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13References
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Pharmacokinetic studies ofBorago officinalis in rabbit
Ma Ying-nan, Yu ya-nan
DepartmentofPharmacology,Heilongjianguniversity of Traditional Chinese Medicine,Heilongiang
Harbin, China
Abstract:Medicine worked in the form of constituents in blood. In order to found the matters of Borago affect body directly, An UPLC-TOF/MS method has been used to analyze the metabolites and the active constituent in blood of the rabbit after fed by Borago. On the assay result, five compositions were found in urine, and one of them appears a strong ionic strength in the chromatogram of the blood.
Key word: Pharmacokinetic; Borago; UPLC-TOF/MS; active constituent
1. Introduction
Borage (Borago officinalis L.) is a large hairy annualherb that is a member of Boraginaceae 1 2 family. 1 Borageseed has a high content of c-linolenic acid and is used as adietary supplement.. 2
Borage juice and tea are used to treatinfluenza, colds, injuries, and ulcers.3-5 Borage istraditionally
used to treat respiratory, cardiovascular, andgastrointestinal diseases.6 In recent studies, it is show
that Borage can be the treatment of jaundice, rheumatism, skin inflammation, can also be used as a
7-9
diaphoretic, diuretic antitumorand analgesic. - In order to clarify theactive composition and acting mechanism of Borage, An UPLC-TOF/MS method has been used to analyze its metabolites and blood in biological samples. UPLC-TOF/MS is a recently developed technology. It can provide a higherpeak capacity, greater resolution, increased sensitivity, and higherspeed of analysis.
2. Materials and methods
2.1. Chemicals
Acetonitrile chromatography pure (Fisher, USA); water distilled water (Guangzhou Watsons Beverage Co. Ltd., China); formic acid as chromatographic pure (DIKMA, USA); other reagents were analytically pure.
2.2 Instrumentation and chromatographic conditions
The UPLC/MS system "LCTPremierTMXE" comprised a WatersUPLC system connected inline with a time-of-flight mass spectrometer(TOF MS) (Waters Ltd. USA). The column utilized was anACQUITY UPLC® HSS T3 2.1x100 mm, 1.8 ^m) maintained at 40°CMobile phases were A( 1% formic acid in acetonitrile) and B (0.1% formic acid). LCgradient condition of Bwas as follows: 0 min, 1% A ; 2 min, 5% A ; 10 min, 30% A ; 12 min, 50% A ; 14 min, 80% A ; 15 min, 100% A. The flow rate was set at 0.4 mL/min. Samples were maintainedat 4 °C throughout.
2.3. Mass spectrometry
Analytes were quantified using TOF MS in negative-ion mode.Source parameters included capillary voltage of 2.0 kV, cone voltageof 60V, source temperature of350°C, desolvation temperatureof 350 °C, cone gas flow of 10 L/h, desolvation gas flow of 700 L/h. Productions were monitored in W- extended mode. In the full scanmode, the mass spectrometer was operated over a range of m/z 100-1000. Injection volumes for samples and standards were 4p,Lwith an auto-injector.
2.4 animals
Six rabbits ( 2.0-2.5kg, male)provided by the Experimental Animal Center of Heilongjiang university of Traditional Chinese Medicine. The animals were housed andcared for under a constant temperature at (22±1 °C) and humidity(50±10%). Foodwaswithheld prohibited for one day before theexperiment while water was provided freely.
2.5 Drug preparation
Themixture was boiled for 30 min three times in water at a volumeof thee times of the dry weight of the Borage. The resulted Borage solution was concentrated, andvacuum dried. And the driedBorage powder wasmixed with distilled water into the suspension of 0.25g/mL.
2.6Sample preparation and identification
Each animalwas orally administrated Borage solution for 10 ml/kg every 4 hour , and for atotal of three doses.
Blood sample was centrifuged at 3000rpm for 10 min, 2L of the upper layer was mixed with 10Lmethanol using vortex-mixing for 1min and then centrifuged at 3,000rpmfor 15min. 5^L of the upper layer was injected intothe UPLC system for analysis.
The whole urine of each rabbit was collected. Each urine sample was centrifuged at 4500rpm for 10 min. Hundredmicrolitres of the upper layer was mixed with 100^L methanolusing vortex-mixing for 1min and then centrifuged at 12,000rpmfor 15min. The upper layer was transferred to another tubeand mixed with 600^L water. The mixture was centrifuged at10,000rpm for 10min. 10^L of the supernatant was injected intothe UPLC system for analysis.
3. Results and discussion
Five compositions of Borage in vivo have been found in the urine by establishing the extracted ion chromatograms. They are the Cymarose (Fig.1), Andrographolide (Fig.2), Eleganin (Fig.3), Indicine (Fig.4), Supinine (Fig.5). In the extracted ion chromatograms of the serum sample by collected in different time after fed the drug, four of them have the weak ionic strength, just one of them has a strong ionic strength at 60 min (Fig.6).
wlc-urine-neg-1 386 (4.665) 100 161 0795
1 : TOF MS ES- wlc-urine-blank-neg-3 386 (4.665)
150.0551
,172.9894
1.11e3 100n
172.9885
1 : TOF MS ES-1.53e3
230.0109
203.0001
173.9946
230.9951 261.0072
!
277.0675
187.0083
136.0393
!
217.0140/
232.0014
285.0602 m/z
100 120 140 160 180 200 220 240 260 280
A. blank urine B. urine with drug
Fig.1 The TOF MS base peak intensity (BPI) chromatograms of the metabolites of Cymarose in urine after feeding Borage to rabbit. 0
wlc-urine-neg-1 684 (8.178) 100 349 1981
1 : TOF MS ES- wlc-urine-blank-neg-3 681 (8.146) ' 1.02e3 100-1 327.0880
187.0061
1: TOF MS ES-219
491.1585
327.0865 305.1035 350.2074
491.1573
257.1207
453.1815
0
A. blank urine
ItWIfHWt'rf
527.2929
492.1571
200 250 300 350 400 450 500 550
B. urine with drug
Fig.2 The TOF MS base peak intensity (BPI) chromatograms of the metabolites of Andrographolide in urine after feeding Borage to rabbit.
wlc-urine-blank-neg-3 782 (9.335)
100n
!
431.2014
!
415.1642
!
417.0697
!
423.2291
0J
!
423.0487
1 : TOF MS ES- .
33 0 tfC-jreoeç-1 Я2 ? 396)
100
!
433.1718
410 415 420 425 430 435 440 A. blank urine B. urine with drug
Fig.3The TOF MS base peak intensity (BPI) chromatograms of the metabolites of Eleganin in urine after feeding Borage to rabbit.
0
0
0
wlc-urine-pos-1 337 (4.087) 100-
8.1274
Ni-
0
226.1086
146.0608
wl
........................if
1 : TOF MS ES+ wlc-urine-blank-pos-1 338 (4.098) 3.81e3 2r.....
0.1797
301.1837 393.1666 509.1506
JL
100 150 200 250 300 350 400 450 500 550 A. blank urine B. urine with drug
• m/z 0
1: TOF MS ES+ 427
206.0489
269.1539 332.1738
' 393.1754 !
J 394.1758 47Z1784
Fig.4The TOF MS base peak intensity (BPI) chromatograms of the metabolites of Indicine in urine after oral Borage to rabbit.
wlc-urine-pos-1 452 (5.444)
10^ „„ L„
186.0688
1: TOF MS ES+ . . . . . .... „ ....
89g wlc-urine-blank-pos-1 451 (5.434)
100 '
1 : TOF MS ES+
217.0978
246.1708
284.1884
285.1923
150 200
A. blank urine
250
lf"'' i"H".....H"
300
366.1329380.1089
M.........^......i"1''.....'"'"'I...... m/z
350
400
333.1090
B. urine with drug
Fig.5The TOF MS base peak intensity (BPI) chromatograms of the metabolites of Supinine in urine after feeding Borage to rabbit.
60-1-neg 100i
8.16 349.2020
1: TOF MS ES-349.2 0.10Da 242
8.82
K .................. ................_ 353.1459_______ _____
0 ,T1TrrTrP'î'r'rrrrrrTriTVFrrl î1TTrl '^f'1 l'lTri P1"1 rpi-rrr'pHi iVrnrTTrrrfTrtiTTi'r^mTprrrrTTri-r^-rfr
5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 Fig.6 HPLC-TOF/MS Chromatograms of Andrographolide in blood after oral Borage for 60 min to rabbit
4. Conclusions
The composition ofcrude drugs is quite complicated, and it is hard to clarify the evidence of efficacy. It can not to be solved the problem of the characteristic of crude drugs by researching composition through purifying the drug merely.
This experiment in analyzing the metabolite and serum of the rabbit after fed by Borage, preliminary studied about the directly active matters in vivo of Borage. It can also lay the foundation for researching the chemical composition of raw Borage.
145.0871
0
This study identified five compositions of Borage in vivo by building the method of UPLC-TOF/MS. The chemical composition of Borage was preliminarily analyzed in this study. In which, the Cymarose Eleganin and Indicine have the function of antitumor. And the Andrographolide has the function of anti-inflammatory. It provided a scientific basis to research the function of antitumor and anti-inflammatory on Borage by the Pharmacokinetic studies ofBorago officinalis in rabbit. This study also provided an evidence of the action of Borage for the further clinical application.
References
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Study of Hypericum attenuatum Choisy and Salvia miltiorrhiza extracts and compatibility on Myocardial Ischemia in Mice
Cao Mingming ji Li
(Heilongjiang University of Chinese Medicine,Harbin,Heilongjiang,China 150040) Fax: 13654669049; E-mail address: x87211@163.com Heilongjiang University of Chinese Medicine Harbin China
Abstract Objective: Discussion the protective effect of Hypericum attenuatum Choisy (H.attenuatum Choisy) and Salvia miltiorrhiza compatibility on Myocardial Ischemia in Mice, which provide the basis for the compatibility of the Chinese medicine. Methods: Mice were randomly divided into H.attenuatum Choisy extract group, Salvia miltiorrhiza extract group , H.attenuatum Choisy and Salvia miltiorrhiza ratio (1:1,1:2,2:1),model group, positive group, blank group, each group have ten rats. Drugs were diluted with an equal dose of normal saline. The model group and the blank group were fed with equivalent dosage of saline. The contents of CK and the LDH-L were detected. The level of SOD and MDA were investigated. Result: H.attenuatum Chosiy and Salvia miltiorrhiza compatibility ratio 1:2group can significantly reduce the LDH,CK level, increased SOD activity, decreased MDA content, and which can significantly prolong the time of myocardial ischemia mice's hypoxia. Conclusion: Such as dose, the experimental conditions compared with the single herb, H.attenuatum Chosiy and the Salvia miltiorrhiza ratio 1:2 has apparent protective effect in mice of myocardial ischemia.
Keywords: H.attenuatum Choisy, Salvia miltiorrhiza, Myocardial Ischemia, Mice
