CN1500486A - Special ingredient extract of tetrandra root and its use - Google Patents

Special ingredient extract of tetrandra root and its use Download PDF

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CN1500486A
CN1500486A CNA021464855A CN02146485A CN1500486A CN 1500486 A CN1500486 A CN 1500486A CN A021464855 A CNA021464855 A CN A021464855A CN 02146485 A CN02146485 A CN 02146485A CN 1500486 A CN1500486 A CN 1500486A
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radix stephaniae
stephaniae tetrandrae
sprst
tetrandrine
special composition
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CN1203853C (en
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陈介甫
周正仁
沈郁强
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Abstract

The present invention discloses special extractives from tetrandra root and their use. The special extractives include tetrandrine, fangchinoline, cyclanoline and oblongine as four alkaloids and other physically active effective components. The medicine prepared with the four alkaloids and other physically active effective components has antiphlogistic effect and the effect of protecting heart against anaerobic damage.

Description

Special composition extract of Radix stephaniae tetrandrae and uses thereof
Technical field
The present invention relates to special composition extract of a kind of Radix stephaniae tetrandrae and uses thereof.
Background technology
The anti-inflammatory of the special composition extract of Radix stephaniae tetrandrae (SPRST) act as its contained Tetrandrine (Tetrandrine; TETRA), the peaceful (Fangchinoline of Radix Stephaniae Tetrandrae promise; FAN) and the result of other active ingredient combined effect, can not reach the special composition extract of Radix stephaniae tetrandrae (SPRST because be equivalent to the Tetrandrine (0.1 μ g/ml) and the Radix Stephaniae Tetrandrae promise peaceful (0.1 μ g/ml) of the contained dosage of the special composition extract of Radix stephaniae tetrandrae (SPRST); 10 μ g/ml) identical effect [annotate: the special composition extract of Radix stephaniae tetrandrae (SPRST) contains 1.3% Tetrandrine (TETRA) and 0.7% Radix Stephaniae Tetrandrae promise peaceful (FAN)].Summing up the special composition extract of Radix stephaniae tetrandrae (SPRST) is the special extracting composition of Chinese medicine of a tool anti-inflammatory effect low cytotoxicity, and it can be via the flow of calcium ions (calcium influx) and living radical (the reactiveoxygen species that suppress G albumen (Gprotein) regulation and control; ROS) generation and prevent that neutrophils from can not show huge adhesion molecule (Mac-1 by overactivity; CD11b/CD18) reach its anti-inflammatory and protection heart anoxia injury (ischemic reperfued injury; I/R) effect.
Radix Stephaniae Tetrandrae is a kind of medicine plant commonly used, in Bencao Shiyi, puts down in writing, and Radix Stephaniae Tetrandrae master aqueous vapor, Radix cocculi laurifolii main air gas, and in property of medicine book on Chinese herbal medicine, put down in writing, it is painful that Radix Stephaniae Tetrandrae is controlled the oblique brothers of wet wind actinal surface, looses to stay expectorant, main lung qi to cough and breathe heavily ...Descendant's research thinks that the Chinese medicinal plant that belongs to Radix Stephaniae Tetrandrae has Radix stephaniae tetrandrae (Stephania tetrandra s.Moore), Radix cocculi laurifolii (Cocculus trilobus (Thunb.) DC.), Radix Aristolochiae Fangchi (Aristolochia weslandi), radix aristolochiae heterophyllae (Aristolochia heterophylla), Sinomenium acutum (Sinomenium acutum).These plants are Radix stephaniae tetrandrae (S.tetrandra) only, and Radix cocculi laurifolii (C.trilobus) belongs to Menispermaceae (Menispermaceae) plant.And Radix Aristolochiae Fangchi (A.weslandi), radix aristolochiae heterophyllae (A.heterophylla) is Aristolochiaceae (Aristolochiaceae) plant.Radix Aristolochiae Fangchi and radix aristolochiae heterophyllae are not menispermaceous plants, are the wrong usefulness of Radix Stephaniae Tetrandrae.Japan then uses Sinomenium acutum medicinal as radix aristolochiae heterophyllae (A.heterophylla).The radix aristolochiae heterophyllae in ancient times (A.heterophylla) is the tuber of Menispermaceae liana Radix stephaniae tetrandrae (S.tetrandra).
Often only use Radix Stephaniae Tetrandrae in early days on the Chinese medicine, not chatting bright is to adopt radix aristolochiae heterophyllae or Radix cocculi laurifolii.And Wenbing Tiaobian, Detailed Analysis of Epidemic Warm Diseases is formed a surname's numbness soup with Radix Stephaniae Tetrandrae compatibility Semen Coicis, silkworm excrement, is used for the treatment of rheumatic arthralgia.
The contained composition of Chinese medicinal plant Radix Stephaniae Tetrandrae is two different quinone quinolines of benzyl (bisbenzylisoquinoline), protoberberine (protoberberine), the morphinan (morphinane) and luxuriant and rich with fragrance types such as (phenanthrene) of alkaloid (alkaloids).Radix stephaniae tetrandrae (S.tetrandra) contains Tetrandrine (Tetrandrine, TETRA), the peaceful (fangchinoline of Radix Stephaniae Tetrandrae promise, FAN), (-)-Cissamine (cyclanoline, CYC), OBL oblongine (oblongine, OBL), remote Buddhist nun believes (menisine), remote Buddhist nun's fourth compositions such as (menisidine).Radix cocculi laurifolii (C.trilobus) contains the Luo Bin that quenches (trilobine), the different Luo Bin of quenching (isotrilobine), the floating shore (magnoflorine) of agate, collection postscript quick (trilobamine), Ke Luobin (coclobine), remote sarin (menisarine), moves sarin compositions such as (normenisarine).Radix Aristolochiae Fangchi of Aristolochiaceae (A.weslandi) and radix aristolochiae heterophyllae (A.heterophylla) then contain Aristolochic Acid (aristolochic acid).
Claim the main effectively pharmacology composition of Radix stephaniae tetrandrae plant that Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) are arranged according to Tang W in 1992 and Eisenbrand G at Chinese Drugs of PlantOriginal works 963-978 page or leaf.Report in nineteen thirty-five J.Bio.Chem the 109th volume 681-685 page or leaf according to Chen KK and Chen AL, Tetrandrine (TETRA) architectural feature is the alkaloid of two different quinone quinolines of benzyl (bis-benzylisoquinoline).People such as Felix JP are known best natural calcium ion channel blockors in Biochemistry the 31st volume 11793-11800 page or leaf report Tetrandrine (TETRA) in 1992, and many scholars report many other modern pharmacology effects respectively, comprise that Huang YT and Hong CY have the effect of the cardiovascular system of adjustment in Cardiovasc Drug Rev the 16th volume 1-15 page or leaf report Tetrandrine (TETRA) in 1998; People such as DeConti RC have antitumaous effect in the 96th page of report of Cancer Res Am Soc ClinOncol in 1975 the 16th volume, and people such as Shen YC has the anti-inflammatory effect in Mol Pharmacol the 55th volume 186-193 page or leaf report Tetrandrine in 1999.And the modern pharmacology effect of Radix Stephaniae Tetrandrae promise peaceful (FAN), people such as Ma JY possess antioxidation in 1 992 years Exp Lung Res the 18th volume 829-843 page or leaf report, and people such as Choi HS has the anti-inflammatory effect in JEthnopharmacol the 69th volume 173-179 page or leaf report in 2000; People such as Kim HS in J Ethnopharmacol in 1997 the 58th volume 117-123 page or leaf report its calcium channel blocking-up and vasorelaxation action than Tetrandrine (TETRA) a little less than.
People such as inventor Yu XC are in calendar year 2001 Life Sci the 68th volume 2863-2872 page or leaf report; discovery contains the effect of Radix stephaniae tetrandrae extract tool protection ischemia (oxygen) heart of 10% Tetrandrine (TETRA), and it is tired quite and does not again have the side effect of typical calcium ion channel blockor (verapamil) with Tetrandrine (TETRA).
Williams FM was in Role of neutrophils in reperfusion injury in 1994, and in:Immunopharmacology of Neutrophils 245-257 page or leaf works is pointed out neutrophils (neutrophil; PMN) activation is adhered (adhesion) and is moved the injury tool critical role of apprentice (transmigration) to ischemia (oxygen) heart.The sticking action of Neutrophils is the early stage important phenomenon of inflammation, people such as Albelda SM must be adhered to endotheliocyte earlier in FASEB J the 8th volume 504-512 page or leaf report Neutrophils in 1994, just can pass blood vessel wall (transmigration) to soak into Inflamed tissue, carry out its defendance function.Ley K reports this adhesion process in Cardiovasc Res the 32nd volume 733-742 page or leaf in 1996 can be subdivided into for three phases: rolling phase (rolling), pot-life (activation) and tight adhesion phase (firm adhesion); The adhesion molecule (adhesion molecules) that the different times Drawing upon is different.Wherein the tight adhesion of participating in huge adhesion molecule (Mac-1 or claim macrophageadhesion molecule-1, or claim CD11b/CD18) (firm adhesion) is the most important.
Summary of the invention
The present invention's purpose provides the special composition extract of a kind of Radix stephaniae tetrandrae.
Another object of the present invention provides a kind of tool anti-inflammatory effect of the special composition extract of Radix stephaniae tetrandrae and medicine of protection heart anoxia injury effect of containing.
The present invention's extract includes that Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
The special composition extract of the present invention's Radix stephaniae tetrandrae, the mobile phase of its high-performance liquid chromatograph collection of illustrative plates are the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution, utilizing linear gradient to dash and carry, the detection wavelength is 280nm; Four kinds of compositions can separate fully in 45 minutes, and its quantitative linearity returns the concentration of agenda formula and correlation coefficient between 12.5～1637 μ g/ml scopes, and detectable limit is calculated according to the S/N=3 regulations; Buffer solution KH 2PO 4-H 2O is with KH 2PO 4Water-soluble, and the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution is H 2O: CH 3CN: KH 2PO 4The ratio allotment of=400ml: 600ml: 5g.
The special composition extract of the Radix stephaniae tetrandrae of a kind of tool anti-inflammatory effect, it comprises, and Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
The special composition extract of Radix stephaniae tetrandrae of a kind of tool protection heart anoxia injury effect, it comprises, and Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
Description of drawings
Fig. 1 is the high-performance liquid chromatograph collection of illustrative plates
(1) OBL oblongine (oblongine, OBL)
(2) (-)-Cissamine (cyclanoline, CYC)
(3) the Radix Stephaniae Tetrandrae promise peaceful (fangchinoline, FAN)
(4) Tetrandrine (tetrandrine, TETRA)
Fig. 2 is that Fu Jiaan (fMLP) also uses inhibition Neutrophils adhesion (adhesion) in human Fibrinogen with the special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN).* expression and Fu Jiaan (fMLP) individual processing group are received relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. Fu Jiaan (fMLP) is also with Radix Stephaniae Tetrandrae promise peaceful (FAN)
2. Fu Jiaan (fMLP) is also with Tetrandrine (TETRA)
3. Fu Jiaan (fMLP) is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Fig. 3 is leukotriene (LTB 4) also be adhered to human Fibrinogen with the special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) with the inhibition Neutrophils.* expression and leukotriene (LTB 4) the individual processing group receives relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. leukotriene is also with Radix Stephaniae Tetrandrae promise peaceful (FAN)
2. leukotriene is also with Tetrandrine (TETRA)
3. leukotriene is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
To be Fu Jiaan (fMLP) move apprentice (transmigration) with the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) and with inhibition Neutrophils (PMN) to Fig. 4.* expression and Fu Jiaan (fMLP) individual processing group are received relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. Fu Jiaan (fMLP) is also with Radix Stephaniae Tetrandrae promise peaceful (FAN)
2. Fu Jiaan (fMLP) is also with Tetrandrine (TETRA)
3. Fu Jiaan (fMLP) is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Fig. 5 is leukotriene (LTB 4) move the apprentice with the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) and with inhibition Neutrophils (PMN).* expression and leukotriene (LTB 4) the individual processing group receives relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. leukotriene is also with Radix Stephaniae Tetrandrae promise peaceful (FAN)
2. leukotriene is also with Tetrandrine (TETRA)
3. leukotriene is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Fig. 6 is Fu Jiaan (fMLP) and the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) and uses and suppress the huge adhesion molecule of Neutrophils (PMN) (Mac-1, the cartogram of performance CD11b/CD18).* expression and Fu Jiaan (fMLP) individual processing group are received relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. matched group (not having any drug treating)
2. Fu Jiaan (fMLP)
3. Fu Jiaan (fMLP) is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. Fu Jiaan (fMLP) is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. Fu Jiaan (fMLP) is also with 5 μ g/ml Tetrandrine (TETRA)
6. Fu Jiaan (fMLP) is also with 10 μ g/ml Tetrandrine (TETRA)
7. Fu Jiaan (fMLP) is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. Fu Jiaan (fMLP) is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Fig. 7 is leukotriene (LTB 4) with the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) and with suppressing the huge adhesion molecule of Neutrophils (PMN) (Mac-1, CD11b/CD18) cartogram of performance.* expression and leukotriene (LTB 4) the individual processing group receives relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. matched group (not having any drug treating)
2. leukotriene (LTB 4)
3. leukotriene (LTB 4) also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. leukotriene (LTB 4) also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. leukotriene (LTB 4) also with 5 μ g/ml Tetrandrine (TETRA)
6. leukotriene (LTB 4) also with 10 μ g/ml Tetrandrine (TETRA)
7. leukotriene (LTB 4) also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. leukotriene (LTB 4) also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Fig. 8 is typical flow cytometry figure, and cell hydrogen peroxide (H is described 2O 2) generation.
1. matched group (not having any drug treating)
2. Fu Jiaan (fMLP)
3. Fu Jiaan (fMLP) is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Fig. 9 is typical flow cytometry figure, and superoxide anion (O in the cell is described 2 *-) generation.
1. matched group (not having any drug treating)
2. Fu Jiaan (fMLP)
3. Fu Jiaan (fMLP) is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Figure 10 is that the special composition extract of Radix stephaniae tetrandrae (SPRST) and Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) suppress superoxide anion (O 2 *-) and hydrogen peroxide (H 2O 2) cartogram of generation.* hydrogen peroxide (the H that excites with Fu Jiaan (fMLP) individual processing group of expression 2O 2) (DCF fluorescent) and superoxide anion (O 2 *-) (EB fluorescent) receive relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. matched group (not having any drug treating)
2. Fu Jiaan (fMLP)
3. Fu Jiaan is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. Fu Jiaan is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. Fu Jiaan is also with 5 μ g/ml Tetrandrine (TETRA)
6. Fu Jiaan is also with 10 μ g/ml Tetrandrine (TETRA)
7. Fu Jiaan is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. Fu Jiaan is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Figure 11 is that 60 minutes time-history analysis (time course monitoring) the special composition extract of pretreatment Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) can suppress the cartogram that the interior pH-value (pH) of Neutrophils cell that Fu Jiaan (fMLP) excited increases suddenly.Data are pH-value (pH) meansigma methods ± meansigma methods standard error (n=6).
1. Fu Jiaan (fMLP)
2. Fu Jiaan is also with calcium ion channel blockor (verapamil)
3. Fu Jiaan is also with Radix Stephaniae Tetrandrae promise peaceful (FAN)
4. Fu Jiaan is also with Tetrandrine (TETRA)
5. Fu Jiaan is also with the special composition extract of Radix stephaniae tetrandrae (SPRST)
Figure 12 is that the special composition extract of pretreatment Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) can limit the cartogram that Neutrophils intracellular calcium ([Ca] i) that Fu Jiaan (fMLP) excited increases suddenly.Data are meansigma methods ± meansigma methods standard error (n=6) that calcium ion concentration changes net value (net increase).
1. Fu Jiaan (fMLP)
2. Fu Jiaan is also with pertussis toxin, PT (PTX)
3. Fu Jiaan is also with calcium ion channel blockor (veraDamil)
4. Fu Jiaan is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. Fu Jiaan is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
6. Fu Jiaan is also with 5 μ g/ml Tetrandrine (TETRA)
7. Fu Jiaan is also with 10 μ g/ml Tetrandrine (TETRA)
8. Fu Jiaan is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
9. Fu Jiaan is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Figure 13 is that the special composition extract of pretreatment Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) can limit leukotriene (LTB 4) cartogram that increases suddenly of the Neutrophils intracellular calcium ([Ca] i) that excited.Data are meansigma methods ± meansigma methods standard error (n=6) that calcium ion concentration changes net value (net increase).
1. leukotriene (LTB 4)
2. leukotriene is also with pertussis toxin, PT (PTX)
3. leukotriene is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. leukotriene is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. leukotriene is also with 5 μ g/ml Tetrandrine (TETRA)
6. leukotriene is also with 10 μ g/ml Tetrandrine (TETRA)
7. leukotriene is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. leukotriene is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Figure 14 is the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) cartogram to the effect of G albumen (G protein).Independent G protein activation agent aluminium fluoride anion (AlF 4 -) can cause intracellular calcium concentration to increase, this effect can be reversed by pertussis toxin, PT (PTX).* expression and G protein activation agent (AlF 4 -) the individual processing group receives relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. aluminium fluoride anion (AlF 4 -)
2. the aluminium fluoride anion is also with pertussis toxin, PT (PTX)
3. the aluminium fluoride anion is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. the aluminium fluoride anion is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. the aluminium fluoride anion is also with 5 μ g/ml Tetrandrine (TETRA)
6. the aluminium fluoride anion is also with 10 μ g/ml Tetrandrine (TETRA)
7. the aluminium fluoride anion is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. the aluminium fluoride anion is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Figure 15 is the special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) cartogram to the effect of G albumen.Independent G protein activation agent (AlF 4 -) can cause the adhesion (adhesion) of cell, this effect can be reversed by pertussis toxin, PT (PTX).* expression and G protein activation agent (AlF 4 -) the individual processing group receives relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' s test) through reaching.
1. aluminium fluoride anion (AlF 4 -)
2. the aluminium fluoride anion is also with pertussis toxin, PT (PTX)
3. the aluminium fluoride anion is also with the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
4. the aluminium fluoride anion is also with the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
5. the aluminium fluoride anion is also with 5 μ g/ml Tetrandrine (TETRA)
6. the aluminium fluoride anion is also with 10 μ g/ml Tetrandrine (TETRA)
7. the aluminium fluoride anion is also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
8. the aluminium fluoride anion is also with 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
Figure 16 is the superoxide anion (O of the special composition extract of Radix stephaniae tetrandrae (SPRST) that extracts of all kinds of solvents to Neutrophils intracellular reactive free radical 2 *-) the statistical figure of inhibitory action.
General Luo Zhi (PMA, the Neutrophils superoxide anion (O that phorbol-12-myristate-13-acetate) is brought out 2 *-) can be by SPRST/H2O/EtOH, SPRST/EtOH and SPRST/CH 2Cl 2The solvent extract of three kinds of process differences such as/EtOH suppresses.Cell excites superoxide anion (O with the general Luo Zhi of 100ng/ml (PMA) in the presence of the special composition extract of the Radix stephaniae tetrandrae that all kinds of solvents extracted (100 μ g/ml) 2 *-) generation of (EB fluorescent), with the result of flow cytometer (flow cytometry) respectively at FL-2 (EB) monitoring.The control group is not sentenced any medicine.Data are with average candle power value (mean channel fluorescence; MCF) expression.* expression and general Luo Zhi (PMA) individual processing group are received relatively tool statistical discrepancy (P＜0.05) of single-factor variation calibrating afterwards (Dunnett ' stest) through reaching.
1. matched group (not having any drug treating)
2. general Luo Zhi (PMA)
3.SPRST/H 2O
4.SPRST/H 2O/EtOH
5.SPRST/EtOH
6.SPRST/EtOH/H 2O
7.SPRST/EtOH/CH 2Cl 2
8.SPRST/CH 2Cl 2
9.SPRST/CH 2Cl 2/EtOH
10.SPRST/CH 2Cl 2/H 2O
Figure 17 is the cytotoxicity assessment (cytotoxicity) of the special composition extract of the Radix stephaniae tetrandrae of various medicines and extraction (SPRST) to Neutrophils.The Neutrophils cytotoxicity that general Luo Zhi (PMA, phorbol-1 2-myristate-13-acetate) and antagonist (staurosporine) thereof are brought out and the toxicity of special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient thereof are relatively.Data are that the meansigma methods of mortality rate is represented.
1. matched group
2. general Luo Zhi (PMA)
3. antagonist (Staurosporine)
4. 1 μ g/ml Radix Stephaniae Tetrandrae promise peaceful (FAN)
5. 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
6. 10 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
7. 1 μ g/ml Tetrandrine (TETRA)
8. 5 μ g/ml Tetrandrine (TETRA)
9. 10 μ g/ml Tetrandrine (TETRA)
10. the 1 special composition extract of μ g/ml Radix stephaniae tetrandrae (SPRST)
11. the 5 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
12. the 10 special composition extracts of μ g/ml Radix stephaniae tetrandrae (SPRST)
13. 1 μ g/ml Tetrandrine (TETRA) is also with 1 μ g/ml Radix Stephaniae Tetrandrae promise peaceful (FAN)
14. 2.5 μ g/ml Tetrandrine (TETRA) are also with 2.5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
15. 5 μ g/ml Tetrandrine (TETRA) are also with 5 μ g/ml Radix Stephaniae Tetrandrae promises peaceful (FAN)
The specific embodiment
The present invention's Radix stephaniae tetrandrae is available from general Chinese medicine market, through being accredited as the tuber of Menispermaceae liana Radix stephaniae tetrandrae (Stephania tetrandra S.Moore).The present invention's extract is through water or to mix the organic solvent extraction of various proper proportions dry with medical material.And the seriality that generally is usually used in Chinese medicine extracts, and also can be applicable to the present invention.Usually the organic solvent of extraction has ethanol (EtOH), dichloromethane (CH 2Cl 2), acetone (acetone).The product of extraction drying, through single organic solvent, or two kinds, the mixed solvent of multiple organic solvent composition are for to separate towards extract with tubing string.General suitable gallbladder body as the filling tubing string has silica gel (silica gel), reaches dragon (diaion), Sui Huadi (sephadex), C-18.So-called single organic solvent, or two kinds, the mixed solvent of multiple organic solvent composition can be selected methanol (MeOH), ethanol (EtOH), dichloromethane (CH for use 2Cl 2), two kinds or three kinds of organic solvents of acetone (acetone), water, toluene (toluene), be towards extract with various ratios.
With the superoxide anion (O of the special composition extract of the Radix stephaniae tetrandrae that all kinds of solvents was extracted (SPRST) to Neutrophils intracellular reactive free radical 2 *-) inhibitory action relatively.
(PMA phorbol-12-myristate-13-acetate) brings out Neutrophils superoxide anion (O with general Luo Zhi 2 *-) produce.Cell is earlier with SPRST/H 2O/EtOH, SPRST/EtOH and SPRST/CH 2Cl 2Preact was 10 minutes under the solvent extract of three kinds of process differences such as/EtOH (100 μ g/ml) existed.Excite superoxide anion (O with the general Luo Zhi of 100ng/ml (PMA) 2 *-) generation of (EB fluorescent), with flow cytometer respectively at 30 minutes result of FL-2 (EB) monitoring; The control group is not sentenced any medicine.The result shows the extract group via alcohol extraction, comprises SPRST/EtOH, SPRST/H 2O/EtOH, SPRST/CH 2Cl 2Three kinds of extracts such as/EtOH all significantly suppress Neutrophils superoxide anion (O 2 *-) generation, as shown in figure 16.SPRST/H wherein 2O/EtOH represents the special composition extract of Radix stephaniae tetrandrae (SPRST), is through water (H 2O) medical material of extraction back is again with ethanol (EtOH) extraction after drying products therefrom.SPRST/EtOH represents that the special composition extract of Radix stephaniae tetrandrae (SPRST) is the product through ethanol (EtOH) extraction after drying gained.SPRST/CH 2Cl 2/ EtOH represents that the special composition extract of Radix stephaniae tetrandrae (SPRST) is through dichloromethane (CH 2Cl 2) extract the medical material of back again with ethanol (EtOH) extraction after drying products therefrom.
The special composition extract of the present invention's Radix stephaniae tetrandrae (SPRST) is the method for extracting preparation by tuber utilization the present invention of Radix stephaniae tetrandrae (Stephaniatetrandra S.Moore).Use HitachiModel L-7100 high-performance liquid chromatograph (HPLC), mobile phase is the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution, utilizing linear gradient to dash and carry, the detection wavelength is 280nm; Four kinds of compositions can separate fully in 45 minutes, and the concentration of its quantitative linearity regression equation and correlation coefficient is between 12.5～1637 μ g/ml scopes, and detectable limit is calculated according to the S/N=3 regulations.Buffer solution KH 2PO 4-H 2O is with 5gKH 2PO 4Be dissolved in 1000ml water, and the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution is H 2O: CH 3CN: KH 2PO 4The ratio allotment of=400ml: 600ml: 5g.
Shown in Fig. 1 high-performance liquid chromatograph collection of illustrative plates, the special composition extract of the present invention's Radix stephaniae tetrandrae (SPRST) contains OBL oblongine (Oblongine, OBL), (-)-Cissamine (cyclanoline, CYC), peaceful (the Fangchinoline of Radix Stephaniae Tetrandrae promise, FAN), Tetrandrine (Tetrandrine, TETRA) four kinds of remarkable compositions, shown in Fig. 1 (B) high-performance liquid chromatograph collection of illustrative plates, can converse by its integral area and to contain OBL oblongine (OBL) in the special composition extract of Radix stephaniae tetrandrae (SPRST), (-)-Cissamine (CYC), Radix Stephaniae Tetrandrae promise peaceful (FAN), 5～20%w/w that four kinds of remarkable compositions of Tetrandrine (TETRA) are total extract.Four kinds significantly become the holdup time (retention time) of branch to be respectively OBL oblongine (OBL) 10.11 (branch), (-)-Cissamine (CYC) 11.71 (branch), peaceful (FAN) 26.69 (branch) of Radix Stephaniae Tetrandrae promise, Tetrandrine (TETRA) 32.69 (branch) in the high-performance liquid chromatograph collection of illustrative plates.And do not contain Aristolochic Acid (aristolochic acid) in the special composition extract of Radix stephaniae tetrandrae (SPRST), because the holdup time of Aristolochic Acid (retention time) (63.12 minutes) after 62 (branches) in the present invention's high-performance liquid chromatograph operating condition, instrumental sensitivity is 2 μ g.Fig. 1 (A) is depicted as the high-performance liquid chromatograph collection of illustrative plates of simple solvent, and being presented at 5 to 50/holdup time (retention time) interior solvent does not have the peak and compose existing; Comparison diagram 1 (A) is to the high-performance liquid chromatograph collection of illustrative plates of Fig. 1 (C), show in the present invention's the special composition extract of Radix stephaniae tetrandrae (SPRST) and do not contain Aristolochic Acid (aristolochic acid), because of the Radix stephaniae tetrandrae certified products of the present invention's uses at the later Aristolochic Acid that do not occur of 62 (branches).Aristolochic Acid is the known toxic substance of the aristolochiaceae plant of Radix stephaniae tetrandrae misuse, and this analysis result is also rejected the chance that aristolochiaceae plant participates in the Radix stephaniae tetrandrae certified products except that the quality control condition as the special composition extract of Radix stephaniae tetrandrae (SPRST).
Because Chinese medicine Radix Stephaniae Tetrandrae and so on is a lot of like plant, Radix stephaniae tetrandrae is the tuber of menispermaceae liana powder anti-(Stephania tet randra S.Moore) among the present invention.And the special composition extract of the present invention's so-called Radix stephaniae tetrandrae (SPRST), contain Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN), (-)-Cissamine (CYC), OBL oblongine four kinds of alkaloids such as (OBL) through experiment confirm, and other has the active ingredient of physiologically active.
As many as its active ingredient Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN), therefore infer has other unknown efficacy component to participate in the effect of the special composition extract of Radix stephaniae tetrandrae (SPRST) jointly to the special composition extract of Radix stephaniae tetrandrae SPRST (only contain 1.35% Tetrandrine (TETRA) and 0.7% Radix Stephaniae Tetrandrae promise is peaceful) to the inhibition effect of G albumen.Because the special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) can effectively suppress the inflammation phenomenon of Neutrophils down in medicine reasonable concentration (1-10 μ g/ml), so the special composition extract of Radix stephaniae tetrandrae and active ingredient thereof can be applicable to clinical treatment or the prevention inflammatory disease of being correlated with that early stage improper activation is caused such as Neutrophils future.
The special composition extract of 1-10 μ g/ml Radix stephaniae tetrandrae (SPRST) can effectively suppress Fu Jiaan (fMLP, N-formyl-methionyl-leucyl-phenylalanine) and leukotriene (LTB 4, leukotriene B 4) institute brings out the adhesion (adhesion) of neutrophils (PMN) and move apprentice (transmigration) effect, and have dose-response relationship (concentration-dependentresponse).The special composition extract of Radix stephaniae tetrandrae (SPRST) contains two known efficacy components: Tetrandrine (Tetrandrine; TETRA) and the peaceful (Fangchinoline of Radix Stephaniae Tetrandrae promise; FAN), the effect that in 1-10 μ g/ml same dose scope, also has the special composition extract of similar Radix stephaniae tetrandrae (SPRST).The present invention confirms that the special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) all can suppress Fu Jiaan (fMLP) and leukotriene (LTB 4) huge adhesion molecule (Mac-1) performance of being brought out and the generation of living radical (ROS); Huge adhesion molecule (Mac-1) performance receptor 1 activity free radical (ROS) regulation and control, and the generation of living radical (ROS) stems from the activation of neutrophils (PMN) cell Nei Enfei oxidase (NADPHoxidase), and desire activates this enzyme then must keep Cytoplasm appropriateness alkalization (alkalization); The special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) and a kind of calcium channel blocker (Verapamil) four not only can be blocked the intracellular calcium increase (calcium influx) that Fu Jiaan (fMLP) brings out, also effectively suppress the Cytoplasm alkalization phenomenon that Fu Jiaan (fMLP) causes, so we infer that effect that the special composition extract of Radix stephaniae tetrandrae (SPRST) waits may be with to block calcium relevant from the G of interdependent (calcium-dependent) albumen pathway; With G protein activation agent (AlF 4 -) the interior calcium ion of the obvious activated cell matter of direct activation G albumen increases and cell adhesion, and this two phenomenon is almost completely by G protein inhibitor pertussis toxin, PT (pertussis toxin; PTX) reverse (reverse); The special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) but also this two phenomenon of part antagonism.Comprehensive these results can infer that the effect of the special composition extract of Radix stephaniae tetrandrae (SPRST) protection heart anoxia injury can give the credit to its and suppress neutrophils (PMN) and adhere and move empty effect, and this effect may be the combined effect achievement of Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) and other active ingredient contained in the special composition extract of Radix stephaniae tetrandrae (SPRST).Because the special composition extract of Radix stephaniae tetrandrae (SPRST) only contains 1.3% Tetrandrine (TETRA) and 0.7% Radix Stephaniae Tetrandrae promise peaceful (FAN), the same effect that can not reach the special composition extract of Radix stephaniae tetrandrae (SPRST) with the Tetrandrine (TETRA) that is equivalent to the special composition extract of Radix stephaniae tetrandrae (SPRST) content and peaceful (FAN) dosage (0.1 μ g/ml) of Radix Stephaniae Tetrandrae promise.Generally speaking the special composition extract of Radix stephaniae tetrandrae (SPRST) is the special extracting composition of Chinese medicine of tool anti-inflammatory effect, and it can prevent that neutrophils (PMN) from can overactivity not showing huge adhesion molecule (Mac-1) and reach the effect of its antiinflammatory and the injury of protection heart anoxia via the generation of flow of calcium ions that suppresses the G protein regulation and living radical (ROS).
Followingly from normal adult periphery blood circulation, isolate neutrophils (PMN), utilize the inflammatory response of neutrophils (PMN) to inquire into the pharmacologically active of the special composition extract of Radix stephaniae tetrandrae (SPRST).The various active testings that mainly carry out are: (1) Neutrophils adhesion (adhesion); (2) Neutrophils moves apprentice (transmigration); (3) performance of the Neutrophils huge adhesion molecule in surface (Mac-1); (4) cytotoxicity (cytotoxicity) assessment.
The special composition extract of Radix stephaniae tetrandrae (SPRST) and its active ingredient Tetrandrine (TETRA), Radix Stephaniae Tetrandrae the promise rather cytotoxicity analysis of (FAN)
As shown in figure 17, throw relatively separately and give the special composition extract of Radix stephaniae tetrandrae (SPRST) or its active ingredient Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) and and with Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae the promise rather cytotoxicity of (FAN) neutrophils of being caused.The result shows that the cytotoxicity of the special composition extract of Radix stephaniae tetrandrae (SPRST) is much smaller than its active ingredient Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN); Be respectively with its cytotoxicity of the highest valid density (10tg/ml): SPRST (0.64 ± 0.14%), TETRA (1.24 ± 0.17%), FAN (0.84 ± 0.11%); But and with TETRA and FAN toxicity then up to 1.5 ± 0.23%.In no any medicine purgation cell natural mortality rate is 0.65 ± 0.28%, even show under the highest useful effect concentration, SPRST there is no remarkable cytotoxicity compared with TETRA, FAN and TETRA merging FAN.
Special composition extract of Radix stephaniae tetrandrae and active ingredient Tetrandrine thereof, the peaceful Neutrophils that suppresses huge adhesion molecule (Mac-1) Drawing upon of Radix Stephaniae Tetrandrae promise are adhered and are moved empty phenomenon
Fu Jiaan (fMLP) and leukotriene (LTB 4) can bring out a large amount of performances of the huge adhesion molecule of Neutrophils (Mac-1), cover surface (Everitt et al., 1996) and be adhered to fibrin.The present invention finds that Neutrophils is through 1 μ M Fu Jiaan (fMLP) and 0.1 μ M leukotriene (LTB 4) after the stimulation, as Fig. 2, Fig. 3, Fig. 4, shown in Figure 5, its to the adhesion amount of Fibrinogen and the amount of moving on foot increase 2-3 doubly more than, special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) have inhibitory action really to this two phenomenon, and present the relevant reaction relation of dosage.Special composition extract of Radix stephaniae tetrandrae and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) do not influence the spontaneous sticking value of this cell separately.Survival rate is still greater than 95% behind these medicines of cell pretreatment, with these medicines and the zero difference of being untreated, so these medicines pair cell and avirulence in the dosage range of 1-10 μ g/ml.(toxicity test is seen Figure 17).
For understanding the inhibition of the special composition extract of Radix stephaniae tetrandrae cell adhesion, whether via suppressing the surperficial huge adhesion molecule (Mac-1 of neutrophils (PMN); CD11b/CD18) performance.The present invention further analyzes Fu Jiaan (fMLP; Fig. 6) and leukotriene (LTB 4The huge adhesion molecule (Mac-1) that Fig. 7) is brought out increases phenomenon can or can not be suppressed by this Radix stephaniae tetrandrae preparation.It is to dye with PE-CD11b fluorescent antibody, records the result with flow cytometer in FL-2; The performance of huge adhesion molecule (Mac-1) is with average candle power value (mean channel fluorescence; MCF) expression, the control group is not sentenced any medicine.It is inhibited that Fig. 6, Fig. 7 of result such as huge adhesion molecule (Mac-1) shows special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) increases phenomenon to huge adhesion molecule (Mac-1), and show that by Fig. 6,7 inhibitory action increases with dosage.
The relation of living radical (ROS) generation and huge adhesion molecule (Mac-1) performance and cell adhesion in the special composition extract of Radix stephaniae tetrandrae
Known neutrophils (PMN) is by adhesion molecule (Mac-1 that the surface showed; CD11b/CD18) adhere and move the apprentice, and the performance of this adhesion molecule is subjected to living radical (the reactive oxygen species that self discharges; ROS) regulate and control.Because of also thinking, Fraticelli scholars such as (1996) comprises superoxide anion (O 2 *-) and hydrogen peroxide (H 2O 2) living radical (ROS) of what is called can stimulate the performance of huge adhesion molecule (Mac-1), and then the adhesion of promotion Neutrophils.Because the Tetrandrine (TETRA) of the special composition extract of Radix stephaniae tetrandrae (SPRST) and Radix Stephaniae Tetrandrae promise peaceful (FAN), in the same dose scope, therefore these two kinds of compositions also have the effect of the special composition extract of similar Radix stephaniae tetrandrae (SPRST), then check adhesion that whether the special composition extract of Radix stephaniae tetrandrae (SPRST) can suppress huge adhesion molecule (Mac-1) performance, cell, move the apprentice.Simultaneously with super oxygen disproportionation (superoxide dismutase; SOD) and hydrogen peroxide (catalase) isoreactivity free radical (ROS) scavenger do positive matched group (positive control) check and whether see through the generation that suppresses living radical (ROS) and reach the adhesion that suppresses huge adhesion molecule (Mac-1) performance and cell, move the apprentice.
This experiment utilizes flow cytometry method synchronous monitoring living radical (ROS) to produce people such as (in Europ J Pharmacol the 343rd volume 79-86 page or leaf report in 1998) ShenYC, and this method is represented superoxide anion (O with EB (ethydium bromide) fluorescent 2 *-) amount, (2 ', 7 '-dichlorofluorescein) fluorescent is represented hydrogen peroxide (H with DCF 2O 2) amount.Living radical (ROS) is just accumulated rapidly after Fu Jiaan (fMLP) or general Luo Zhi (PMA) stimulation immediately as a result.The EB and the DCF fluorescent value of being brought out as Fig. 8, Fu Jiaan shown in Figure 9 (fMLP) peaked about 20-40 minute.Special composition extract of Radix stephaniae tetrandrae and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) are led to a phenomenon to living radical (ROS) inhibitory action are arranged really, and present the relevant reaction relation of dosage as shown in figure 10.And the special composition extract of 10 μ g/ml Radix stephaniae tetrandraes is shown in the 30th minute as Fig. 8, figure 0 and still can imitates the generation that suppresses these two kinds of living radicals (ROS); Contrast super oxygen disproportionation (SOD) and hydrogen peroxide (catalase, CATS), they also significantly suppress the increase of DCF (Fig. 8) and EB (Fig. 9) fluorescent, simultaneously the huge adhesion molecule (Mac-1) that also Fu Jiaan (fMLP) is brought out increases and cell adhesion all has obvious inhibition effect, and suppresses effect then better people such as (roll up 79-86 page or leaf report in Europ JPharmacol the 343rd in 1998) inventor Shen YC with super oxygen disproportionation (SOD) and hydrogen peroxide (CATS).
The influence of pH-value (pH) variation in the cell that the special composition extract of Radix stephaniae tetrandrae brings out Fu Jiaan (fMLP)
Living radical (ROS) generation that Fu Jiaan (fMLP) can bring out the neutrophils cell is a kind of reaction of calcium ion dependency, pH-value (pH) in the of short duration rising cell simultaneously is with the activity of the manufacturings enzyme grace phenanthrene oxidase (NADPH oxidase) of keeping living radical (ROS).(the luxuriant and rich with fragrance oxidase NADPH of grace oxidase, nicotinamide adenine dinucleotidephosphate oxidase).
As shown in figure 11 the active Neutrophils of Fu Jiaan (fMLP) time, pH-value (pH) in the cell that also raises fast and significantly, and kept nearly 60 minutes.As shown in figure 11, special composition extract of Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN), and calcium ion channel blockor (verapamil) all can effectively suppress this phenomenon, hints that the message mechanism of certain calcium ion dependency is subjected to the influence of these medicines.
The special composition extract of Radix stephaniae tetrandrae suppresses sticking action and intracellular calcium changes and the relation of huge adhesion molecule (Mac-1) performance
Shown in Figure 12,13, throw separately and give Fu Jiaan (fMLP; Figure 12) and leukotriene (LTB 4Figure 13) all but the interior calcium ion of activated cell rolls up.Because Tetrandrine (TETRS) is effective natural calcium ion pipeline antagonist (Felix et al., 1992), the variation of intracellular calcium can participate in the Neutrophils sticking action (Lawson of huge adhesion molecule (Mac-1) Drawing upon again; Maxfield, 1995), therefore further inquire into the special composition extract of Radix stephaniae tetrandrae (SPRST) and change whether relevant with adherent phenomenon to calcium ion.
Special composition extract of pretreatment Radix stephaniae tetrandrae (SPRST) and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) can limit Neutrophils the intracellular calcium ([Ca that Fu Jiaan (fMLP) is excited as shown in Figure 12 and Figure 13 ++] i) increase suddenly.Show that the special composition extract of Radix stephaniae tetrandrae can non-selective inhibition Fu Jiaan (fMLP) and leukotriene (LTB 4) calcium ion increases suddenly in institute's activated cell, and present concentration correlation effect relation, thereby get rid of the probability that the special composition extract of Radix stephaniae tetrandrae acts on single-minded acceptor (receptor).
It mainly is the pathway that activates G albumen (G-protein) by acceptor that intracellular calcium increases suddenly, gives G protein activation agent aluminium fluoride anion (AlF as Figure 14, independent throwing shown in Figure 15 4 -) also can cause intracellular calcium to increase (Figure 14) and cell adhesion (Figure 15), and can be sentenced pertussis toxin, PT (the pertussis toxin of 120 minutes G protein blocking agent 500ng/ml in advance; PTX) reverse.Cell is if the special composition extract of Radix stephaniae tetrandrae (SPRST) of pretreatment 5-10 μ g/ml and active ingredient Tetrandrine (TETRA) thereof, Radix Stephaniae Tetrandrae promise peaceful (FAN) also can effectively suppress and have statistical significance to this two phenomenon.
The special composition extract of Radix stephaniae tetrandrae (SPRST) effectively suppress Fu Jiaan (fMLP, N-formyl-methionyl-leucyl-phenylalanine) and leukotriene (LTB 4, leukotrieneB 4) institute brings out neutrophils (PMN) adhesion (adhesion) and move apprentice (transmigration), and dose-response relationship (concentration-dependent response) is arranged.The special composition extract of Radix stephaniae tetrandrae (SPRST) contains two known efficacy components: Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN), in the same dose scope, these two kinds of compositions also have the effect of the special composition extract of similar Radix stephaniae tetrandrae (SPRST).(Mac-1 CD11b/CD18) adheres and moves the apprentice, and the performance of this adhesion molecule is subjected to living radical (the reactive oxygen species that self discharges because the huge adhesion molecule that known neutrophils (PMN) surface is showed; ROS) regulate and control.
The present invention confirms that the special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) all can suppress Fu Jiaan (fMLP) and leukotriene (LTB 4) huge adhesion molecule (Mac-1) performance of being brought out and the generation of living radical (ROS); Huge adhesion molecule (Mac-1) performance receptor 1 activity free radical (ROS) regulation and control, and the generation of living radical (ROS) stems from the activation of neutrophils (PMN) cell Nei Enfei oxidase (NADPH oxidase), and the desire activation luxuriant and rich with fragrance oxidase of grace (NADPH oxidase) then must be kept Cytoplasm appropriateness alkalization (alkalization).The special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) and a kind of calcium channel blocker Fu Paer (Verapamil) four not only can block the intracellular calcium increase (calcium influx) that Fu Jiaan (fMLP) brings out, and also effectively suppress the Cytoplasm alkalization phenomenon that Fu Jiaan (fMLP) causes.
The effect of the active ingredient that therefore can infer the special composition extract of Radix stephaniae tetrandrae (SPRST) and be contained may be relevant with the G albumen pathway of blocking-up calcium ion interdependent (calcium-dependent); With G protein activation agent (AlF 4 -) the interior calcium ion of the obvious activated cell matter of direct activation G albumen increases and cell adhesion, and this two phenomenon is almost completely by G protein inhibitor pertussis toxin, PT (pertussis toxin; PTX) reverse (reverse); The special composition extract of Radix stephaniae tetrandrae (SPRST), Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) but also this two phenomenon of part antagonism.
Comprehensive The above results can be inferred the effect of the special composition extract of Radix stephaniae tetrandrae (SPRST) protection heart anoxia injury, is to suppress neutrophils (PMN) via this special composition extract to adhere and move empty effect; This effect may be the Tetrandrine (TETRA) contained in the special composition extract of Radix stephaniae tetrandrae (SPRST) and the combined effect achievement of Radix Stephaniae Tetrandrae promise peaceful (FAN) and other active ingredient.Because, can not reach and the identical effect of the special composition extract of Radix stephaniae tetrandrae (SPRST) with the content of the Tetrandrine (TETRA) of 0.1 μ g/ml relative quantity and Radix Stephaniae Tetrandrae promise peaceful (FAN) simulation interior Tetrandrine of the special composition extract of Radix stephaniae tetrandrae (SPRST) (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) [the Radix Stephaniae Tetrandrae promise that SPRST contains 1.3% Tetrandrine (TETRA) and 0.7% is (FAN) rather].Sum up; the special composition extract of Radix stephaniae tetrandrae (SPRST) is the special extract composition of the Chinese medicine of tool anti-inflammatory effect, it can be via the generation of flow of calcium ions that suppresses the G protein regulation and reactive oxygen free radical anti-neutrophils can overactivity do not show huge adhesion molecule (Mac-1) and reach the effect of its anti-inflammatory and the injury of protection heart anoxia.
Desire of the present invention is inquired into the possibility mechanism of the Radix stephaniae tetrandrae special composition extract of extract (SPRST) tool Cardioprotective.At first suppose the function of the special composition extract of Radix stephaniae tetrandrae (SPRST) tool protection ischemia (oxygen) heart injury, prevent neutrophils (polymorphonuclear neutrophil in (or inhibition) blood with it; PMN) activation and produce inflammatory response substantial connection is arranged.Therefore the present invention isolates neutrophils (PMN) from normal adult periphery blood circulation, utilizes the inflammatory response of neutrophils (PMN) to inquire into the pharmacological mechanism of the special composition extract of Radix stephaniae tetrandrae (SPRST).
The special composition extract of 1-10 μ g/ml Radix stephaniae tetrandrae (SPRST) effectively suppresses Fu Jiaan (fMLP) and leukotriene (LTB 4) institute brings out neutrophils (PMN) adhesion (adhesion) and move apprentice (transmigration), and dose-effect relationship (concentration-dependentresponse) is arranged.
The special composition extract of Radix stephaniae tetrandrae (SPRST) contains two known efficacy components: Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN), in 1-10 μ g/ml same dose scope, Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) also have the effect of the special composition extract of similar Radix stephaniae tetrandrae (SPRST).
,、： ( 1 ) ( 2 ) ( TETRA ) ( FAN ) ( 3 ) ( HPLC ) ( SPRST ) ( 4 ) ( flow cytometry ) ( SPRST ) ( 5 ) ( SPRST ) 。
The present invention's compositions is to contain the special composition extract of Radix stephaniae tetrandrae (SPRST) of Tetrandrine (TETRA), Radix Stephaniae Tetrandrae promise peaceful (FAN) and other active ingredient, or be main constituent via the special composition extract of the prepared Radix stephaniae tetrandrae of method for making of the present invention (SPRST), form constituent with drug effect according to the addition salts that adds tolerable acid in various excipient, carrier or diluent and the pharmacy in case of necessity.That these preparations can be is for oral administration, rectum is thrown with it because of body dosage form, liquid dosage form, or the injection type of parenteral use, or directly applies the ointment dosage form in affected part.These solid dosage formss are to receive and so on and to collapse class binding agents such as powder, ethanol, glycerol in adding starch, carboxylic methyl cellulose according to known formulation method, or magnesium stearate, lactose and make lozenge or filling in capsule or make suppository and so on solid preparation.The right to use is adjusted acid-base value with aqueous solution, the saline solution of the present composition with the phosphoric acid salt buffer makes its pH value reach suitable degree, and makes injection or other liquor in interpolation auxiliary agent, emulsifying agent.The mixed various bases of the addition salts of tolerable acid also can be made into ointment dosage form according to known formulation method in the present composition or the pharmacy.Compositions with the present invention is that the prepared pharmaceutical compositions of main constituent can apply to the drug effect that mammal produces main constituent, and the dosage of generally offeing medicine can need allotment with symptom, is generally everyone each 50～300mg, every day 3 times.
The special composition extract of Radix stephaniae tetrandrae produce embodiment
Embodiment 1
100g is through the Radix stephaniae tetrandrae of pulverizing, and 95% ethanol (EtOH) solution that is soaked in 1500ml extracts in 80 ℃, and each 5 hours, totally 5 times.Get extract that 40ml extracts gained at every turn as analytic liquid, all the other extract concentrating under reduced pressure that will respectively take second place extract again.
Embodiment 2
100g is soaked in the dichloromethane (CH of 1500ml through the Radix stephaniae tetrandrae of pulverizing 2Cl 2) solution extracts in 80 ℃, each 5 hours, totally 5 times.Get the extract that 40ml extracts gained at every turn and extract with 95% ethanol (EtOH), all the other extract concentrating under reduced pressure that will respectively take second place extract the solution of back again as analytic liquid.
Embodiment 3
100g is through the Radix stephaniae tetrandrae of pulverizing, and 95% ethanol (EtOH) solution that is soaked in 1500ml extracts in 80 ℃, and 5 hours with the extract concentrating under reduced pressure.
Embodiment 4
100g is through the Radix stephaniae tetrandrae of pulverizing, and 95% ethanol (EtOH) solution that is soaked in 1500ml extracts in 80 ℃, and each 5 hours, totally 5 times.Get extract that 40ml extracts gained at every turn as analytic liquid, all the other extract concentrating under reduced pressure that will respectively take second place extract with methanol (MeOH) again.
Embodiment 5
Medicine is dissolved in the stock solution of making 10mM in the 0.1N HCl solution earlier
The special composition extract of the present invention's Radix stephaniae tetrandrae (SPRST), and contained Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) are the method for extracting preparations by tuber utilization the present invention of Radix stephaniae tetrandrae (Stephania tetrandra S.Moor).Medicine is dissolved in the stock solution of making 10mM in the 0.1N HCl solution earlier
SPRST/H 2O/EtOH represents that the special composition extract of Radix stephaniae tetrandrae is through water (H 2O) with ethanol (EtOH) extraction after drying products therefrom, other by that analogy again for the medical material of extraction back.
Embodiment 6
Use Hitachi Model L-7100 high-performance liquid chromatograph, adopt cosmosil5C18-AR-II, the C of 4.6 * 250mm 18Reverse chromatography tubing string, mobile phase are the fine (CH of second 3CN)-dihydric phosphate (KH 2PO 4) buffer solution, utilizing linear gradient to dash and carry, the detection wavelength is 280nm; With above-mentioned four kinds of compositions as the extrapolation standard; Four kinds of compositions can separate fully in 45 minutes, and the concentration of its quantitative linearity regression equation and correlation coefficient is between 12.5～1637 μ g/ml scopes, and detectable limit is calculated according to the S/N=3 regulations, four kinds of standard substance Tetrandrine (TETRA; Tetrandrine), the peaceful (FAN of Radix Stephaniae Tetrandrae promise; Fangchinoline), (-)-Cissamine (CYC; Cyclanoline), OBL oblongine (OBL; Oblongine) be respectively 0.95,0.95,0.95 and 1.69 μ g/ml.
A＝KH 2PO 4-H 2O(5g∶1000ml)
B＝H 2O-CH 3CN-KH 2PO 4(400ml∶600ml∶5g)
Embodiment 7
The lozenge dosage form of compositions
Compositions 50mg
Lactose 30mg
Starch 4mg
Magnesium stearate 6mg
Semen Maydis powder 10mg
Can make the lozenge dosage form that contains compositions according to above-mentioned prescription.
Neutrophils (neutrophil; PMN) preparation
Gather venous blood from adult healthy volunteers, (20unit/ml) work as anticoagulant with heparin (heparin).Adopt decigram (Ficoll) gradient centrifugation to separate a Neutrophils, centrifugal back with hypotonic solution the removal (Boyum rolls up 269-274 page or leaf report in Tissue Antigens the 4th in 1974) of rising broken of residual erythrocyte.Be summarized as follows: in the blood and the 3%dextran of 50ml centrifuge tube mixing equal-volume, leave standstill in the room temperature treated most of erythrosedimentation in 20-30 minute after, the blood plasma of leukocyte is rich at 4 ℃ in the careful upper strata of collecting, centrifugal 15 minutes of 250 * g, the collecting cell precipitate is broken up with phosphate buffer normal saline solution (PBS, phosphate buffered saline) suspend again (about 8ml).This cell suspending liquid injected gently decigram (Ficoll) solution that 6ml, density are 1.077g/ml is housed in advance (Histopaque 1077, and Sigma) upper strata is noticed that these two layers of solution can not have slightly to mix, in order to avoid follow-up leukocyte separates not exclusively.In 20 ℃, 400 * g continuous centrifugal 40 minutes is removed the upper strata all liq again, and left cell precipitation thing is a neutrophils, mixes a little erythrocyte.Breaing up the cell precipitation thing was suspended 30 seconds with 0.2% sodium chloride (NaCl) of 6ml, 1.8% sodium chloride (NaCl) that adds rapidly 6ml is opened solution answer etc., this moment, erythrocyte almost completely rose broken, again in 20 ℃, centrifugal 3 minutes of 120 * g, gained cell precipitation thing is with phosphate buffer normal saline solution (PBS, phosphate buffered saline) cleans once, get ten thousand cells of 1-2 at last and beat sheet with the centrifugal pelleter of cell specimen (cytospin), through golden Sha (Giemsa) solution-dyed, examine under a microscope cellular morphology, do the cell purity check, almost completely be multinuclear Neutrophils (PMNs), purity is generally greater than 95%.Neutrophils through preparing be suspended at last writing brush gram balance liquid (Hank ' s balanced salt solution; HBSS) standby in the solution, general cell concentration is adjusted into 1-2 * 10 6/ ml.Below all experiments except that specifying, medicine is 1: 100 (10 μ l in 1.0ml) with the volume ratio that cell suspending liquid reacts.
The mensuration of Neutrophils adhesion (adhesion)
With Fu Jiaan (fMLP) or leukotriene (LTB 4) activate the adhesion of Neutrophils and measure as follows: earlier with phosphate buffer normal saline solution (phosophate buffered saline; PBS) the 250 μ l Fibrinogens (fibrinogen) that are deployed into 50 μ g/ml concentration are inserted in the 24 hole tissue cultures dish, training to put after two hours approximately in room temperature makes Fibrinogen be covered in card, absorb the unnecessary liquid in upper strata and restrain balance liquid (HBSS) cleaning once, with 1% bovine serum albumin (bovine serumalbumin with writing brush; BSA) do card and cut off (blocking) processing, in order to avoid cell and card produce non-selective adhesion.Clean secondary with the writing brush gram balance liquid (HBSS) that contains 0.1% tween-20 (tween-20) through cutting off (blocking) after one hour, the writing brush gram balance liquid (HBSS) that does not contain tween-20 (tween-20) cleans once, so just finishes culture plate and handles before.
Prepare Neutrophils suspension 1.0ml (5 * 10 5/ ml) add gently in the culture plate, in advance with the special composition extract medicines (ultimate density 1-10 μ g/ml) such as (SPRST) of Radix stephaniae tetrandrae, in 37 ℃ of cultivations 10 minutes, again with 1 μ M Fu Jiaan (fMLP) or 0.1 μ M leukotriene (LTB 4) in 37 ℃ of reactions 15 minutes.Adhesive cell is not absorbed fully, and (contains 1mM Ca with 500 μ l temperature phosphate buffer normal saline solutions (PBS, phosphate buffered saline) gently 2+) clean twice.The cell that adheres to card dyeed in room temperature 10 minutes with stain (rose bengal) (Jenget al., the 1996) solution of 0.25% cell membrane.Absorb unnecessary dyeing liquor, card is cleaned twice with phosphate buffer normal saline solution (PBS), cultivating in 37 ℃ with 50% alcoholic solution of 250 μ l at last went dissolving to be adsorbed in the stain (rose bengal) of cell membrane in 30 minutes, treat that all stains are fully after the stripping, get 200 μ l and in 96 porose discs, measure solution O D value in wavelength 570nm with little dish analyser (micro-platereader (EL311sx)), deduction solution background light absorption value is measured value, at last with OD 570* 100 quantizating index as the cell adhesion degree.Or Neutrophils indicates with two carboxylic vinegar fluoresceins (BCECF-AM) fluorescent agent earlier, when experiment finishes, detect the machine of reading (Cytofluor 2300, Millipore R) and set the cell concentration that exciting light 485nm fluorescent 530nm reads cell fluorescence intensity (BCECF) the representative adhesion that adheres to card with fluorescent.
Neutrophils moves the mensuration of apprentice (transmigration)
People such as modification Krull M measure Neutrophils in J Immunol the 162nd volume 4834-4841 page or leaf report in 1999 and move empty method; Earlier diameter 6.5mm wide be the empty dish (Transwell of moving of 5 μ m apertures; Corning Costar, USA) the human Fibrinogen (fibrinogen of internal gasket (insert) lining last layer; 201 μ g/ml, 100 μ l) with basis as the Neutrophils adhesion.Before experiment is carried out earlier Neutrophils with BCECF-AN (2 ', 7 '-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester) fluorescent agent indicates, and then with the special composition extract of test drug Radix stephaniae tetrandrae (SPRST), Radix Stephaniae Tetrandrae promise peaceful (FAN), Tetrandrine (TETRA) in 37 ℃ do 10 minutes earlier after, get 100 μ l and insert and move on foot in the internal gasket of dish upper strata, and add inflammation inductive substance 1.0 μ M Fu Jiaan (fMLP) or 0.1 μ M leukotriene (LTB in lower floor 4), in 37 ℃ of incubators, cultivated 60 minutes, last neutrophilia club moves because of inducing of inflammation material and enters lower floor on foot, its quantity can be come quantitatively by fluorescence intensity (BCECF), detect the machine of reading (cytofluor2300, Millipore R) setting exciting light 485nm fluorescent 530nm with fluorescent and read fluorescence intensity.
The measurement of Neutrophils surface huge adhesion molecule (adhesion molecule) Mac-1 performance
The performance of Mac-1 is to be applied after adopting scholars' (Endemann equals JPharmacol Exp Ther the 276th volume 5-12 page or leaf report in 1996) such as Endemann method slightly modified.The Neutrophils cell suspending liquid of handling through the special composition extract of test drug Radix stephaniae tetrandrae (SPRST), Radix Stephaniae Tetrandrae promise peaceful (FAN) and Tetrandrine (TETRA) is with the general Luo Zhi of 100ng/ml (PMA), 1 μ M Fu Jiaan (fMLP) or 0.1 μ M leukotriene (LTB 4) effect 20-40 minute, centrifuging and taking cell precipitation (pellet), be suspended in again 1ml ice and contain 10% hyclone and the 10mM sodium axide (NaN that deactivates through heat 3) in the phosphate buffer normal saline solution (PBS, phosphate buffered saline).The fluorescence staining that below carries out huge adhesion molecule (Mac-1) carries out on ice in steps fully.Above-mentioned cell suspending liquid and huge adhesion molecule (Mac-1) primary antibody (mouse anti-human CD11b, IgG 1Pharmingen) cultivated 60 minutes, or with non-specificity antibody (IgG1, Sigma) replace huge adhesion molecule (Mac-1) primary antibody and do antibody background value contrast usefulness, clean twice with glacial phosphoric acid buffer normal saline solution (PBS)-FCS-azide, (goat anti-mouse, IgG) the lucifuge effect is 30 minutes with the secondary antibody of FITC fluorescent sign again for the cell that has huge adhesion molecule (Mac-1) primary antibody.Clean secondary with the phosphate buffer normal saline solution that contains 5% hyclone (PBS, phosphate bufferedsaline).Last cell suspension is in 1% para-formaldehyde fluidic cell dedicated buffering liquid (sheath nuid), with flow cytometer (FACSort; Becton Dickinson) the performance amount of the huge adhesion molecule of analysis of cells (Mac-1).Data are to represent huge adhesion molecule (Mac-1) content with the fluorescent value that records (meanchannel fluorescence), and at Cell Quest Software is auxiliary to be calculated down.
Intracellular reactive oxygen-derived free radicals (ROS) comprises superoxide anion (O 2 *-) and hydrogen peroxide (H 2O 2) assay
Carry out intracellular reactive oxygen with the flow cytometry method and (comprise superoxide anion (O according to scholars such as Robinson (Robinson equals Flow Cytometry 437-442 page or leaf report in 1994) deliver 2 *-) and hydrogen peroxide (H 2O 2) mensuration.Get 1.5ml cell suspending liquid (2 * 10 6/ ml) with two chlorine fluorescein (DCFH-DA) (2 ', the 7 '-dichlorofluorescin diacetate of the 20mM of 1.5 μ l; Molecular Probe) bathed 10 minutes in 37 ℃ of temperature, or the 10mM hydrogen phenanthrene that adds 1.5 μ l diamine (the HE) (hydroethidium that mutters; MolecularProbe) temperature was bathed 10 minutes.The hydrogen phenanthrene diamine (HE) of muttering itself can directly enter cell, can the intercalation of DNA when being formed the bromine phenanthrene by the superoxide ion oxidation and mutter diamine (EB) (ethidium bromide), can detect fluorescent in wavelength 590nm (FL2).Through above-mentioned pair of chlorine fluorescein (DCFH-DA) and the hydrogen phenanthrene cell that diamine (HE) indicated of muttering, with the special composition extract of test drug Radix stephaniae tetrandrae (SPRST), Radix Stephaniae Tetrandrae promise rather (FAN), Tetrandrine (TETRA) pretreatment 10 minutes, again through Fu Jiaan (fMLP), leukotriene (LTB 4) or general Luo Zhi (PMA) excite and produce a large amount of hydrogen peroxide (H 2O 2) and superoxide anion (O 2 *-), produce two chlorine fluoresceins (DCF) (2 ', 7 '-dichlorofluorescein) and bromine phenanthrene mutter (ethidiumbromide) fluorescent of diamine (EB) and the anti-hydrogen peroxide (H that pushes away of variation of monitoring fluorescence intensity by above-mentioned reaction 2O 2) and superoxide anion (O 2 *-) relative amount, with the fluorescent value representation of flow cytometer detecting.
Carrying out of the preparation of the special composition extract of Radix stephaniae tetrandrae and reaction
Special composition extract of Radix stephaniae tetrandrae (SPRST) and contained Tetrandrine (TETRA) and Radix Stephaniae Tetrandrae promise peaceful (FAN) thereof are dissolved in the stock solution (stocksolution) of making 10mM in the 0.1N HCl solution earlier, face and do serial dilution with phosphate buffer normal saline solution (PBS, phosphate bufferedsaline) solution again before the experiment and become 1.0 μ M-0.1mM.For the research of G albumen, cell elder generation is with G protein inhibitor pertussis toxin, PT (the pertussis toxin of 500ng/ml; PTX) 37 ℃ of temperature were bathed 120 minutes, added G albumen stimulant AlF again 4 -(10mM NaF adds 10 μ M AlCl 3) or other stimulants.
The mensuration that intracellular calcium concentration changes
(MolecularProbes USA) bathed 45 minutes in 37 ℃ of temperature the benzyl furan 2 (Fura-2/AM) of human Neutrophils suspension elder generation and 5 μ M; Because of benzyl furan 2 (Fura-2/AM) is very easy to enter cell for carboxylate, after being become benzyl furan 2 (Fura-2/AM), lose fat-solublely by the acidolysis enzyme hydrolysis, just can't penetrate any born of the same parents' device or cell membrane again and be accumulated in the Cytoplasm.Temperature is bathed the writing brush gram balance liquid (HBSS of back with no calcium; Hank ' s balanced saline solution) clean secondary, the writing brush gram balance liquid (HBSS) with calcic (2mM) suspends again again, and the adjustment cell number is 1 * 10 7/ ml, get this cell suspending liquid 2ml insert the four sides printing opacity the sample cup in (cuvettes; Sarsted, the fluorescent that Germany), moves into and be equipped with lucifuge, constant temperature, reaches self-poking arrangement is detectd the machine of reading (Hitachi F-4500; Hitachi Instruments CA) rose again 5 minutes.Add medicine training to be measured subsequently and put 10 minutes, add stimulant AlF again 4 -(10mM NaF adds 10 μ M AlCl 3) or A23187 (1 μ M) or safe big true (thapsigargin) (1 μ M) or Fu Jiaan (fMLP) (1 μ M) with Ca in the activated cell 2+Release.The Ca that discharges in benzyl furan 2 (Fura-2/AM) itself and the cell 2+Be incorporated into suitable wavelength and excite down the generation fluorescent, detect with fluorescent and read the variation of machine record fluorescent.Detect the machine of reading and when measuring fluorescent, can almost send simultaneously wavelength 340nm (E continuously from light source 340) and 380nm (E 380) exciting light, in wavelength 510nm (Em 510) fluorescent of per 0.2 second record.Automatically the fluorescence intensity of continuous record gained is converted into ratio (R) with F-4500 software, and, calculates Ca in the cell automatically according to the formula that people such as Grynkiewicz (1985) derive 2+Actual concentration, formula is as follows:
[Ca 2+] I＝K d(R-R min)(Sf 380)/(R max-R)(Sb 380)
K dBe 224nM, promptly benzyl furan 2 (Fura-2/AM) is in 37 ℃ dissociation constant.
R is for examining product in E 340And E 380Exciting light purgation fluorescence intensity ratio.
R MaxMake benzyl furan 2 (Fura-2/AM) combine measured fluorescence intensity ratio for adding 0.2% digitalin (digitonin) dissolved cell film with all calcium ions in the solution.
R MinRemove the fluorescence intensity ratio that records when benzyl furan 2 (Fura-2/AM) becomes free state behind the calcium ion in all solution for adding 25mM ethylene glycol diamine tetraacethyl (EGTA).
S F380Be E 380Excite the fluorescence intensity constant of free state benzyl furan 2 (Fura-2/AM) generation.
S B380Be E 380Excite the fluorescence intensity constant of combined state benzyl furan 2 (Fura-2/AM) generation.
The mensuration of pH-value (pH) in the cell
We revise Boyer ﹠amp; The method of Hedly (in Flow Cytometry 135-148 page or leaf report in 1994) is as follows: cell indicates (37 ℃ of temperature were bathed 30 minutes) with two carboxylic vinegar fluoresceins (BCECF-AM) (2 μ g/ml) earlier, the back is with phosphate buffer normal saline solution (PBS, phosphatebuffered saline) clean unnecessary two carboxylic vinegar fluoresceins (BCECF-AM), adjusting cell concentration then is 1 * 10 6Cells/ml.Cell is handled medicine earlier and was added Fu Jiaan (fMLP) (1 μ M) constant temperature in CO2 gas incubator (37 ℃) cultivation in 10 minutes again.With the variation of flow cytometer in different time monitoring Cytoplasm fluorescent.In order to record the pH-value (pH) in the cell, adopt fluorescence intensity ratio (ratio) the interior pH-value of cell (pH) that converts.Two carboxylic vinegar fluoresceins (BCECF-AM) are at fluorescence intensity and the pH-value in the cell (pH) positive correlation of wavelength 525-535nm, it is high more to be that fluorescent carries strong pH value, so surveyed the fluorescent value divided by getting a ratio (R) in the measured fluorescent value of wavelength 640nm (with the incoherent fluorescent value of pH-value (pH)) with wavelength 525nm, this R value can avoid testing the irradiation of irradiation line, cell became uneven, instrument is unstable and be written into the even interference of fluorescent agent density unevenness.The buffer that must not wait with one group of acid number of high potassium buffer preparation (pH) value height (6.8-7.8) is with replacement phosphate buffer normal saline solution (PBS in addition, phosphatebuffered saline) is the cell experiment buffer, to make inspection amount line, principle is as follows: when cell suspension adds Buddhist nun's phase suffering (nigericin) (a kind of H in different pH-values (pH) value buffer balance +/ K +The agent of ion duct) 3 minutes, Buddhist nun's phase suffering this moment (nigericin) can make the inside and outside K of cell +And H +Ion concentration ratio is equal, i.e. [[K +] In/ [K +] OutwardEqual [H +] In/ [H +] OutwardTherefore [K at once +] In=[K +] OutwardThe time, [H +] InCan equal [H +] OutwardTherefore the cell of the interior correspondence of the cell of this moment is the respective value of pH-value in the cell (pH) value in the fluorescence intensity (X value) of 525nm as long as record the pH-value (pH) (Y value) of buffer, so record the corresponding series fluorescent value of serial buffer gained, can try to achieve the inspection amount line (Y=aX+b) of the corresponding fluorescent value of a pH-value (pH) in pH-value (pH) 6.5-7.8
The cytotoxicity assessment
Select active drug activity (1-10 μ g/ml), when finishing, each experiment dying dead cell (can't discharge dyestuff) with trypan blue dyestuff (trypan blue) solution, at microscopically counting dead cell number (dying blue person), be the method for cytotoxicity assessment with not dying blue person (living cells) joint account mortality rate.
Statistical analysis
Experimental data is with meansigma methods ± meansigma methods standard error (mean ± S.E.M.) expression of measurement.Come the statistical significance of testing data respectively with list or double factor analysis of variance (AVOVA) according to the experiment needs.When analysis of variance was meaningful, whether the meansigma methods that Yi Dana examines and determine between (post-hoc Dunnett ' stest) comparison and the control group afterwards was variant, was considered as the difference of statistical significance with P＜0.05.Concentration-response effect (concentration-dependency) relation is to be Student ' s t-test with the linear regression slope of concentration-reaction to zero, has been considered as the difference of statistical significance with P＜0.05.

Claims (4)

1, the special composition extract of a kind of Radix stephaniae tetrandrae, it includes:
Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
2, according to the special composition extract of the described a kind of Radix stephaniae tetrandrae of claim 1, the mobile phase of its high-performance liquid chromatograph collection of illustrative plates is the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution, utilizing linear gradient to dash and carry, the detection wavelength is 280nm; Four kinds of compositions can separate fully in 45 minutes, and its quantitative linearity returns the concentration of agenda formula and correlation coefficient between 12.5～1637 μ g/ml scopes, and detectable limit is calculated according to the S/N=3 regulations; Buffer solution KH 2PO 4-H 2O is with KH 2PO 4Water-soluble, and the fine (CH of second 3CN)-biphosphate potassium salt (KH 2PO 4) buffer solution is H 2O: CH 3CN: KH 2PO 4The ratio allotment of=400ml: 600ml: 5g.
3, the special composition extract of the Radix stephaniae tetrandrae of a kind of tool anti-inflammatory effect, it comprises, and Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
4, the special composition extract of Radix stephaniae tetrandrae of a kind of tool protection heart anoxia injury effect, it comprises, and Tetrandrine, Radix Stephaniae Tetrandrae promise are peaceful, (-)-Cissamine, four kinds of alkaloids of OBL oblongine, and other has the active ingredient of physiologically active.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100396686C (en) * 2006-08-03 2008-06-25 西安皓天生物工程技术有限责任公司 Preparation method of Tetrandrine and Fangchino-kine
CN102382119A (en) * 2011-10-19 2012-03-21 陕西省西安植物园 Extraction method of tetrandrine and demethyltetrandrine
CN104906098A (en) * 2015-05-14 2015-09-16 天津中医药大学 Applications of tetrandrine in preparation of medicines

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100396686C (en) * 2006-08-03 2008-06-25 西安皓天生物工程技术有限责任公司 Preparation method of Tetrandrine and Fangchino-kine
CN102382119A (en) * 2011-10-19 2012-03-21 陕西省西安植物园 Extraction method of tetrandrine and demethyltetrandrine
CN102382119B (en) * 2011-10-19 2014-06-25 陕西省西安植物园 Extraction method of tetrandrine and demethyltetrandrine
CN104906098A (en) * 2015-05-14 2015-09-16 天津中医药大学 Applications of tetrandrine in preparation of medicines

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