CC BY
8Study on the constituents in rat CSF after oral administration of Kai-xinsan
LIU Xue-wei, Ye Yang LIU Shuang, Wang rui, HUANG Shu-ming*
(Research Institute of Chinese Medicine,Heilongjiang University of Chinese Medicine,Harbin
150040,China)
Abstract.
Object:To analyse the constituents in rat cerebrospinal fluid after oral administration of KXS-60%E.Method:A UPLC-TOF-MS method for analyzing the constituents in KXS-60%E and in rat CSF after oral administration of KXS-60%E,has been established.The constituents in rat CSF after oral administration of KXS-60%E were pointed out compared with the control rat CSF. Result: 3 compounds were detected in KXS-60%E in rat CSF, all of them were original form in KXS-60%E.Conclusion:The constituents absorbed into CSF were the direct basis of action of the drug protecting neural ,Further study on these constituents may find out the effective compounds in KXS-60%E and clarify the mechanism on treatment of AD
[Key Words] KXS-E60%; UPLC-TOF-MS;CSF; AD
1.Introduction
KXS is a famous classic formaula, formed by Ginseng, Poria Radix ,Polygalae and Rhizoma
Acori Talarinowii,the formula of KXS and analogous prescription have been used to treat
[1-21
Neurodegenerative diseases such as Alzheme's Disease in Chinese1 -1 Medicine. Recently, studies revealed that KXS compound as well as each single herb in the formula can improve learning and memory functions of dementia animal models, and the mechanism of action may be closely related to many ways such as reducing the reaction of peroxidation, neutralizing free radicals, protecting vascular endothelial cell, minimizing toxicity of Ap and protecting the neurons [3-61. Recently,our studies have revealed that the cerebrospinal fluid(CSF) of rat after oral administration of KXS-60%E have a protective effect of hippocampal by culturing PC12 cells[71, which indicates that the effective material basic in KXS-60%E is to be expected .However,it remains unclear as to which constituents are responsible for the neural protective effect of the drug ,so what are these constituents and how about the information are these constituents became the basic and key problem in our study.in our study, :A high -precision and high-sensitivity UPLC-TOF-MS method for analyzing the trace amounts of constituents in rat CSF has been established, by which we can clarify the direct basis of action of the drug protecting neural and provide a a kind of method for analyseing Chinese herbal prescription.
2.Materials and methods
2.1 Materials
The preparation of KXS-60%E power was prepared by the optimized process in preliminary study, The Male Wistar rats (200-240 g) were obtained from the Laboratory Animal Center of Heilongjiang University of Chinese Medicine; Ultra performance liquid chromatograph/mass spectrometer (UPLC-MS) was provide in Research Institute of Chinese Medicine,Heilongjiang University of TCM,all the laboratory reagents were chromatographic grade; An improved injector was made by ourself in order to drow CSF.
2.2 Methods
2.2.1 Preparation of samples for analysis in vitro The preparation of KXS-60%E power(100mg), Ginseng power(53.4mg), Poria Radix power(3.3mg) ,Polygalae(27.5mg) and Rhizoma Acori Talarinowii power(15.8mg) were dissolved espectively in the 60% methanol in 25ml schott and centrifuged at 13,000 rpm for 10 min at 4°C, the supernatant was used for UPLC.
2.2.2. Preparation for control and treated CSF sample According to our previous research method, 7 wistar rats was were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium (0.15mL/100g body weight) , the rat was fixed in brain solid positioner and clothing hair in
armpit and occipital was cut out so 0.15-0.2ml control CSF was draw out in posterior cistern by an improved injector.7 days later, KXS-60%E was orally administrated to rats (0.375 g/mL,1mL/100g body\weight) once a day for 3 three-day, the treated CSF was collected in the same method, and then centrifuged at 13,000 rpm for 10 min at 4°C. The CSF was applied to a pre-activated OASIS HLB solidphase extraction C18 column. The column was washed with 1mL of water and 2mL of 100% methanol. The 100% methanol elutes were collected and dried under nitrogen gas at 45°. The residues were redissolved in 200p,L of methanol and filtered through a 0.22^m-filter, the filtrate was used as UPLC sample.
2.2.3 Instrumentation and conditions The chromatographic condition was shown in Table1;Waters Micromass TOF-microTM (Manchester, UK) equipped with an electrospray ion source operating in either positive ion or negative ion mode, and with Masslynx data analysis system. The mass spectrometric data was collected in full scan mode from 100 to1500 (m/z). Table1 Solvent gradient program of UPLC analysis
Time (min) Flow (ml/min) A (%) B (%)
0.0 0.300 5.0 95.0
5.0 0.300 30.0 70.0
12.0 0.300 50.0 50.0
15.0 0.300 80.0 20.0
16.0 0.300 100.0 0.0
2.2.4 The identification of ion peaks in KXS-60%E in vitro The origin of peaks in KXS-60%E were pointed out compared with the peaks of each single herb in the formula and the control solution.
2.2.5The identification of constituents in CSF sample after oral KXS-60%E administration
The constituents in treated CSF after oral KXS-60%E were identified by contrasting the control rat CSF,Similarly,whether the constituents were original comform was identified by compared with KXS-60%E in vitro.
3. Results and discussions
3.1 The identification of ion peaks in KXS-60%E The chromatogram of the reference compounds provide the information of their retention time and mass spectra for the comparison with those of peaks detected.94 compounds were detected. 35 of the compounds were attributed to the Rhizoma Ginseng, 44 of the compounds were Radix Polygalae while 6 of were attributed to Rhizoma Acori Tatarinowii and 6 of them were Poria according to respective retention time and mass spectra. The chromatogram was shown in Fig.1.
3.2 The analysis of Constituents in CSF after oral KXS-60%E administration By comparing the UPLC-MS of CSF after oral KXS-60%E administration with that of control CSF, 3 peaks were detected in CSF in ESI- model,including peak 28 (TR=5.93, m/z=881,figer 3 a),peak 29 (TR=5.98, m/z=845,figer 3 b) and peak 57 (TR=9.03, m/z=1123,figer 3 c),however,no peaks were detceted in ESI+ model(figer 2,c,d).
O OO 2 OO 4.00 6 OO 8 OO 10 00 12 00 14 00 16.00
ContrüHESN 100
(a)
0.00 2.00 Dnxtm 100i
0.00 2.00
TOFMSES-7.97e3
JLâ^r
XU
4.00
ß.OO 10.00 12.00
Time
14.00 ' ' 16.00 TOFMSES-8.81&3
29
57
Control-(ESM 100i
(c)
0.00 2.00 Druq-(ESI+) 100i
(d)
Time
TOFMSES+
1.21
A
6.00
8.00
10.00 12.00
S
14.00
TOFMSES+ 2.82e4
^Time 16.00
4.00
6.00
8.00
10.00
12.00 14.00
0.00
2.00
ifl ,11 .yfyi'i
.00
6.00
Jl
L
10.00
12.00
14.00
Time 16.00
Fig.2 Total ion current chromatograms of KXS in negative and positive ESI mode. (a)control CSF in ESI-(b) CSF after oral administration in ESI- (c) control CSF in ESI+(b) CSF after oral administration in ESI+
(a)
2658586
I ( 447 1567 267 8471)
I ¿01 0411.
LÀ
802 5629 8454954
Drua-ESI- (5.98)
(b)
255.8268 1443 802 558t 265 8624
200 400
1200 1400
éi
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r265 8595 195.818
39121601 584 8025 537.227«
iwàtÀwumà
0 200 400
1000 1200 1400 " 0 200 400 600
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,47381408 1921
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Figer 3 The ESI-MS of constituents in CSF after oral administration in negative model
(a:neak 28,b:peak 29m c:neak 57i
4.C0IlClUSi0Il
Our preliminary studies have shown that drug-containing CSF with KXS in rats can improve the PC12 cell injury significantly induced by serum-free culture and promote cell survival adherent.Which indicates KXS was nutritious and protective to PC12 cells. These results suggest that the drug-containing CSF with KXS may exist active substances with neural growth factor-like effects[7]. Therefore,this also shows that the material basis of its efficacy exists inevitable objectively. The basic problem to be solved is that whether the active substance (group) is the prototype of the product or new metabolic substance of KXS and how about it's basic information.
As the content has been low when the drug absorbed into the bloodstream,coupled with the special structure of the Blood Brain Barrier makes it difficult to through drugs,which leading drug content in cerebrospinal fluid of animals is minimal and causing lower response peaks. We found three iconic migration component in the drug-containing CSF that had gavaged KXS.We also found thatthe three compounds were both derived from the prototype components of Ginseng in KXS according to its chromatographic characteristics and contrast to composition in vitro. These three compounds may be substance that have more direct neuroprotective effect,and at present,the structural and activity identification of them is being developedactively.
References
1.Bian Huimin,Guo Haiying,Huang Yufang,et al. Effect of Kaixin-san on learning and memory of mice in four animal models.Chinese Journal of Experimental Traditional Medical Formulae,1999,5(5):51-53.
2.Mai weili, Wang Qiong, Sun lihua, et al.Effect of Kaixinsan on learning and memory of mice after sleep deprivation. LiSHiZhen-Medicine And Materta Research. 2010,22(10):2331-2333.
3.Huang yufang, Bian huimin, Gong jiening,et al. Effect of Kaixingsan on SOD and MDA contents in four animal models. Journal Of NanJing University Of TCM, 1999, 15 (3) : 151.
4 Huang fang,Huang zijie,Wang jiali.The study of Anti-lipid peroxidation of each component of KXS in vitro [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2007, 13 (1)55-57.
5 Zeng yidan,Huang fang,Wang jiali. The modern pharmacological research of KXS [J]. Strait Pharmaceutical Journal, 2006, 18(5):12-14.
6 Wen wei,Zhang chao,Hang shuming,et al.The improvement of KXS Containing serum on Ap-induced injury in PC12 cells [J]. Information on Traditional Chinese Medicine, 2012, 29(4):80-81.
7 Li fudong,Wang yanhua,Huang shuming,et al. The growth-promoting effect of KXS drug-containing cerebrospinal fluid on serum-free culture PC12 cell [J]. Journal of Traditional Chinese Medicine, 2012, 40(6):12-14.
Studies on Amide Alkaloids of theViscum coloratum (Kom.) Nakai f.
Rubroaurantiacum Kitag
Yong-hui Sun1, Ying-li Ma 2, Yong Ling 2
(1.Research Institute of Chinese medicine, Heilongjiang university of Chinese medicine; 2.Dept of TCM, Heilongjiang university of Chinese medicine, harbin 150040,China )
Abstract: Using chromatography technique two constituents were isolated from Viscum coloratum (Kom.) Nakaif. Rubroaurantiacum Kitag for the first time. On the basis of chemical methods and spectroscopic analysis their structure were characterized as N-butyl amido cinnamyl amine and N-cinnamoyl spermidine (new compound) .
