation method of suitable pharmaceutical composition is completely in the knowledge of drug field technical staff.For example, can as pharmacy
Diluent of the excipient of receiving including pharmaceutical field routine, carrier, filler, adhesive, wetting agent, disintegrant, absorption promote
Into agent, surfactant, absorption carrier and lubricant etc., if necessary, flavouring agent, anti-corrosion can also be added in pharmaceutical composition
Agent and sweetener etc..
Tablet, pulvis, granule, capsule, oral liquid, paste, creme, injection breast can be made in aforementioned pharmaceutical compositions
The diversified forms such as agent, aseptic powder needle for injection.The medicine of above-mentioned various formulations can be according to the conventional method system of pharmaceutical field
It is standby.
The beneficial effects of the invention are as follows：
Double rhizoma nardostachyos ketenes A compounds (Dinardokanshone A, DNK-A) of the present invention, belong to from Valerianaceae rhizoma nardostachyos and planting
Isolate and purify and obtain in the rhizome of thing rhizoma nardostachyos (Nardostachys chinensis Batal.), dropped by 1 sequiterpene and 1
Sequiterpene is formed by connecting by carbon carbon, is a noval chemical compound for having no document report through SciFinder database retrievals, has and promote
Into the function of serotonin transporter (SERT) activity, depression, anxiety disorder, schizophrenia, obsession, nerve can be used as to move back
The medicine of the diseases such as row disease, drug addiction and digestive system function disorder, there is important drug development to be worth.
Brief description of the drawings
Fig. 1 is the chemical structural formula of the double rhizoma nardostachyos ketenes A of compound；
Fig. 2 is humidifications of the double rhizoma nardostachyos ketenes A of compound to SERT activity, wherein, 2 μM of positive control drug
Fluoxetine, * * p<0.01, * * * P<0.001；
Fig. 3 is the double rhizoma nardostachyos ketenes A of compound1H-NMR spectrum；
Fig. 4 is the double rhizoma nardostachyos ketenes A of compound13C-NMR spectrograms；
Fig. 5 is the HSQC correlation spectrograms of the double rhizoma nardostachyos ketenes A of compound；
Fig. 6 is the double rhizoma nardostachyos ketenes A of compound1H-1H COSY figures related to HMBC；
Fig. 7 is the related figures of NOESY of the double rhizoma nardostachyos ketenes A of compound；
Fig. 8 is the judgment rule schematic diagram of the double rhizoma nardostachyos ketenes A absolute configurations of compound；
Fig. 9 is the low resolution ESI-MS mass spectrograms of the double rhizoma nardostachyos ketenes A of compound；
Figure 10 is CD and the UV figure of the double rhizoma nardostachyos ketenes A of compound Chang.
Embodiment
It is below in conjunction with the accompanying drawings and specific real in order to make those skilled in the art be better understood from technical scheme
Mode is applied to be described in further detail technical solution of the present invention.
Laboratory apparatus and reagent：Fourier transform nuclear magnetic resonance spectrometer (Bruker companies of Switzerland, AVIII types 400MHz
And 600MHz)；Color developing agent：10% sulfuric acid ethanol.
Embodiment 1
The preparation (extraction separation process) of the double rhizoma nardostachyos ketenes A of active ingredient：
Rhizoma nardostachyos pharmaceutical decocting piece is purchased from Anhui Jiren Pharmacy Co., Ltd.'s (lot number：110709, specification：1Kg/ bags, the place of production：Four
River), about 20Kg, rhizoma nardostachyos rhizome 20Kg 70% alcohol steep 3 times, every time 48 it is small when, merge extracting solution, be concentrated under reduced pressure, obtain slightly
Extract medicinal extract 3Kg；Then put on 70% ethanol heat and state the dregs of a decoction 3 times, every time 2 it is small when, merge extracting solution, be concentrated under reduced pressure, obtain slightly
Extract medicinal extract 400g；Merge crude extract twice and obtain total medicinal extract 3.4Kg；The total medicinal extract of gained crude extract, after moisture dissipates, use successively etc.
Petroleum ether, ethyl acetate, the n-butanol of volume are extracted, and obtain petroleum ether part 320g, ethyl acetate extract 1Kg, positive fourth
Alcohol position 600g, water layer 1.2Kg；Petroleum ether part 320g (is mixed into sample silica gel 100-200 mesh 400g, column silicon through silica gel column chromatography
Glue 200-300 mesh 3.3Kg, petroleum ether：Ethyl acetate=100:0~100:50 solvent system gradient elutions), obtain 22 fractions
Fr.1~22；Fraction Fr.5 (petroleum ethers：Ethyl acetate=100:2 solvents elute fraction), through silica gel column chromatography (oil repeatedly
Ether：Ethyl acetate=100:0~100:2 gradient elutions), isolated this pair of rhizoma nardostachyos ketenes A compounds (about 20.0mg).
Above-mentioned double rhizoma nardostachyos ketenes A compounds (Fig. 1), white, needle-shaped crystals (dichloromethane), in various development systems,
For single spot, 10% sulfuric acid ethanol shows purple dot；UV(MeOH)λmax:202nm；CD (MeOH, c=2.0 × 10-3)λ(Δ
ε):306(+2.65)、267(-3.40)、238(-0.02)、213(-1.22)nm；Provided in low resolution ESI-MS mass spectrums (Fig. 9)
Molecular ion peak：(-)-ESI-MS m/z 393.21[M-H]-、(+)-ESI-MS:m/z 395.44[M+H]+, thus it is speculated that its molecule
Measure as 394, compose (Fig. 3~4) data with reference to nuclear-magnetism hydrogen carbon, thus it is speculated that its molecular formula is C27H38O2。
1H-NMR(CDCl3, 600MHz, Fig. 3) spectrum in, δH0.91 (3H, s), 0.93 (3H, d, J=6.6Hz), 1.04 (3H,
S) and 1.74 (6H, s) are 5 methyl hydrogen proton signals；δH4.74,4.73 (each 1H, brs) is 2 of exocyclic double bond
Terminal hydrogen proton signal；δH6.46 (1H, t, J=3.6Hz), 5.43 (1H, s) are 2 double bond alkene Hydrogen Proton signals；δH6.92
(1H, s) is associated without any carbon atom signal in hsqc spectrum, is an active hydrogen proton signal.
13C-NMR(CD3OD, 150MHz, Fig. 4) spectrum in, give 27 carbon signals, with reference to hydrogen compose, thus it is speculated that exist in structure
4 groups of double bond (δC163.2、150.5、142.8、136.5、129.5、123.2、117.2、108.8)；δC196.5 be 1 α, β-no
Saturated ketone carbonyl carbon signals.Nuclear magnetic data is shown in Table 1.
Comprehensive HSQC, HMBC spectrogram information (Fig. 5~6), the hydrogen atom (δ on 8 carbonH5.43) with 6,7,10,15 carbon
Atom (δC43.6th, 29.3,47.1,21.5) related, 13 methyl hydrogen atom (δH1.74) with 4,11,12 carbon atom (δC46.7th, 150.5 it is, 108.8) related, 14 methyl hydrogen atom (δH0.91) with 4,5,6,7 carbon atom (δC46.7、33.7、
43.6th, 29.3) related, there are A, B ring sequiterpene fragment as shown in Figure 1 in prompting structure；Hydrogen atom (δ in 6' secondary carbonsH2.58th, 2.16) with 7', 8', 10' carbon atom (δC196.5th, 117.2 it is, 136.5) related, the reactive hydrogen atom on 9' carbon
(δH6.92) with 8', 9', 10' carbon atom (δC117.2nd, 163.2 it is, 136.5) related, 11' methyl hydrogen atom (δH1.04) with
5', 6', 10' carbon atom (δC37.8th, 49.8 it is, 136.5) related, 12' tertiary carbon methyl hydrogen atom (δH0.93) with 3', 5'
Carbon atom (δC26.0th, 37.8) related, there are 12 carbon of C, D ring as shown in Figure 1 in prompting structure to drop sequiterpene fragment；Because of 6
Hydrogen atom (δ on the carbon of positionH1.60th, 1.11) with 8' carbon atom (δC117.2) there are correlation, A, B, C, D ring are passed through into C7-C8'
Key connection is got up.
Nuclear magnetic data (the CDCl of 1. pairs of rhizoma nardostachyos ketenes A compounds of table3,1H-NMR in 600MHz,13C-NMR in
150MHz)
In summary nuclear magnetic data (including1H-1H COSY compose, Fig. 6) gained information, with reference to ESI-MS can release this pair it is sweet
The planar structure of loose ketenes A compounds, can show that relative configuration is as shown in Figure 1 by two-dimentional NOESY spectrum informations (Fig. 7).
In the circular dichroism spectra (CD) (Figure 10) of the compound, 306 (Δ ε:+ 2.65) positive Cotton effects are provided at nm,
267(Δε:-3.40)、238(Δε:- 0.02) and 213 (Δ ε:- 1.22) negative Cotton effects are provided at nm.Ring according to Huang
The research of dragon, C.Djerassi, L.Velluz, H.B.Kagan et al., what n → π * transition of α, β-unsaturation cyclic ketones was presented
Cotton effects are related with the conformation that ring is taken, hexamethylene -2- ketenes and the amyl- 2- ketenes of ring contrast in nature, but both
π → π * transition of conjugated double bond follows same spiral rule.Therefore, as shown in figure 8, with reference to NOESY and CD spectrum informations, judge
Each chiral carbon absolute configuration is respectively 4S, 5S, 7R, 10R, 4'S and 5'R.
To sum up, determine that its structure is as follows according to the physicochemical property of double rhizoma nardostachyos ketenes A and Spectral Characteristic, text is had no for one
Offer the noval chemical compound of report, be named as (4S, 5S, 7R, 10R, 4'S, 5'R)-bis- rhizoma nardostachyos ketenes A [(4S, 5S, 7R, 10R, 4'S,
5'R)-dinardokanshone A,DNK-A]。
Embodiment 2
The double rhizoma nardostachyos ketenes A (Dinardokanshone A, DNK-A) of compound of the present invention are to serotonin transporter
(SERT) influence
Using the hSERT-HEK293 cell lines of stable transfection, with 4- (4- (dimethylamino) phenyl) -1-
Methylpyridinium (APP+) is fluorogenic substrate, and the double rhizoma nardostachyos ketenes A of detection compound live SERT in high intension system
The influence of property.
1) laboratory apparatus and reagent
Laboratory apparatus：
High intension Operetta systems and Columbus data managements and analysis system (PerkinElmer), super-clean bench, is moved
Liquid rifle (1000 μ L, 200 μ L, 20 μ L, 10 μ L, 2.5 μ L, Eppendorf companies of the U.S.)
Reagent and material：
Human embryonic kidney cell line HEK293 (the American Type Culture Collection committee of Chinese Academy of Sciences cell bank), hSERT pcDNA3
Plasmid (Addgene, plasmid 15483), MEM culture mediums (Gibco), APP+ (Sigma), 33342 (Cell of Hoechst
Signaling Technology), 96 orifice plates (Costar 3605)
2) experimental implementation process
Initially set up and identify stable expression hSERT-HEK293 cell lines and { pacify the people source 5- such as of heap of stone, Li Jing, golden blast
The foundation of hydroxytryptamine transporter stable expression cell line and its function investigation [J] military medicines 2011,35 (9):681-684}.
Using APP+ as fluorogenic substrate, function { Fowler A, Seifert N, the Acker V.et based on high intension system detectio SERT
al.A nonradioactive high-throughput/high-content assay for measurement of the
human serotonin reuptake transporter function in vitro[J].Journal of
Biomolecular Screening,2006,11(8):1027-1034}
Specific steps：
(1) precision weighs double rhizoma nardostachyos ketenes A, and the mother liquor of 20mM is configured to DMSO, is diluted with without phenol red MEM bases training base
Medicine is to 10.0 μM, 1.0 μM, 0.1 μM.
(2) 1.0 × 10 are pressed4The density of cells/well is inoculated with the hSERT-HEK293 cells of stable transfection into 96 orifice plates,
37 DEG C, 5%CO2Under the conditions of cultivate 24h.
(3) test and set up blank control group, 2.0 μM of Fluoxetine groups of positive control, double A10.0 μM of rhizoma nardostachyos ketenes,
1.0 μM, 0.1 μM of group.Cell discards culture medium, is washed 2 times with PBS buffer, and each sample to be tested is added according to 80 μ L/ pore volumes,
Each 3 multiple holes of concentration, at 37 DEG C, 5%CO2Under the conditions of lucifuge be incubated 2-3h.
(4) after the completion of being incubated, 20 μ L APP+ are added per hole, are incubated 10 minutes.
(5) liquid in hole is discarded, is washed 2 times with PBS buffer, 1.0 μ g/mL Hoechst, 50 μ L, lucifuge are added per hole
It is incubated 20min.
(6) liquid in orifice plate is discarded, PBS is washed 2-3 times, using the intracellular fluorescence intensity of high intension system detectio
Hoechst 33342 Excitation：360-400nm, Emission：410-480nm
APP+ Excitation：460-490nm, Emission：505-550nm
3) data analysis：
Graphical analysis is carried out using Columbus data managements and analysis system, according to 33342 fluorescence identifyings of Hoechst
Nuclear pattern determines cell, determines SERT transport activities according to intracellular APP+ fluorescence intensities, calculates relative intensity of fluorescence
=(intracellular APP+ fluorescence intensitiesMedicine group/ intracellular APP+ fluorescence intensitiesControl group) carry out ANOVA analyses.
4) experimental result
The double rhizoma nardostachyos ketenes A of experimental result (Fig. 2) display significantly increase SERT transport activities (F (4,47)=436.7, p<
0.0001) and it is in amount-effect dependence, Dunnett's Multiple Comparison post hoc Test confirm 3 surveys
The double rhizoma nardostachyos ketenes A of amount of reagent significantly enhance SERT activity, 10.0 μM of (q=6.47, p compared with the control group<0.001),1.0
μM (q=6.97, p<0.001), 0.1 μM of (q=3.42, p<0.01), 2.0 μM of Fluoxetine of positive controls and blank pair
Compared according to group and significantly suppress SERT activity (q=28.99, p<0.001).
In summary, the serotonin transporter (SERT) is a kind of Na for having high affinity to 5-HT+/Cl-Rely on
Type turns transmembrane transporter, is predominantly located at 5-HT serotonergic neurons, and 5-HT is reuptaked from nerve synapse gap and enters the presynaptic
Neuron, directly affects synaptic cleft 5-HT concentration, changes the amount and acting duration of postsynaptic receptor mediation signal, so that
Participate in a variety of Physiological Psychology functions (such as mood, appetite, sleep, memory, study)；In addition, divide on intestinal epithelial cell film
The a large amount of SERT of cloth, key player is played in gastrointestinal function disease, is such as clinically used to treat slow Constipation, intestines
The functional gastrointestinal diseases such as road irritable syndrome, functional distension achieve curative effect.In addition, in placenta tissue, genital tract, bone
The organs such as marrow, kidney, lung, the heart, adrenal gland, liver, parathyroid gland, thyroid gland, pancreas and small intestine are distributed, and prompt SERT to participate in more
Kind physiological function.
SERT is the important target spot of clinical medicine research and development, and the serotonin reuptake transporter accelerating agent (SSRE) reported so far has thiophene
Nai Puting (tianeptine), clinic is mainly used for antidepression and antianxiety.Tianeptine includes the action character of human body：It is right
Mental state disorder has certain effect, between sedating antidepressants and excitability antidepressants；It is especially right to Somatic discomfort
There is obvious effect in anxiety and the disorderly related upset,gastro-intestinal of mental state；The personality that occurs to alcoholism patient during abstinence from alcohol and
Conduct disorder has certain effect；Moreover, Tianeptine to following aspect without ill-effect：Sleep and vigilance；Cardiovascular system；Courage
Alkali energy system (nonreactive cholinergic symptoms)；Drug habit.Tianeptine belongs to a kind of SSRE, its action character has prompted 5- hydroxyl colors
The characteristics of amine reuptake accelerating agent (SSRE) is in clinical practice and advantage.
The present invention is transported by the way that isolated double rhizoma nardostachyos ketenes A from rhizoma nardostachyos rhizome are carried out with the external serotonin that influences
The research of body (SERT) activity, it is found that double rhizoma nardostachyos ketenes A can significantly increase SERT transport activities, so as to prompt double rhizoma nardostachyos ketenes
A can by adjusting a variety of nervous systems of SERT active treatments and gastronintestinal system function disease, can prepare be used for treat depression,
The nervous system diseases such as anxiety disorder, schizophrenia, obsession, nerve degenerative diseases, drug addiction and slow transporting
Applied in terms of the medicine of the digestive system function disorders such as constipation, irritable bowel syndrome and functional distension.
Embodiment 3
Preparation method：Double rhizoma nardostachyos ketenes A, newborn sugar and starch are uniformly mixed according to the above ratio, cross 200 mesh sieves, it is equal with water
Mixture after wetting, is dried re-sieving, adds magnesium stearate by even wetting, and then by mixture tabletting, every weighs 250mg,
Active component content is 10mg.
Embodiment 4
Capsule：Double rhizoma nardostachyos ketenes A 20mg
Galactolipin 188mg
2 mg of magnesium stearate
Preparation method：Double rhizoma nardostachyos ketenes A are uniformly mixed with galactolipin according to the above ratio, cross 200 mesh sieves, it is mixed what is obtained
Compound, adds magnesium stearate, loads No. 2 capsules, to obtain the final product.
Above-mentioned reference embodiment carries out this pair of rhizoma nardostachyos ketenes A compound and preparation method and application detailed
Description, is illustrative rather than limited, several embodiments can be included according to limited scope, therefore do not departing from
Changing and modifications under present general inventive concept, should belong within protection scope of the present invention.
Claims (5)
- A kind of 1. double rhizoma nardostachyos ketenes A compounds, it is characterised in that：For (2'R, 4aR, 4'aR, 5S, 8'S, 8'aS) -1- hydroxyls - 4a, 4', 5,8'a- tetramethyl -8'- (propane -1- alkene -2- bases) -1', 2', 4a, 4'a, 5,5', 6,6', 7,7', 8', 8'a- ten Dihydro-[2,2'- double naphthalenes] -3 (4H) -one, has structure shown in formula (I)：
- 2. the preparation method of double rhizoma nardostachyos ketenes A compounds according to claim 1, it is characterised in that：Comprise the following steps that：(1) rhizoma nardostachyos rhizome per 20Kg with 70% alcohol steep 3 times, every time 48 it is small when, merging extracting solution, is concentrated under reduced pressure, obtains and slightly carry Thing medicinal extract 3Kg；Then put on 70% ethanol heat and state the dregs of a decoction 3 times, every time 2 it is small when, merge extracting solution, be concentrated under reduced pressure, obtain and slightly carry Thing medicinal extract 400g；Merge crude extract twice and obtain total medicinal extract 3.4Kg；(2) the gained total medicinal extract of crude extract, after moisture dissipates, respectively with isometric petroleum ether, ethyl acetate and n-butanol successively Extraction, obtains petroleum ether part 320g, ethyl acetate extract 1Kg, n-butanol portion 600g, water layer 1.2Kg；(3) by petroleum ether part 320g through silica gel column chromatography, wherein, mix sample silica gel 100-200 mesh 400g, column silica gel 200-300 Mesh 3.3Kg, petroleum ether：Ethyl acetate=100:0~100:50 solvent system gradient elutions, obtain 22 fraction Fr.1~22；(4) fraction Fr.5 is petroleum ether：Ethyl acetate=100:2 solvents elute fraction, through silica gel column chromatography repeatedly, wherein, stone Oily ether：Ethyl acetate=100:0~100:2 gradient elutions, isolated this pair of rhizoma nardostachyos ketenes A compounds.
- 3. double rhizoma nardostachyos ketenes A compounds described in claim 1 are in terms of promotion serotonin transporter active medicine is prepared Using.
- 4. double rhizoma nardostachyos ketenes A classes compounds described in claim 1 prepare treatment depression, anxiety disorder, schizophrenia, Obsession, nerve degenerative diseases, drug addiction or digestive system function disorders medicine in terms of application.
- 5. the pharmaceutical composition with double rhizoma nardostachyos ketenes A class compounds described in claim 1, it is characterised in that：Comprising treatment and/ Or double rhizoma nardostachyos ketenes A compounds and the optional pharmaceutically acceptable excipient of prevention effective dose.
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