CN107595892A - Application of the toad cake extract in terms of T type calcium ion channel blockor medicines are prepared - Google Patents
Application of the toad cake extract in terms of T type calcium ion channel blockor medicines are prepared Download PDFInfo
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- CN107595892A CN107595892A CN201710756136.9A CN201710756136A CN107595892A CN 107595892 A CN107595892 A CN 107595892A CN 201710756136 A CN201710756136 A CN 201710756136A CN 107595892 A CN107595892 A CN 107595892A
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- toads
- type calcium
- dried venom
- bufalin
- spirit
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Abstract
The invention discloses a kind of bufo gargarizans Cantor toad cake extract, dried venom of toads spirit (English name：Bufalin), the application in terms of T type calcium ion channel blockor medicines are prepared.The compound produces blocking effect by non-G G-protein linked receptors and non-tyrosine kinase receptor approach to T types calcium channel, can build the T types calcium ion channel blockor comprising dried venom of toads spirit to carry out clinical analgesic application.
Description
Technical field
The present invention relates to biochemical field, and in particular to a kind of clever (English name of toad cake extract dried venom of toads：Bufalin) exist
Prepare the application in terms of T- type calcium ion channel blockors.
Background technology
The dried venom of toads is China's tradition rare Chinese medicine,《Chinese Pharmacopoeia》Version clear stipulaties this product in 2010 is Bufonidae animal China
Bufo or the drying secretion of Bufo melanostictus.The traditional Chinese medical science thinks there is detumescence, clearing heat and detoxicating, the effect of analgesic, is mainly used in tooth
Bitterly, the diseases such as scrofula, abscess of throat, vomiting and diarrhoea.Modern pharmacology research shows that the dried venom of toads has antitumor, analgesia, local anaesthesia, anti-inflammatory etc.
Effect.The dried venom of toads is widely used in clinic at present, in treatment angiocardiopathy (such as angina pectoris), antitumor and analgesia side
Face is evident in efficacy.Dried venom of toads spirit (bufalin) is a kind of compound extracted from the dried venom of toads, molecular formula C24H34O4, average molecular matter
Measure as 386.5.A series of reports show that bufalin has the effect such as cardiac stimulant, immunological regulation, antitumor, anti-inflammatory, analgesia.Before
Research show that bufalin has clear and definite analgesic effect in animal nerve pathologic pain model, and without habituation and tolerance
Property, persistent can be as the medicine of new pain therapy.But up to the present, it produces the target spot and machine of analgesic activity
Make not clear.
Voltage sensitive calcium channel is divided into three families:L-type calcium channel (or Cav1), neuron N-, P/Q- and
R- types (or Cav2) and T- types calcium channel (or Cav3).Wherein, T- types calcium channel is a kind of gated ion of low pressure
Passage, there is unique electrophysiological characteristics：After one small depolarising, cell membrane fast approaching resting membrane electric potential can be made, and then
Neuronal excitability is adjusted, participates in pain nerve signal transmission.So far, molecular biology has identified three kinds of low pressure gates
T- type calcium channels subunit, respectively Cav3.1, Cav3.2 and Cav3.3.The abnormal low pressure gate T- type calcium of studies have shown that leads to
Road expression can cause the neurogenic pains such as epilepsy, glioma and cancer pain, diabetic pain, trigeminal neuralgia.This
Outside, pharmacology, science of heredity and function assessment research all show that peripheral neurons T- types calcium channel can promote and amplify from periphery
Pain signal.Therefore, membrane excitability is changed based on low pressure gate T- types calcium channel, and then influences the pain sensation, can be considered as to control
Treat the potential therapy target of acute and chronic pain.
However, whether toad cake extract dried venom of toads spirit (bufalin) act to T- types calcium channel, at present simultaneously
Without any report.
The content of the invention
Present invention aims at provide the toad cake extract dried venom of toads clever (bufalin) to prepare T- type calcium ion channel blockors
The application of aspect.
In order to solve the above problems, technical scheme provided by the invention is：The dried venom of toads spirit (bufalin) prepare T- types calcium from
Application in terms of subchannel blocking agent medicine.
The optimal technical scheme of the present invention, the dried venom of toads clever (bufalin) are played by suppressing T- types calcium channel
Effect in nervous system disease, particularly it is used for the application for preparing analgesic.
The optimal technical scheme of the present invention, dried venom of toads spirit suppress DRG neuron T- type calcium channel electric currents, have dosage
Dependence, its effective dose (IC50) it is 17 μM.
The second aspect of the present invention provides a kind of pharmaceutical composition, is T- type calcium channel inhibitors, it, which contains, has
Imitate the dried venom of toads clever (bufalin) of composition and pharmaceutically acceptable auxiliary material.
Relative to scheme of the prior art, the advantage of the invention is that：Present invention is disclosed the dried venom of toads clever (bufalin) to exist
The application in terms of T- type calcium ion channel blockors is prepared, the dried venom of toads clever (bufalin) can lead to by suppressing T- type calcium ions
Road plays the clinical treatment in terms of the nervous system disease (such as easing pain).
Brief description of the drawings
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Fig. 1：Mouse dorsal root ganglion neurons T- types calcium channel detects and checking；Wherein Figure 1A be recorded it is small
Diameter DRG neurons T- type calcium channel electric currents.Figure 1B is various concentrations NiCl2To minor diameter DRG neurons T- types calcium from
The statistical chart of the inhibitory action of subchannel.
Fig. 2：Dried venom of toads spirit can dose-dependent inhibition T- type calcium channel electric currents；Fig. 2A is the chemical constitution of dried venom of toads spirit
Figure.Fig. 2 B show that dried venom of toads spirit suppresses the current diagram of T- type calcium channels in 100 μM of concentration.Fig. 2 C are dried venom of toads spirit suppression
The amount effect curve of T- types calcium channel processed.
Fig. 3：Had quick access to information non-acceptor and non-protein kinase pathways of the dried venom of toads suppress T- type calcium channels；Fig. 3 A are to divide in electrode
G- protein inhibitors GDP- β-S, protein kinase A inhibitor PKI 6-22 are not given, and after tyrosine kinase inhibitor PKI-PA,
Suppression of the dried venom of toads spirit to T- type calcium channels is not influenceed.Fig. 3 B are that cell incubates bath Na+/K+- ATP enzyme blocking agent unabain, and
Dried venom of toads spirit is not influenceed to T- type Calcium Currents；Fig. 3 C are Fig. 3 B column statistical chart.
Embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate
The present invention and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done according to the condition of specific producer
Further adjustment, unreceipted implementation condition is usually the condition in normal experiment.
Embodiment
The present embodiment uses the recording method of voltage clamp in whole-cell patch-clamp, records T- type calcium channel electric currents, grinds
Study carefully influence of the dried venom of toads clever (bufalin) to mouse dorsal root ganglion neurons T- type calcium channel electric currents, and illustrate its mechanism.
1) detection of mouse dorsal root ganglion neurons T- types calcium channel and checking
Experiment material
Kunming mouse, body weight 18-22g, provided by University Of Suzhou's animal center, animal quality certification number：SYXK (Soviet Union)
2007-0035。NiCl2Purchased from Sigma companies.
Experimental program
Microsurgery takes DRG, enzyme process separation DRG neurons, using Whole-cell recording minor diameter DRG neurons T-
Type calcium channel electric current, 5-7 cell of every group of record.Embodiment have detected NiCl2, and to mouse minor diameter, (cell space diameter is less than
30 microns) influences of dorsal ganglion unit T- type calcium channel electric currents.As a result it is as shown in Figure 1.Whole-cell patch-clamp result shows,
10 μM and 100uM NiCl2It is able to suppress minor diameter DRG neuron T- type calcium channels electric current 65.3% and 82.6%.
Figure 1A is the minor diameter DRG neuron T- type calcium channel electric currents that recorded.DRG neurons are clamped down in -110mV, stimulation side
For ripple to -40mV, stimulation time-histories is 40ms.Figure 1B be various concentrations NiCl2 (10 μM, n=5；100 μM, n=6) to minor diameter DRG
The statistical chart of the inhibitory action of neuron T- type calcium channels.**p<0.01；***p<0.001vs.control.
2) dried venom of toads clever (bufalin) can dose-dependent inhibition T- type calcium channel electric currents.
Experiment material
Dried venom of toads spirit (bufalin) (lot number：SLBQ5349V) it is purchased from Sigma Co., USA.
Experimental program：
Embodiment application Whole-cell recording mouse minor diameter DRG neuron T- type calcium channel electric currents, every group
Record 5-7 cell.Detect influence of the dried venom of toads clever (bufalin) to DRG neuron T- type calcium channel electric currents.As a result as schemed
Shown in 2, whole-cell patch-clamp result shows that the dried venom of toads clever (bufalin) can suppress DRG neuron T- type calcium channel electric currents,
And there is dose dependent.Fig. 2A is the dried venom of toads clever (bufalin) chemical structural drawing.Fig. 2 B show that the dried venom of toads is clever (bufalin)
Suppress the current diagram of T- type calcium channels in 100 μM of concentration.After dried venom of toads spirit (bufalin) elution, electric current can part
Recover.DRG neurons are clamped down in -70mV, stimulate square wave use -110mV to -40mV, stimulation time-histories is 40ms.Fig. 2 C show toad
Crisp spirit (bufalin) can dose-dependent inhibition T- type calcium channel electric currents.Concentration dependency curves use sigmoidal
Hill equations [Y=1/ (1+10(log IC50-X)nH], wherein X is the logarithm value of concentration, and IC50 is median effective dose, through Prism
5.0 (Graph-Pad Software Inc., CA, USA) softwares are fitted.Bracket inner digital is that each concentration dried venom of toads is clever (bufalin)
The cell record number of effect.As a result it is 41.13 ± 3.17% (n=5), effective dose 50 (IC to show its maximal percentage inhibition50)
For 17 μM.
3) dried venom of toads clever (bufalin) suppresses T- types calcium channel independent of G-protein coupling receptor, protein-tyrosine
Kinases receptors and Na+/K+- ATP enzyme approach.
Experiment material：
G-protein blocking agent GDP- β-S, Na are used in experiment+/K+- atpase inhibitor unabain (ouabain), receptor tyrosine
Kinase blocking agent protein kinase inhibitor peptide A (PKI-PA) and protein kinase A inhibitor PKI 6-22
It is purchased from SIGMA companies of the U.S..
Experimental program：
Embodiment blocks G- albumen, receptor tyrosine kinase and albumen respectively by GDP- β-S, PKI-PA and PKI 6-22
The signal path of kinases A mediations, to detect the mechanism that the dried venom of toads clever (bufalin, 100 μM) suppresses T- type calcium channels.Such as figure
G- protein inhibitor GDP- β-S (1mM), tyrosine kinase inhibitor PKI-PA (1 μM) are given shown in 3A, in recording electrode respectively
And protein kinase A inhibitor PKI 6-22 (1 μM), it can not block 100 μM of dried venom of toads spirits (bufalin) to lead to T- type calcium ions
Suppression (GDP- β-S the inhibiting rates %=35.9 ± 5.7, n=6 in road；PKI-PA inhibiting rates %=37.2 ± 4.9, n=8；PKI
6-22 inhibiting rates %=36.5 ± 3.8, n=6).This result shows the dried venom of toads clever (bufalin) to low-voltage T- type Calcium Currents
Inhibitory action, neither by receptor-independent pathway (non-G- albumen lotus root joins acceptor and non-tyrosine kinase receptor), also independent of
In protein kinase A phosphorylation.As shown in Figure 3 B, it is extracellular give Na+/K+-ATP enzyme blocking agent unabains (ouabain) (1 μM,
N=6 DRG neuron T- type calcium channels electric currents (ouabain inhibiting rates %=1.7 ± 2.1, n=6)) are not changed, and
100 μM of dried venom of toads spirits (bufalin) then can further suppress ouabain and incubate the DRG neuron T- type calcium channel electric currents bathed
(bufalin inhibiting rates %=37.2 ± 5.6, n=6).Show that direct blocking of the dried venom of toads clever (bufalin) to T- type calcium channels is made
With being not rely on Na+/K+- atpase activity.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
For personnel it should be appreciated that the present invention is not limited by examples detailed above, simply illustrating described in examples detailed above and specification is of the invention
Principle, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these change and
Improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its is equal
Thing defines.
Claims (4)
- A kind of 1. application of toad cake extract dried venom of toads spirit in terms of T- type calcium ion channel blockor medicines are prepared.
- 2. purposes according to claim 1, it is characterised in that dried venom of toads suppression T- type calcium channels of having quick access to information are used for Prepare analgesic.
- 3. purposes according to claim 1, it is characterised in that dried venom of toads spirit suppresses DRG neuron T- types calcium channel electricity Stream, has dose dependent, its effective dose (IC50) it is 17 μM.
- 4. a kind of T- types calcium channel inhibitor, it is characterised in that its dried venom of toads for containing active ingredient is clever and can pharmaceutically connect The auxiliary material received.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102247335A (en) * | 2011-06-02 | 2011-11-23 | 江苏省中医药研究院 | Bufalin dry powder inhalant as well as preparation method and application thereof |
| CN105125592A (en) * | 2015-10-12 | 2015-12-09 | 北京诺康达医药科技有限公司 | Medicine containing toad venom lipid-soluble substances and preparation method thereof |
| CN106344588A (en) * | 2016-10-13 | 2017-01-25 | 苏州大学 | Application of bufalin in preparation of L-type calcium channel blocker |
-
2017
- 2017-08-29 CN CN201710756136.9A patent/CN107595892A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102247335A (en) * | 2011-06-02 | 2011-11-23 | 江苏省中医药研究院 | Bufalin dry powder inhalant as well as preparation method and application thereof |
| CN105125592A (en) * | 2015-10-12 | 2015-12-09 | 北京诺康达医药科技有限公司 | Medicine containing toad venom lipid-soluble substances and preparation method thereof |
| CN106344588A (en) * | 2016-10-13 | 2017-01-25 | 苏州大学 | Application of bufalin in preparation of L-type calcium channel blocker |
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Application publication date: 20180119 |
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