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Effect of Ginkgo biloba leaf extract on cerebral cortex amino acid levels in cerebral ischemia model rats
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J Tradit Chin Med 2018 October 15; 38(5): 676-684 ISSN 0255-2922 . © 2018 JTCM. This is an open access article under the CC BY-NC-ND license.
RESEARCH ARTICLE TOPIC
Effect of Ginkgo biloba leaf extract on cerebral cortex amino acid levels in cerebral ischemia model rats
Cui Yiran, Wu Hongwei, Liu Mengting, Qin Haijiao, Liu Xin, Yang Hongjun aa RESULTS: Compared with the MCAO group, behavioral performance, neurological deficit score, and cerebral infarct volume were significantly improved in the MCAO_D group (P < 0.05, P < 0.01). Compared with the sham group, the levels of 17 amino acids in the cerebral cortex were markedly changed in the MCAO group. The levels of Alanine (Ala), Isoleucine (Ile), Glutamic acid (Glu), Serine (Ser), Valine (Val), Phenylalanine (Phe), Proline (Pro), Threonine (Thr), Lysine (Lys), Tyrosine (Tyr), Hydroxyproline (Hyp), Arginine (Arg), Leucine (Leu), Tryptophan (Trp), and Glycine (Gly) were increased (P < 0.001, P < 0.05), while levels of Gln and Tau were decreased (P < 0.001, P < 0.05). Compared with the MCAO group, Ginkgo biloba extract treatment in the MCAO_D group significantly down-regulated the levels of 11 amino acids, especially those of Arg, Thr, and Ser in 12 or 24 h.
Cui Yiran, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China; Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Wu Hongwei, Liu Mengting, Yang Hongjun, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Qin Haijiao, College of Science, Central South University of Forestry and Technology, Changsha 410004, China Liu Xin, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China Correspondence to: Associate Prof. Liu Xin, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China. [email protected]; Prof. Yang Hongjun, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. [email protected] Telephone: +86-10-18811072630; +86-10-13910000292; Accepted: September 12, 2017
CONCLUSION: Injection of Ginkgo biloba leaf extract has a therapeutic effect on model rats with MCAO-induced cerebral ischemia by acting on amino acids in the cerebral cortex. This effect might be associated with the regulation of amino acid metabolism in the cerebral cortex.
Abstract OBJECTIVE: To investigate the effect of Ginkgo biloba leaf extract on amino acid levels in the cerebral cortex of cerebral ischemia model rats induced by middle cerebral artery occlusion (MCAO).
© 2018 JTCM. This is an open access article under the CC BY-NC-ND license.
METHODS: A rat model of cerebral ischemia was established by MCAO. Male rats were divided into a negative control group (Control), a sham-operated group (Sham), an ischemic group (MCAO), and an ischemic group treated with Ginkgo biloba leaf extract (MCAO_D). All groups were divided into two subgroups with occlusion times of 12 and 24 h, respectively. The levels of 18 endogenous amino acids in the cerebral cortex were quantified by triple quadrupole-liquid chromatography-mass spectrometry. JTCM | www. journaltcm. com
Keywords: Cerebral ischemia; Ginkgo biloba leaf extract; Amino acids; Metabolism
INTRODUCTION Cerebral ischemic stroke, a leading cause of death and disability worldwide, is a severe neurological disorder that is usually induced by a temporary or permanent decrease in the blood supply to the brain.1An ischemic 676
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stroke is a dynamic process in which perfusion or diffusion change throughout the evolution of the infarct. In the course of its occurrence and development, cerebral infarction is closely related to changes in the amino acid metabolism.2 Exploring the characteristics of amino acid metabolism can help identify biomarkers of the disease, discover targets for drug therapy, and explain mechanisms of drug treatment. Ginkgo biloba seeds were first mentioned in Compendium of Materia Medica and are used against cough, asthma, enuresis, and alcohol misuse.3 Ginkgo biloba leaf extract has been widely used as a phytochemical and dietary supplement to improve conditions involving impaired blood circulation, for example, in the prevention and treatment of cardiovascular disease and cerebral insufficiency disorders, such as peripheral and cerebrovascular disorders, and ischemic and Alzheimer-type dementia.4,5 Ginkgo biloba leaf extract injection can affect the levels of amino acids in the cerebral cortex of cerebral ischemic rats.6 However, in this study, the number of amino acids examined was small and the time period of reperfusion was short. It is, therefore, necessary to further study the effect of Ginkgo biloba leaf extract on amino acid metabolism to comprehensively reveal its therapeutic characteristics. Thus, in this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion (MCAO), and explored the effect of Ginkgo biloba leaf extract on amino acid levels in the cerebral cortex.
(USA). Other reagents used include, chloral hydrate (Sinopharm Chemical Reagent Co., Ltd.); 2,3,5-triph-enyltet razolium chloride (TTC) (Nanjin Green syn-chemical Technology Co., Ltd., Nanjing, China); 0.9% NaCl injection solution (Shijiazhuang siyao Co., Ltd., No. 1409143203, Shijiazhuang, China); 4% polyoxymethylene (Beijing Solarbio Science﹠Technology Co., Ltd., No. 20150424, Beijing, China). Model building and drug administration Male SD rats were randomly divided into eight groups: 12 h-control, sham, MCAO, and MCAO_D groups and 24 h-control, sham, MCAO, and MCAO_D groups. Twenty-three rats were allocated to each group. Focal cerebral ischemia was induced by intra-luminal MCAO as described previously.7 Briefly, rats were anesthetized and placed on a heating pad to maintain rectal temperature at 37 ℃ . A nylon filament was advanced from the right external carotid artery into the lumen of the internal carotid artery to occlude the right MCA for 12 h (12 h groups) or 24 h (24 h groups). The filament was subsequently withdrawn to allow reperfusion. Cerebral blood flow was closely monitored by laser Doppler flowmetry during the operation. Sham-treated rats were subjected to the same surgical procedure without the insertion of the nylon filament. For the 12 h MCAO_D group, rats were intraperitoneally injected with Ginkgo biloba leaf extract at 1.575 mg/kg (equivalent to a clinical dose) at 15 min and 6 h after modeling. The drug was prepared at a concentration of 11.25 mg/ml in 0.9% NaCl before use. For the 24 h MCAO_D group, rats were intraperitoneally injected with Ginkgo biloba leaf extract at 1.575 mg/kg at 15 min, 6 h, and 12 h after modeling. The other groups (control, sham, MCAO) were given 0.9% NaCl by intraperitoneal injection. The volume of all injections was 14.4 mL/kg.
MATERIALS AND METHODS Animals Seven-week-old male Sprague-Dawley (SD) rats specific-pathogen-free (SPF) with body weight of 240-260 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [Beijing, China, License No. SCXK (Jing) 2012-0001]. All animals were kept in a SPF facility at (25 ± 2) ℃ under a normal light cycle with free access to normal diet and water. Animals were acclimatized for 3 d before use in experiments. This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed.
Evaluation of neurological deficit Before killing animals, Bederson's neurological examination scoring system was used to evaluate neurological deficit in animals.8 Scoring standard: 0: no observable deficit and normal activity (normal); 1: not fully extending the left forepaw (mild deficit); 2: circling towards the contralateral side (moderate deficit); 3: unable to walk and righting reflex impairment (severe deficit).
Drugs and reagents The Ginkgo biloba leaf extract was made by Ji Sheng Medical Co., (Taibei, China Taiwan) and contained 17.50 mg of Ginkgo biloba leaf extract and 4.20 mg of ginkgo flavone glycosides (Production code HA079). Standard references of alanine (Ala), isoleucine (Ile), glutamic acid (Glu), L-serine (Ser), valine (Val), L-phenylalanine (Phe), proline (Pro), L-threonine (Thr), hydroxyproline (Hyp), L-arginine (Arg), tryptophan (Trp), glycine (Gly), lysine (Lys), tyrosine (Tyr), leucine (Leu), glutamine (Gln), taurine (Tau), and γ-aminobutyric aid (GABA) were purchased from Sigma JTCM | www. journaltcm. com
Measurement of cerebral infarction by TTC staining After the final evaluation of neurological deficit, eight rats from each group were sacrificed and the brains were removed. The cerebellum, olfactory bulb and the remainder of the lower brain stem were removed and a 2-mm thick brain slice at the intersection of the optic nerves was rapidly dissected. Slices were fixed in 4% paraformaldehyde for 30 min, and stained in 2% TTC in phosphate buffer for 10 min at 37 ℃ without light. 677
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After staining with TTC, the normal tissue stained a rose red colored, and the infarct tissue was white. Pictures were taken using a Ben Q scan 5560 scanner (Taiwan, China). The total area of the brain slice and the infarct area were calculated using image analysis software, and the percentage of the infract area to total area was calculated.
was determined by analysis of standard solutions of different concentrations. The intra-day and inter-day precision was determined by the assay of the standard solution at different concentrations on a single day and on three continuous days. Five determinations of each amino acid concentration were analyzed and precision and accuracy were expressed as relative standard deviation (RSD % ) and deviation from the theoretical value. The stability of amino acids in cortex tissue was tested by analysis of five samples stored in 4 ℃ for 36 h. To determine the recovery, three samples were prepared and analyzed for amino acid content. Recovery of amino acids was calculated using the following equation: recovery = [(measured content-sample content)/addition content] × 100%.
Determination of amino acids in rat cortex by LC-MS An Agilent 6460 triple quadrupole (QQQ)-liquid chromatography-mass spectrometry (LC-MS) system (Agilent Technologies, Palo Alto, CA, USA) was utilized to determine amino acid levels. High pressure liquid chromatography was performed using an Agilent 1260 uHPLC system (Agilent Technologies, Palo Alto, CA, USA). The mass analyzer used was an Agilent 6460 triple quadrupole with an ESI interface (ESI-QQQ/MS, Agilent Technologies, Palo Alto, CA, USA). Brain cortex tissue samples were weighted, and added with 10 times volume (1g /10 mL) of different concentrations of methanol (20% , 50% , 100% ). The tissue was then homogenized using a high throughput homogenizer (CIENIZ SC VT2-48, Ningbo Xingzhi Biotechnology Co., Ltd., Ningbo, China) at 70 Hz for 45 s. Twenty microliters of grinding fluid was added to 980 μL initial mobile phase. After vortexing for 1 min, the sample was centrifuged at 13 500 × g for 5 min at 4 ℃ and then the supernatant was collected and 5 μL of the supernatant were injected into the LC-MS system. According to the detection of amino acids by mass spectrometry, we selected the best extraction solvent (methanol) concentration. To select the volume of extraction solvent, we added different volumes of solvent (5, 10, 20, 50 times) to Control and MCAO groups samples, and detected the amino acid response intensity. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) either in positive or negative ionization mode. Amino acids were separated by an ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm). Gradient elution was performed using water containing 0.1% formic acid-water (Solvent A) and acetonitrile containing 0.1% formic acid and 1% ammonium formate (0.1 mol/L) (Solvent B). Ten percent Solvent B (10% B) was employed for 1 min and was increased to 40% B for 7 min at a flow rate of 0.25 mL/ min. The 10% B was used for 2 min to re-equilibrate. Mass analysis of compounds was performed using positive ion mode. Other parameters were: nebulizer gas flow, 45.0 psi; dry gas flow, 11.0 L/min; electrospray voltage of the ion source, 4000 V, 52 nA; capillary temperature, 350 ℃ ; Collision Induced Dissociation (CID), 32 eV. The external standard method was used to establish a methodology, including inter-and intra-day precision, linearity range, repeatability, stability and accuracy of sample determination. The linearity of 18 amino acids JTCM | www. journaltcm. com
Statistical analysis SIMCA-P (version 11.5, Umertrics, Umea, Sweden) software was used to carry out supervised pattern recognition analysis by partial least squares discriminate analysis (PLS-DA).9 The differences between the sham and MCAO groups, and between MCAO and MCAO_D groups were observed. Metabolic markers between the groups were screened by variable importance in the projection values (VIP) (VIP > 1).10,11 Data are expressed as mean ± standard error of mean (SEM) or as percentages and were analyzed for statistical significance using one-way analysis of variance. Tests were performed using SPSS 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA); a P value less than or equal to 0.05 was considered statistically significant.
RESULTS Effects of Ginkgo biloba leaf extract on MCAO-induced cerebral ischemia The effects of Ginkgo biloba leaf extract on neurological function scores after cerebral ischemia are shown in Figure 1A. These two indicators of neurological behavior score and cerebral infarct in control and sham group were zero. Compared with the sham group, neurological behavior was strongly affected in the 12 and 24 h MCAO groups, as indicated by neurological scores measured (P < 0.001). Significant improvements in neurological scores were observed in the 24 h MCAO_D group rats treated with Ginkgo biloba leaf extract compared with the 24 h MCAO group (P < 0.05). The cerebral infarct area and volume were also reduced in drug-treated rats (Figure 1B and Figure 2). Determination of 18 amino acids in brain cortex tissue Brain tissue was extracted with methanol at a ratio of 1 ∶20. To optimize the methanol concentration for extraction, 20%-100% methanol was used to extract 678
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a
a b
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50 40 30
a b
The intra- and inter-day precision and accuracy of the assays were assessed by analyzing samples five times per day for three continuous days. In this assay, the intraand inter-day precision of various amino acids ranged from 0.92%-2.60% and 2.1%-3.8% , respectively. To calculate the repeatability of the method, the RSD of each amino acid was calculated. The RSDs of amino acids were 2.4%-4.9%, which showed that the method gave good reproducibility. The stability of amino acids from rat brain cortex was investigated using samples stored in 4 ℃. RSDs of amino acids ranged from 2.6% to 5.2%. The extraction recovery was calculated by comparing the peak areas of the brain cortex sample spiked with standards with those of the standard solutions. According to the determined values, three different concentrations were added to the amino acid mixed standard solution and the recovery was investigated. Average recovery of amino acids from brain cortex tissue was 75.4%105.4% (RSD 5.2%-11.1% , n = 6), 88.0%-122.5% (RSD 5.0%-10.2%, n = 6), and 98.4%-125.4% (RSD 4.6%-10.2% , n = 6) for high, middle, and low level samples, respectively.
B
a c
20
1 0
A
Infarct area (%)
Neurological deficits score
Cui YR et al. / Research Article
10
0 24 h 12 h 24 h Control Sham MCAO MCAO_D Figure 1 Ginkgo biloba leaf extract treatment reduced neurological deficits and cerebral infarction in middle cerebral artery occlusion rats A: The results of neurological deficits score shown that Ginkgo biloba leaf extract (1.575 mg/kg, injected) can improve neurological function in middle cerebral artery occlusion rats after 12 and 24 h ischaemic injury. B: TTC staining results and histogram shown that Ginkgo biloba leaf (1.575 mg/kg, injected) extract can decrease the volume of cerebral infraction in middle cerebral artery occlusion rats. These two indicators of neurological behavior score and cerebral infarct in control and sham group were zero. All values are expressed as the mean ± standard error of mean. MCAO group vs sham group, aP < 0.001; MCAO-D group vs MCAO group, bP < 0.05, cP < 0.01. 12 h
brain cortex tissue samples. According to amino acid detection by LC-MS, 20% methanol was determined to be the best concentration. Full scan production ion spectra of amino acids were investigated in MRM mode and monitoring ions are shown in Table 1. Amino acid calibration curves for the 18 amino acids were constructed by plotting peak area (Y), versus their concentration (X). Linearity was assessed by weighted (1/x) linear regression of calibration curves generated in triplicate on three consecutive days. Typical equations of the calibration curves are shown in Table 2. Good linearity was obtained over the concentration range from 10 to 300 pg/μL for Ala, γ-GABA, Ser, Thr and Lys, from 1 to 30 pg/μL for Ile and Gly, from 30-1000 pg/μL for Glu and Tau, with correlation coefficients r > 0.99, from 5 to 100 pg/μL for Val, Pro, Arg and Leu, from 1.5 to 50 pg/μL for Phe, from 15 to 500 pg/μL for Gln, from 0.3 to 10 pg/μL for Tyr and Hyp, and from 10 to 150 pg/μL for Trp.
Control
Data processing and analysis of amino acid metabolism According to the mass data and the amino acid standard curves, we calculated the amino acid contents in each group. First, one-way ANOVA was used to analyze amino acid data. P < 0.05 indicated significant difference between groups. Then SIMCA-P software (version 11.5) with supervised pattern recognition was applied to analyze the data. We used amino acid content as statistical variables and built modeling by PLS-DA. The loading scatter plot showed differences between different time points among groups. The VIP value of each amino acid variable (VIP>1) and the results of analysis of variance (P < 0.05) can determine the relevant biomarkers. Compared with the sham group, the levels of Ala, Ile, Glu, Ser, Val, Phe, Pro, Thr, Lys, Tyr, Hyp, Arg, Leu, Trp and Gly were significantly increased in the MACO group (P < 0.001, P < 0.05), while the levels of Gln
Sham
MCAO MCAO_D Control Sham MCAO 24 h 12 h Figure 2 Triph-enyltet razolium chloride staining of the brain The normal tissue stained a rose red colored, and the infarct tissue was white. JTCM | www. journaltcm. com
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Cui YR et al. / Research Article Table 1 Monitoring ions of various amino acids in MRM mode No.
Name
Parention
Fragmention
No.
Name
Parention
Fragmention
1
Ala
90.07
44.27
10
Thr
120.02
74.13
2
γ-GABA
104.09
87.01
11
Lys
147.08
84.15
Ile
132.12
86.16
12
Tyr
182.16
165.10
4
Glu
148.08
84.07
13
Tau
125.9
108.07
5
Ser
106.01
60.10
14
Hyp
132.09
86.15
6
Val
118.00
71.90
15
Arg
175.14
70.15
7
Phe
166.11
120.07
16
Leu
132.06
86.16
8
Gln
147.04
84.07
17
Trp
205.11
188.12
9
Pro
116.09
70.11
18
Gly
76.00
30.15
3
Table 2 Calibration curves of various amino acids No.
Name
Range (pg/μL)
calibration curves
r value
1
Ala
10-300
Y = 57.384X-1803.36
0.999
2
γ-GABA
10-300
Y = 578.654X-3470.35
0.996
Ile
1-30
Y = 5897.31X+408.95
0.992
4
Glu
30-1000
Y = 3352.66X-11922.7
0.999
5
Ser
10-300
Y = 654.991X-2613.2
0.999
6
Val
5-100
Y = 2015.06X-450.45
0.999
7
Phe
1.5-50
Y = 15907X-110.009
0.999
8
Gln
15-500
Y = 34481.2X-588028
0.967
9
Pro
5-100
Y = 5047.65X+6909.01
0.885
10
Thr
10-300
Y = 2387.16X-146750
0.996
11
Lys
10-300
Y = 2453.57X-118549
0.995
12
Tyr
0.3-10.0
Y = 2155.66X+16868.4
0.969
13
Tau
30-1000
Y = 602.901X+2814.95
0.996
14
Hyp
0.3-10.0
Y = 6345.06X-290.138
0.999
15
Arg
5-100
Y = 6624.64X-3314.27
0.993
16
Leu
5-100
Y = 4940.3X-1094.83
0.999
17
Trp
10-150
Y = 4009.86X-913.613
0.997
18
Gly
1-30
Y = 4846.25X+1015.69
0.996
3
Notes: Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: L-serine ; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threonine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid.
and Tau were significantly decreased (P < 0.001, P < 0.05). For the 12 h groups, Ginkgo biloba leaf extract treatment in the MCAO_D group significantly decreased the levels of Ala, Ser, Gln, Thr, Arg and Tau (P < 0.001, P < 0.01, P < 0.05) and increased the levels of Phe and Tyr (P < 0.001, P < 0.05) compared with the MACO group. In the 24 h groups, Ginkgo biloba leaf extract treatment in the MCAO_D group significantly reduced the levels of Ile, Ser, Phe, Pro, Thr, Lys, Tyr, Tau, Hyp, Arg, Leu and Gly compared with the MACO group (P < 0.001, P < 0.01, P < 0.05) (Figures 3-4). PLS-DA was used to analyze the influence of amino acJTCM | www. journaltcm. com
id levels in each group. A PLS-DA discriminant model was constructed to analyze the amino acid levels in the sham, MACO and MACO_D groups (Q2 value: 12 h groups, 0.934, and 24 h groups, 0.696). The PLS-DA score map showed that the sham, MACO and MACO_D groups were separated, indicating that levels of the 18 amino acids changed significantly during the process of cerebral ischemia (Figure 5). At the same time, the amino acids with a VIP value >1 in the MACO_D group at different time points were selected and compared with those of the MACO group. There were seven distinct amino acids in the 12 h groups and nine in the 24 h groups (Table 3).
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A
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Figure 3 Change in amino acid levels at 12 h A: Ala; B: GABA; C: IIe; D: Glu; E: Ser; F: Val; G: Phe; H: Gln; I: Pro; J: Thr; K: Lys; L: Tyr; M: Tau; N: Hydroxyproline; O: Arg; P: Leu; Q: Trp; R: Gly. Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: Lserine; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threonine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid. Ⅰ: Control; Ⅱ: Sham; Ⅲ: MCAO; Ⅳ: MCAO_D. The red rings indicate amino acids whose levels are changed by Ginkgo biloba leaf extract treatment. All values are expressed as the mean ± standard error of mean. aP < 0.05, eP < 0.01, dP < 0.001 vs control group; bP＜ 0.001 vs sham group; gP < 0.05, fP < 0.01, cP < 0.001 vs MCAO group.
PLS-DA analysis and statistical analysis between different groups were compared. By analyzing the VIP > 1 intersection at different times, it was found that five amino acids, Phe, Tau, Arg, Thr and Ser, were changed in the 12 and 24 h MACO groups. Differences between MACO and MACO_D groups gave a comprehensive analysis and showed that Ginkgo biloba leaf extract treatment had corrective action on Arg, Thr and Ser at 12 and 24 h, and on Phe at 24 h, but no corrective action on Tau.
and 24 h. The levels of Ala, Ile, Glu, Ser, Val, Phe, Pro, Thr, Lys, Tyr, Hyp, Arg, Leu, Trp and Gly were significantly increased (P < 0.05), while Gln and Tau were remarkably decreased in the MACO group compared with the sham group. The changing levels of Ala, Glu, Gly, Phe, Tyr, Trp, Tau, Val, Ile, Leu and Lys are consistent with those from previous reports.21,22 Ginkgo biloba leaf extract can reduce the levels of Ala, Ser, Gln, Thr, Arg and Tau, and increase the levels of Phe and Tyr in the cortex of rats with 12 h of cerebral ischemia. In rats with ischemia for 24 h, the extract can decrease the levels of Ile, Ser, Phe, Pro, Thr, Lys, Tyr, Tau, Hyp, Arg, Leu and Gly in brain cortex. A comprehensive analysis of the results by VIP value > 1 combined with multiple groups of variance analysis found that Ginkgo biloba leaf extract has a corrective action on Arg, Thr
DISCUSSION In this study, we investigated the effect of Ginkgo biloba leaf extract on the metabolism of 18 amino acids in the rat cerebral cortex after cerebral ischemia for 12 JTCM | www. journaltcm. com
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A
B
C
a
b
a
E
a
a
I
J
a
a
c
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K
a
e
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t[2]
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－4 0 2 4 6 8 10 －9 －7 －5 －3 － 0 1 t[1] t[1] Figure 5. The PLS-DA score map of sham, MACO and MACO_D groups at 12 and 24 h A: 12; B: 24h. ■: sham group, ●: MACO group, ▲: MACO_D group. －10 －8 －6 －4 －2
and Ser in the 12 and 24 h groups and on Phe in the 24 h group, while no corrective effect was observed for Tau. These results indicate that its effect on Arg, Thr and Ser in the cerebral cortex of rats with cerebral ischJTCM | www. journaltcm. com
P
Figure 4 Change in amino acid levels at 24 h R A: Ala; B: GABA; C: IIe; D: Glu; E: Ser; F: Val; G: Phe; H: Gln; I: Pro; J: Thr; K: Lys; L: Tyr; M: Tau; N: Hydroxyproline; O: Arg; P: Leu; Q: Trp; R: Gly. Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: L-serine ; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threob nine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid. Ⅰ: Control; Ⅱ: Sham; Ⅲ: MCAO; Ⅳ: MCAO_D. The red rings indicate amino acids whose levels are changed by Ginkgo biloba leaf extract treatment. All values are expressed as the mean ± SEM. fP < 0.05, cP < 0.001 vs control group; hP < 0.05, gP < 0.01, aP < 0.001 vs sham group; dP < 0.05, bP < 0.01, eP < 0.001 vs MCAO group. 4
0
b
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b
f
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M g
a
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9
emia is persistent and stable. When astrocytes are damaged, L-serine from glucose catabolism is transformed into D-serine at the presynaptic membrane, and D-serine is a coactivator of the 682
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Cui YR et al. / Research Article Table 3 Amino acids with a VIP > 1 in the MACO and MACO_D groups at different times 12 h
No.
VIP
Amino acid
1
1.51542
Phe
2
1.39369
3
No.
VIP
Amino acid
1
1.63125
Hyp
Tau
2
1.14266
Arg
1.19382
Arg
3
1.13571
Gly
4
1.14123
Thr
4
1.11857
Phe
5
1.12901
Ile
5
1.10829
Thr
6
1.11886
Gln
6
1.06567
Tyr
7
1.10749
Ser
7
1.06441
Tau
8
1.02366
Ala
9
1.01392
Ser
N-methyl-D-aspartate receptor (NMDAR).23,24 After exocytosis, D-serine activates NMDARs in the postsynaptic membrane, which causes calcium overload resulting in damage.25 Arg is a nitric oxide donor, which is generated by nitric oxide synthase. Nitric oxide is a signaling molecule that can regulate cellular function and is involved in cardiovascular system, and peripheral and central nervous system physiology. Nitric oxide has a dual role in the process of ischemic brain injury, indicating that the concentration of L-Arg may play a dual role in ischemic injury.26 Thr deficiency can inhibit the production of immunoglobulins and T and B lymphocytes, which then affects immune function. 27 In conclusion, Ginkgo biloba leaf extract may improve MCAO-induced cerebral ischemia in rats by regulating Arg, Thr and Ser in the cerebral cortex. However, it is necessary to further explore the roles that amino acids play in the process of cerebral ischemia.
3
4 5
6
7
8
ACKNOWLEDGMENTS 9
We are grateful to Guo Na from the Experimental Research Center of the China Academy of Chinese Medical Sciences for technical assistance. This study was supported by the Key Program of National Natural Science Foundation of China (81330086), the General Program of National Natural Science Foundation of China (81573726, 81403210) and the Fundamental Research Funds for the Central Universities (No. 2017-JYB-JS-054).
10
11
REFERENCES 1
2
24 h
12
Albert-Weissenberger C, Siren AL, Kleinschnitz C. Ischemic stroke and traumatic brain injury: the role of the kallikrein-kinin system. Prog Neurobiol 2013; 101-102(1): 65-82. Cheung BM, Li C. Diabetes and Hypertension: Is There a Common Metabolic Pathway? Curr Atheroscler Rep 2012; 14(2): 160-166.
JTCM | www. journaltcm. com
13
14 683
van Beek TA, Montoro P. Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals. J Chromatogr A 2009; 1216 (11): 2002-2032. Kleijnen J, Knipschild P. Ginkgo biloba. Lancet 1992; 340(8828): 1136-1139. Chen F, Li L, Xu F, et al. Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpene lactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of administration. Br J Pharmacol 2013; 170 (2): 440-457. Hu B, Mei YW, Sun SG, et al. Effect of Ginkgo Biloba extract on amino acids balance in cortex of rats during cerebral ischemia and reperfusion. Nao yu shen jing ji bing za zhi 2001; 9 (1): 1-3. Longa EZ, Weinstein PR, CarlsonS, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke1989; 20 (1): 84-91. Desland FA, Afzal A, Warraich Z, et al. Manual versus automated rodent behavioral assessment: comparing efficacy and ease of bederson and garcia neurological deficit scores to an open field video-tracking system. J Cent Nerv Syst Dis 2014; 6: 7-14. Wang-Sattler R, Yu Z, Herder C, et al. Novel biomarkers for pre-diabetes identified by metabolomics. Mol Syst Biol 2012; 8: 615-625. Smith CA, Want EJ, O'Maille G, et al. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 2006; 78 (3): 779-787. Werner E, Croixmarie V, Umbdenstock T, et al. Mass spectrometry-based metabolomics: accelerating the characterization of discriminating signals by combining statistical correlations and ultrahigh resolution. Anal Chem 2008; 80 (13): 4918-4932. Zhuang LX, Li YH, Jiang GH. Treatment and nursing of apoplexy. Shanghai: Shanghai Scientific and Technological Literature Press 2005; 71-89. Gao G, Wong L, Han SP, et al. Determination of amino acids in cerebroprotein hydrolysates with AQC pre-column derivatization by fluorescent. Zhong guo sheng wu zhi ping za zhi 2003; 16 (6): 365-367. Mathers DA, McCarthy SM, Cooke JE, Ghavanini AA, October 15, 2018 | Volume 38 | Issue 5 |
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Puil E. Effects of the beta-amino acid antagonist TAG on thalamocortical inhibition. Neuropharmacology 2009; 56 (8): 1097-1105. Liu HY, Gao WY, Wen W, et al. Taurine modulates calcium influx through L-type voltage-gated calcium channels in isolated cochlear outer hair cells in guinea pigs. Neurosci Lett 2006; 399 (1-2): 23-26. Li Y, Li C, Yan ZY. Monitoring the change of D-serine in the ischemia rats by microdialysis-capillary electrophoresis experiment. Fen xi hua xue 2009; 37 (A03): 251-251. Kagiyama T, Glushakov AV, Sumners C, et al. Neuroprotective action of halogenated derivatives of L-phenylalanine. Stroke 2004; 35 (5): 1192-1196. Uríková A, Babusíková E, Dobrota D, et al. Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion-induced oxidative stress products formation in rat forebrain. Cell Mol Neurobiol 2006; 26 (7-8): 1343-1353. Yan FL, Zheng Y, Zhao FD. Effects of ginkgo biloba extract EGb761 on expression of RAGE and LRP-1 in cerebral microvascular endothelial cells under chronic hypoxia and hypoglycemia. Acta Neuropathol 2008; 116 (5): 529-535. Zheng XJ, Xu Y. Research progress of Ginkgo biloba extract in the treatment of acute cerebral infarction. Zhong Xi Yi Jie He Xin Nao Xue Guan Za Zhi 2014; 12 (5): 617-618.
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22
23
24
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26
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Gao J, Yang GL, Chen C, et al. Determination endogenous amino acids in brain tissues after cerebral ischemia by RRLC-QQQ. Zhong Guo Zhong Yao Za Zhi 2013; 38 (5): 748-752. Guo ZL, Zhu Y, Su XT, et al. Danhong injection dose-dependently varies amino acid metabolites and metabolic pathways in the treatment of rats with cerebral ischemia. Acta Pharmacol Sin 2015; 36(6): 748-757. Panatier A, Theodosis DT, Mothet JP, et al. Glia derived d-serine controls NMDA receptor activity and synaptic memory. Cell 2006; 125 (4): 775-784. Gustafson EC, Stevens ER, Wolosker H, et al. Endogenous d-serine contributes to NMDA-receptor-mediated light-evoked responses in the vertebrate retina. J Neurophysiol 2007; 98 (1): 122-130. Wolosker H, Dumin E, Balan L, et al. D-Amino acids in the brain: D-serine in neurotransmission and neurodegeneration. FEBS J 2008; 275 (14): 3514-3526. Yang RS, Wu X, Ma WK. Effect of L-Arginine-NO pathway in myocardial ischemia and reperfusion in isolated langendorff rat heart model. Jiangsu Yi Yao Za Zhi 2002; 28 (9): 668-670. Ralph DM, Robinson SR, Campbell MS, et al. Histidine, cystine, glutamine, and threonine collectively protect astrocytes from the toxicity of zinc. Free Radical Biol Med 2010; 49(4): 649-657.
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J Tradit Chin Med 2018 October 15; 38(5): 676-684 ISSN 0255-2922 . © 2018 JTCM. This is an open access article under the CC BY-NC-ND license.
RESEARCH ARTICLE TOPIC
Effect of Ginkgo biloba leaf extract on cerebral cortex amino acid levels in cerebral ischemia model rats
Cui Yiran, Wu Hongwei, Liu Mengting, Qin Haijiao, Liu Xin, Yang Hongjun aa RESULTS: Compared with the MCAO group, behavioral performance, neurological deficit score, and cerebral infarct volume were significantly improved in the MCAO_D group (P < 0.05, P < 0.01). Compared with the sham group, the levels of 17 amino acids in the cerebral cortex were markedly changed in the MCAO group. The levels of Alanine (Ala), Isoleucine (Ile), Glutamic acid (Glu), Serine (Ser), Valine (Val), Phenylalanine (Phe), Proline (Pro), Threonine (Thr), Lysine (Lys), Tyrosine (Tyr), Hydroxyproline (Hyp), Arginine (Arg), Leucine (Leu), Tryptophan (Trp), and Glycine (Gly) were increased (P < 0.001, P < 0.05), while levels of Gln and Tau were decreased (P < 0.001, P < 0.05). Compared with the MCAO group, Ginkgo biloba extract treatment in the MCAO_D group significantly down-regulated the levels of 11 amino acids, especially those of Arg, Thr, and Ser in 12 or 24 h.
Cui Yiran, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China; Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Wu Hongwei, Liu Mengting, Yang Hongjun, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Qin Haijiao, College of Science, Central South University of Forestry and Technology, Changsha 410004, China Liu Xin, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China Correspondence to: Associate Prof. Liu Xin, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China. [email protected]; Prof. Yang Hongjun, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. [email protected] Telephone: +86-10-18811072630; +86-10-13910000292; Accepted: September 12, 2017
CONCLUSION: Injection of Ginkgo biloba leaf extract has a therapeutic effect on model rats with MCAO-induced cerebral ischemia by acting on amino acids in the cerebral cortex. This effect might be associated with the regulation of amino acid metabolism in the cerebral cortex.
Abstract OBJECTIVE: To investigate the effect of Ginkgo biloba leaf extract on amino acid levels in the cerebral cortex of cerebral ischemia model rats induced by middle cerebral artery occlusion (MCAO).
© 2018 JTCM. This is an open access article under the CC BY-NC-ND license.
METHODS: A rat model of cerebral ischemia was established by MCAO. Male rats were divided into a negative control group (Control), a sham-operated group (Sham), an ischemic group (MCAO), and an ischemic group treated with Ginkgo biloba leaf extract (MCAO_D). All groups were divided into two subgroups with occlusion times of 12 and 24 h, respectively. The levels of 18 endogenous amino acids in the cerebral cortex were quantified by triple quadrupole-liquid chromatography-mass spectrometry. JTCM | www. journaltcm. com
Keywords: Cerebral ischemia; Ginkgo biloba leaf extract; Amino acids; Metabolism
INTRODUCTION Cerebral ischemic stroke, a leading cause of death and disability worldwide, is a severe neurological disorder that is usually induced by a temporary or permanent decrease in the blood supply to the brain.1An ischemic 676
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stroke is a dynamic process in which perfusion or diffusion change throughout the evolution of the infarct. In the course of its occurrence and development, cerebral infarction is closely related to changes in the amino acid metabolism.2 Exploring the characteristics of amino acid metabolism can help identify biomarkers of the disease, discover targets for drug therapy, and explain mechanisms of drug treatment. Ginkgo biloba seeds were first mentioned in Compendium of Materia Medica and are used against cough, asthma, enuresis, and alcohol misuse.3 Ginkgo biloba leaf extract has been widely used as a phytochemical and dietary supplement to improve conditions involving impaired blood circulation, for example, in the prevention and treatment of cardiovascular disease and cerebral insufficiency disorders, such as peripheral and cerebrovascular disorders, and ischemic and Alzheimer-type dementia.4,5 Ginkgo biloba leaf extract injection can affect the levels of amino acids in the cerebral cortex of cerebral ischemic rats.6 However, in this study, the number of amino acids examined was small and the time period of reperfusion was short. It is, therefore, necessary to further study the effect of Ginkgo biloba leaf extract on amino acid metabolism to comprehensively reveal its therapeutic characteristics. Thus, in this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion (MCAO), and explored the effect of Ginkgo biloba leaf extract on amino acid levels in the cerebral cortex.
(USA). Other reagents used include, chloral hydrate (Sinopharm Chemical Reagent Co., Ltd.); 2,3,5-triph-enyltet razolium chloride (TTC) (Nanjin Green syn-chemical Technology Co., Ltd., Nanjing, China); 0.9% NaCl injection solution (Shijiazhuang siyao Co., Ltd., No. 1409143203, Shijiazhuang, China); 4% polyoxymethylene (Beijing Solarbio Science﹠Technology Co., Ltd., No. 20150424, Beijing, China). Model building and drug administration Male SD rats were randomly divided into eight groups: 12 h-control, sham, MCAO, and MCAO_D groups and 24 h-control, sham, MCAO, and MCAO_D groups. Twenty-three rats were allocated to each group. Focal cerebral ischemia was induced by intra-luminal MCAO as described previously.7 Briefly, rats were anesthetized and placed on a heating pad to maintain rectal temperature at 37 ℃ . A nylon filament was advanced from the right external carotid artery into the lumen of the internal carotid artery to occlude the right MCA for 12 h (12 h groups) or 24 h (24 h groups). The filament was subsequently withdrawn to allow reperfusion. Cerebral blood flow was closely monitored by laser Doppler flowmetry during the operation. Sham-treated rats were subjected to the same surgical procedure without the insertion of the nylon filament. For the 12 h MCAO_D group, rats were intraperitoneally injected with Ginkgo biloba leaf extract at 1.575 mg/kg (equivalent to a clinical dose) at 15 min and 6 h after modeling. The drug was prepared at a concentration of 11.25 mg/ml in 0.9% NaCl before use. For the 24 h MCAO_D group, rats were intraperitoneally injected with Ginkgo biloba leaf extract at 1.575 mg/kg at 15 min, 6 h, and 12 h after modeling. The other groups (control, sham, MCAO) were given 0.9% NaCl by intraperitoneal injection. The volume of all injections was 14.4 mL/kg.
MATERIALS AND METHODS Animals Seven-week-old male Sprague-Dawley (SD) rats specific-pathogen-free (SPF) with body weight of 240-260 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [Beijing, China, License No. SCXK (Jing) 2012-0001]. All animals were kept in a SPF facility at (25 ± 2) ℃ under a normal light cycle with free access to normal diet and water. Animals were acclimatized for 3 d before use in experiments. This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed.
Evaluation of neurological deficit Before killing animals, Bederson's neurological examination scoring system was used to evaluate neurological deficit in animals.8 Scoring standard: 0: no observable deficit and normal activity (normal); 1: not fully extending the left forepaw (mild deficit); 2: circling towards the contralateral side (moderate deficit); 3: unable to walk and righting reflex impairment (severe deficit).
Drugs and reagents The Ginkgo biloba leaf extract was made by Ji Sheng Medical Co., (Taibei, China Taiwan) and contained 17.50 mg of Ginkgo biloba leaf extract and 4.20 mg of ginkgo flavone glycosides (Production code HA079). Standard references of alanine (Ala), isoleucine (Ile), glutamic acid (Glu), L-serine (Ser), valine (Val), L-phenylalanine (Phe), proline (Pro), L-threonine (Thr), hydroxyproline (Hyp), L-arginine (Arg), tryptophan (Trp), glycine (Gly), lysine (Lys), tyrosine (Tyr), leucine (Leu), glutamine (Gln), taurine (Tau), and γ-aminobutyric aid (GABA) were purchased from Sigma JTCM | www. journaltcm. com
Measurement of cerebral infarction by TTC staining After the final evaluation of neurological deficit, eight rats from each group were sacrificed and the brains were removed. The cerebellum, olfactory bulb and the remainder of the lower brain stem were removed and a 2-mm thick brain slice at the intersection of the optic nerves was rapidly dissected. Slices were fixed in 4% paraformaldehyde for 30 min, and stained in 2% TTC in phosphate buffer for 10 min at 37 ℃ without light. 677
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After staining with TTC, the normal tissue stained a rose red colored, and the infarct tissue was white. Pictures were taken using a Ben Q scan 5560 scanner (Taiwan, China). The total area of the brain slice and the infarct area were calculated using image analysis software, and the percentage of the infract area to total area was calculated.
was determined by analysis of standard solutions of different concentrations. The intra-day and inter-day precision was determined by the assay of the standard solution at different concentrations on a single day and on three continuous days. Five determinations of each amino acid concentration were analyzed and precision and accuracy were expressed as relative standard deviation (RSD % ) and deviation from the theoretical value. The stability of amino acids in cortex tissue was tested by analysis of five samples stored in 4 ℃ for 36 h. To determine the recovery, three samples were prepared and analyzed for amino acid content. Recovery of amino acids was calculated using the following equation: recovery = [(measured content-sample content)/addition content] × 100%.
Determination of amino acids in rat cortex by LC-MS An Agilent 6460 triple quadrupole (QQQ)-liquid chromatography-mass spectrometry (LC-MS) system (Agilent Technologies, Palo Alto, CA, USA) was utilized to determine amino acid levels. High pressure liquid chromatography was performed using an Agilent 1260 uHPLC system (Agilent Technologies, Palo Alto, CA, USA). The mass analyzer used was an Agilent 6460 triple quadrupole with an ESI interface (ESI-QQQ/MS, Agilent Technologies, Palo Alto, CA, USA). Brain cortex tissue samples were weighted, and added with 10 times volume (1g /10 mL) of different concentrations of methanol (20% , 50% , 100% ). The tissue was then homogenized using a high throughput homogenizer (CIENIZ SC VT2-48, Ningbo Xingzhi Biotechnology Co., Ltd., Ningbo, China) at 70 Hz for 45 s. Twenty microliters of grinding fluid was added to 980 μL initial mobile phase. After vortexing for 1 min, the sample was centrifuged at 13 500 × g for 5 min at 4 ℃ and then the supernatant was collected and 5 μL of the supernatant were injected into the LC-MS system. According to the detection of amino acids by mass spectrometry, we selected the best extraction solvent (methanol) concentration. To select the volume of extraction solvent, we added different volumes of solvent (5, 10, 20, 50 times) to Control and MCAO groups samples, and detected the amino acid response intensity. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) either in positive or negative ionization mode. Amino acids were separated by an ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm). Gradient elution was performed using water containing 0.1% formic acid-water (Solvent A) and acetonitrile containing 0.1% formic acid and 1% ammonium formate (0.1 mol/L) (Solvent B). Ten percent Solvent B (10% B) was employed for 1 min and was increased to 40% B for 7 min at a flow rate of 0.25 mL/ min. The 10% B was used for 2 min to re-equilibrate. Mass analysis of compounds was performed using positive ion mode. Other parameters were: nebulizer gas flow, 45.0 psi; dry gas flow, 11.0 L/min; electrospray voltage of the ion source, 4000 V, 52 nA; capillary temperature, 350 ℃ ; Collision Induced Dissociation (CID), 32 eV. The external standard method was used to establish a methodology, including inter-and intra-day precision, linearity range, repeatability, stability and accuracy of sample determination. The linearity of 18 amino acids JTCM | www. journaltcm. com
Statistical analysis SIMCA-P (version 11.5, Umertrics, Umea, Sweden) software was used to carry out supervised pattern recognition analysis by partial least squares discriminate analysis (PLS-DA).9 The differences between the sham and MCAO groups, and between MCAO and MCAO_D groups were observed. Metabolic markers between the groups were screened by variable importance in the projection values (VIP) (VIP > 1).10,11 Data are expressed as mean ± standard error of mean (SEM) or as percentages and were analyzed for statistical significance using one-way analysis of variance. Tests were performed using SPSS 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA); a P value less than or equal to 0.05 was considered statistically significant.
RESULTS Effects of Ginkgo biloba leaf extract on MCAO-induced cerebral ischemia The effects of Ginkgo biloba leaf extract on neurological function scores after cerebral ischemia are shown in Figure 1A. These two indicators of neurological behavior score and cerebral infarct in control and sham group were zero. Compared with the sham group, neurological behavior was strongly affected in the 12 and 24 h MCAO groups, as indicated by neurological scores measured (P < 0.001). Significant improvements in neurological scores were observed in the 24 h MCAO_D group rats treated with Ginkgo biloba leaf extract compared with the 24 h MCAO group (P < 0.05). The cerebral infarct area and volume were also reduced in drug-treated rats (Figure 1B and Figure 2). Determination of 18 amino acids in brain cortex tissue Brain tissue was extracted with methanol at a ratio of 1 ∶20. To optimize the methanol concentration for extraction, 20%-100% methanol was used to extract 678
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3
a
a b
2
50 40 30
a b
The intra- and inter-day precision and accuracy of the assays were assessed by analyzing samples five times per day for three continuous days. In this assay, the intraand inter-day precision of various amino acids ranged from 0.92%-2.60% and 2.1%-3.8% , respectively. To calculate the repeatability of the method, the RSD of each amino acid was calculated. The RSDs of amino acids were 2.4%-4.9%, which showed that the method gave good reproducibility. The stability of amino acids from rat brain cortex was investigated using samples stored in 4 ℃. RSDs of amino acids ranged from 2.6% to 5.2%. The extraction recovery was calculated by comparing the peak areas of the brain cortex sample spiked with standards with those of the standard solutions. According to the determined values, three different concentrations were added to the amino acid mixed standard solution and the recovery was investigated. Average recovery of amino acids from brain cortex tissue was 75.4%105.4% (RSD 5.2%-11.1% , n = 6), 88.0%-122.5% (RSD 5.0%-10.2%, n = 6), and 98.4%-125.4% (RSD 4.6%-10.2% , n = 6) for high, middle, and low level samples, respectively.
B
a c
20
1 0
A
Infarct area (%)
Neurological deficits score
Cui YR et al. / Research Article
10
0 24 h 12 h 24 h Control Sham MCAO MCAO_D Figure 1 Ginkgo biloba leaf extract treatment reduced neurological deficits and cerebral infarction in middle cerebral artery occlusion rats A: The results of neurological deficits score shown that Ginkgo biloba leaf extract (1.575 mg/kg, injected) can improve neurological function in middle cerebral artery occlusion rats after 12 and 24 h ischaemic injury. B: TTC staining results and histogram shown that Ginkgo biloba leaf (1.575 mg/kg, injected) extract can decrease the volume of cerebral infraction in middle cerebral artery occlusion rats. These two indicators of neurological behavior score and cerebral infarct in control and sham group were zero. All values are expressed as the mean ± standard error of mean. MCAO group vs sham group, aP < 0.001; MCAO-D group vs MCAO group, bP < 0.05, cP < 0.01. 12 h
brain cortex tissue samples. According to amino acid detection by LC-MS, 20% methanol was determined to be the best concentration. Full scan production ion spectra of amino acids were investigated in MRM mode and monitoring ions are shown in Table 1. Amino acid calibration curves for the 18 amino acids were constructed by plotting peak area (Y), versus their concentration (X). Linearity was assessed by weighted (1/x) linear regression of calibration curves generated in triplicate on three consecutive days. Typical equations of the calibration curves are shown in Table 2. Good linearity was obtained over the concentration range from 10 to 300 pg/μL for Ala, γ-GABA, Ser, Thr and Lys, from 1 to 30 pg/μL for Ile and Gly, from 30-1000 pg/μL for Glu and Tau, with correlation coefficients r > 0.99, from 5 to 100 pg/μL for Val, Pro, Arg and Leu, from 1.5 to 50 pg/μL for Phe, from 15 to 500 pg/μL for Gln, from 0.3 to 10 pg/μL for Tyr and Hyp, and from 10 to 150 pg/μL for Trp.
Control
Data processing and analysis of amino acid metabolism According to the mass data and the amino acid standard curves, we calculated the amino acid contents in each group. First, one-way ANOVA was used to analyze amino acid data. P < 0.05 indicated significant difference between groups. Then SIMCA-P software (version 11.5) with supervised pattern recognition was applied to analyze the data. We used amino acid content as statistical variables and built modeling by PLS-DA. The loading scatter plot showed differences between different time points among groups. The VIP value of each amino acid variable (VIP>1) and the results of analysis of variance (P < 0.05) can determine the relevant biomarkers. Compared with the sham group, the levels of Ala, Ile, Glu, Ser, Val, Phe, Pro, Thr, Lys, Tyr, Hyp, Arg, Leu, Trp and Gly were significantly increased in the MACO group (P < 0.001, P < 0.05), while the levels of Gln
Sham
MCAO MCAO_D Control Sham MCAO 24 h 12 h Figure 2 Triph-enyltet razolium chloride staining of the brain The normal tissue stained a rose red colored, and the infarct tissue was white. JTCM | www. journaltcm. com
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MCAO_D
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Cui YR et al. / Research Article Table 1 Monitoring ions of various amino acids in MRM mode No.
Name
Parention
Fragmention
No.
Name
Parention
Fragmention
1
Ala
90.07
44.27
10
Thr
120.02
74.13
2
γ-GABA
104.09
87.01
11
Lys
147.08
84.15
Ile
132.12
86.16
12
Tyr
182.16
165.10
4
Glu
148.08
84.07
13
Tau
125.9
108.07
5
Ser
106.01
60.10
14
Hyp
132.09
86.15
6
Val
118.00
71.90
15
Arg
175.14
70.15
7
Phe
166.11
120.07
16
Leu
132.06
86.16
8
Gln
147.04
84.07
17
Trp
205.11
188.12
9
Pro
116.09
70.11
18
Gly
76.00
30.15
3
Table 2 Calibration curves of various amino acids No.
Name
Range (pg/μL)
calibration curves
r value
1
Ala
10-300
Y = 57.384X-1803.36
0.999
2
γ-GABA
10-300
Y = 578.654X-3470.35
0.996
Ile
1-30
Y = 5897.31X+408.95
0.992
4
Glu
30-1000
Y = 3352.66X-11922.7
0.999
5
Ser
10-300
Y = 654.991X-2613.2
0.999
6
Val
5-100
Y = 2015.06X-450.45
0.999
7
Phe
1.5-50
Y = 15907X-110.009
0.999
8
Gln
15-500
Y = 34481.2X-588028
0.967
9
Pro
5-100
Y = 5047.65X+6909.01
0.885
10
Thr
10-300
Y = 2387.16X-146750
0.996
11
Lys
10-300
Y = 2453.57X-118549
0.995
12
Tyr
0.3-10.0
Y = 2155.66X+16868.4
0.969
13
Tau
30-1000
Y = 602.901X+2814.95
0.996
14
Hyp
0.3-10.0
Y = 6345.06X-290.138
0.999
15
Arg
5-100
Y = 6624.64X-3314.27
0.993
16
Leu
5-100
Y = 4940.3X-1094.83
0.999
17
Trp
10-150
Y = 4009.86X-913.613
0.997
18
Gly
1-30
Y = 4846.25X+1015.69
0.996
3
Notes: Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: L-serine ; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threonine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid.
and Tau were significantly decreased (P < 0.001, P < 0.05). For the 12 h groups, Ginkgo biloba leaf extract treatment in the MCAO_D group significantly decreased the levels of Ala, Ser, Gln, Thr, Arg and Tau (P < 0.001, P < 0.01, P < 0.05) and increased the levels of Phe and Tyr (P < 0.001, P < 0.05) compared with the MACO group. In the 24 h groups, Ginkgo biloba leaf extract treatment in the MCAO_D group significantly reduced the levels of Ile, Ser, Phe, Pro, Thr, Lys, Tyr, Tau, Hyp, Arg, Leu and Gly compared with the MACO group (P < 0.001, P < 0.01, P < 0.05) (Figures 3-4). PLS-DA was used to analyze the influence of amino acJTCM | www. journaltcm. com
id levels in each group. A PLS-DA discriminant model was constructed to analyze the amino acid levels in the sham, MACO and MACO_D groups (Q2 value: 12 h groups, 0.934, and 24 h groups, 0.696). The PLS-DA score map showed that the sham, MACO and MACO_D groups were separated, indicating that levels of the 18 amino acids changed significantly during the process of cerebral ischemia (Figure 5). At the same time, the amino acids with a VIP value >1 in the MACO_D group at different time points were selected and compared with those of the MACO group. There were seven distinct amino acids in the 12 h groups and nine in the 24 h groups (Table 3).
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A
C
B
c
b
b
D d
b
a
E b
G
F
b
c
c
b
H
a b
f
e
I b
d
b
M b
K
J
b
b
b
c
N
b
L
O g
g
P b
c e
Q b
R b
Figure 3 Change in amino acid levels at 12 h A: Ala; B: GABA; C: IIe; D: Glu; E: Ser; F: Val; G: Phe; H: Gln; I: Pro; J: Thr; K: Lys; L: Tyr; M: Tau; N: Hydroxyproline; O: Arg; P: Leu; Q: Trp; R: Gly. Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: Lserine; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threonine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid. Ⅰ: Control; Ⅱ: Sham; Ⅲ: MCAO; Ⅳ: MCAO_D. The red rings indicate amino acids whose levels are changed by Ginkgo biloba leaf extract treatment. All values are expressed as the mean ± standard error of mean. aP < 0.05, eP < 0.01, dP < 0.001 vs control group; bP＜ 0.001 vs sham group; gP < 0.05, fP < 0.01, cP < 0.001 vs MCAO group.
PLS-DA analysis and statistical analysis between different groups were compared. By analyzing the VIP > 1 intersection at different times, it was found that five amino acids, Phe, Tau, Arg, Thr and Ser, were changed in the 12 and 24 h MACO groups. Differences between MACO and MACO_D groups gave a comprehensive analysis and showed that Ginkgo biloba leaf extract treatment had corrective action on Arg, Thr and Ser at 12 and 24 h, and on Phe at 24 h, but no corrective action on Tau.
and 24 h. The levels of Ala, Ile, Glu, Ser, Val, Phe, Pro, Thr, Lys, Tyr, Hyp, Arg, Leu, Trp and Gly were significantly increased (P < 0.05), while Gln and Tau were remarkably decreased in the MACO group compared with the sham group. The changing levels of Ala, Glu, Gly, Phe, Tyr, Trp, Tau, Val, Ile, Leu and Lys are consistent with those from previous reports.21,22 Ginkgo biloba leaf extract can reduce the levels of Ala, Ser, Gln, Thr, Arg and Tau, and increase the levels of Phe and Tyr in the cortex of rats with 12 h of cerebral ischemia. In rats with ischemia for 24 h, the extract can decrease the levels of Ile, Ser, Phe, Pro, Thr, Lys, Tyr, Tau, Hyp, Arg, Leu and Gly in brain cortex. A comprehensive analysis of the results by VIP value > 1 combined with multiple groups of variance analysis found that Ginkgo biloba leaf extract has a corrective action on Arg, Thr
DISCUSSION In this study, we investigated the effect of Ginkgo biloba leaf extract on the metabolism of 18 amino acids in the rat cerebral cortex after cerebral ischemia for 12 JTCM | www. journaltcm. com
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Cui YR et al. / Research Article
A
B
C
a
b
a
E
a
a
I
J
a
a
c
a
K
a
e
H
d
a
d
b
a
O
N
a
a
a
e b
h
A
B
2 t[2]
t[2]
－2
0
－2
－4 0 2 4 6 8 10 －9 －7 －5 －3 － 0 1 t[1] t[1] Figure 5. The PLS-DA score map of sham, MACO and MACO_D groups at 12 and 24 h A: 12; B: 24h. ■: sham group, ●: MACO group, ▲: MACO_D group. －10 －8 －6 －4 －2
and Ser in the 12 and 24 h groups and on Phe in the 24 h group, while no corrective effect was observed for Tau. These results indicate that its effect on Arg, Thr and Ser in the cerebral cortex of rats with cerebral ischJTCM | www. journaltcm. com
P
Figure 4 Change in amino acid levels at 24 h R A: Ala; B: GABA; C: IIe; D: Glu; E: Ser; F: Val; G: Phe; H: Gln; I: Pro; J: Thr; K: Lys; L: Tyr; M: Tau; N: Hydroxyproline; O: Arg; P: Leu; Q: Trp; R: Gly. Ala: alanine; Ile: isoleucine; Glu: glutamic acid; Ser: L-serine ; Val: valine; Phe: L-phenylalanine; Pro: proline; Thr: L-threob nine, Hyp: hydroxyproline; Arg: L-arginine; Trp: tryptophan; Gly: glycine; Lys: lysine; Tyr: tyrosine; Leu: leucine; Gln: glutamine; Tau: taurine; GABA: γ-aminobutyric aid. Ⅰ: Control; Ⅱ: Sham; Ⅲ: MCAO; Ⅳ: MCAO_D. The red rings indicate amino acids whose levels are changed by Ginkgo biloba leaf extract treatment. All values are expressed as the mean ± SEM. fP < 0.05, cP < 0.001 vs control group; hP < 0.05, gP < 0.01, aP < 0.001 vs sham group; dP < 0.05, bP < 0.01, eP < 0.001 vs MCAO group. 4
0
b
b
b
f
Q
2
L
f
M g
a
G
F
d
a
D
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9
emia is persistent and stable. When astrocytes are damaged, L-serine from glucose catabolism is transformed into D-serine at the presynaptic membrane, and D-serine is a coactivator of the 682
October 15, 2018 | Volume 38 | Issue 5 |
Cui YR et al. / Research Article Table 3 Amino acids with a VIP > 1 in the MACO and MACO_D groups at different times 12 h
No.
VIP
Amino acid
1
1.51542
Phe
2
1.39369
3
No.
VIP
Amino acid
1
1.63125
Hyp
Tau
2
1.14266
Arg
1.19382
Arg
3
1.13571
Gly
4
1.14123
Thr
4
1.11857
Phe
5
1.12901
Ile
5
1.10829
Thr
6
1.11886
Gln
6
1.06567
Tyr
7
1.10749
Ser
7
1.06441
Tau
8
1.02366
Ala
9
1.01392
Ser
N-methyl-D-aspartate receptor (NMDAR).23,24 After exocytosis, D-serine activates NMDARs in the postsynaptic membrane, which causes calcium overload resulting in damage.25 Arg is a nitric oxide donor, which is generated by nitric oxide synthase. Nitric oxide is a signaling molecule that can regulate cellular function and is involved in cardiovascular system, and peripheral and central nervous system physiology. Nitric oxide has a dual role in the process of ischemic brain injury, indicating that the concentration of L-Arg may play a dual role in ischemic injury.26 Thr deficiency can inhibit the production of immunoglobulins and T and B lymphocytes, which then affects immune function. 27 In conclusion, Ginkgo biloba leaf extract may improve MCAO-induced cerebral ischemia in rats by regulating Arg, Thr and Ser in the cerebral cortex. However, it is necessary to further explore the roles that amino acids play in the process of cerebral ischemia.
3
4 5
6
7
8
ACKNOWLEDGMENTS 9
We are grateful to Guo Na from the Experimental Research Center of the China Academy of Chinese Medical Sciences for technical assistance. This study was supported by the Key Program of National Natural Science Foundation of China (81330086), the General Program of National Natural Science Foundation of China (81573726, 81403210) and the Fundamental Research Funds for the Central Universities (No. 2017-JYB-JS-054).
10
11
REFERENCES 1
2
24 h
12
Albert-Weissenberger C, Siren AL, Kleinschnitz C. Ischemic stroke and traumatic brain injury: the role of the kallikrein-kinin system. Prog Neurobiol 2013; 101-102(1): 65-82. Cheung BM, Li C. Diabetes and Hypertension: Is There a Common Metabolic Pathway? Curr Atheroscler Rep 2012; 14(2): 160-166.
JTCM | www. journaltcm. com
13
14 683
van Beek TA, Montoro P. Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals. J Chromatogr A 2009; 1216 (11): 2002-2032. Kleijnen J, Knipschild P. Ginkgo biloba. Lancet 1992; 340(8828): 1136-1139. Chen F, Li L, Xu F, et al. Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpene lactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of administration. Br J Pharmacol 2013; 170 (2): 440-457. Hu B, Mei YW, Sun SG, et al. Effect of Ginkgo Biloba extract on amino acids balance in cortex of rats during cerebral ischemia and reperfusion. Nao yu shen jing ji bing za zhi 2001; 9 (1): 1-3. Longa EZ, Weinstein PR, CarlsonS, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke1989; 20 (1): 84-91. Desland FA, Afzal A, Warraich Z, et al. Manual versus automated rodent behavioral assessment: comparing efficacy and ease of bederson and garcia neurological deficit scores to an open field video-tracking system. J Cent Nerv Syst Dis 2014; 6: 7-14. Wang-Sattler R, Yu Z, Herder C, et al. Novel biomarkers for pre-diabetes identified by metabolomics. Mol Syst Biol 2012; 8: 615-625. Smith CA, Want EJ, O'Maille G, et al. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 2006; 78 (3): 779-787. Werner E, Croixmarie V, Umbdenstock T, et al. Mass spectrometry-based metabolomics: accelerating the characterization of discriminating signals by combining statistical correlations and ultrahigh resolution. Anal Chem 2008; 80 (13): 4918-4932. Zhuang LX, Li YH, Jiang GH. Treatment and nursing of apoplexy. Shanghai: Shanghai Scientific and Technological Literature Press 2005; 71-89. Gao G, Wong L, Han SP, et al. Determination of amino acids in cerebroprotein hydrolysates with AQC pre-column derivatization by fluorescent. Zhong guo sheng wu zhi ping za zhi 2003; 16 (6): 365-367. Mathers DA, McCarthy SM, Cooke JE, Ghavanini AA, October 15, 2018 | Volume 38 | Issue 5 |
Cui YR et al. / Research Article
15
16
17
18
19
20
21
Puil E. Effects of the beta-amino acid antagonist TAG on thalamocortical inhibition. Neuropharmacology 2009; 56 (8): 1097-1105. Liu HY, Gao WY, Wen W, et al. Taurine modulates calcium influx through L-type voltage-gated calcium channels in isolated cochlear outer hair cells in guinea pigs. Neurosci Lett 2006; 399 (1-2): 23-26. Li Y, Li C, Yan ZY. Monitoring the change of D-serine in the ischemia rats by microdialysis-capillary electrophoresis experiment. Fen xi hua xue 2009; 37 (A03): 251-251. Kagiyama T, Glushakov AV, Sumners C, et al. Neuroprotective action of halogenated derivatives of L-phenylalanine. Stroke 2004; 35 (5): 1192-1196. Uríková A, Babusíková E, Dobrota D, et al. Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion-induced oxidative stress products formation in rat forebrain. Cell Mol Neurobiol 2006; 26 (7-8): 1343-1353. Yan FL, Zheng Y, Zhao FD. Effects of ginkgo biloba extract EGb761 on expression of RAGE and LRP-1 in cerebral microvascular endothelial cells under chronic hypoxia and hypoglycemia. Acta Neuropathol 2008; 116 (5): 529-535. Zheng XJ, Xu Y. Research progress of Ginkgo biloba extract in the treatment of acute cerebral infarction. Zhong Xi Yi Jie He Xin Nao Xue Guan Za Zhi 2014; 12 (5): 617-618.
JTCM | www. journaltcm. com
22
23
24
25
26
27
684
Gao J, Yang GL, Chen C, et al. Determination endogenous amino acids in brain tissues after cerebral ischemia by RRLC-QQQ. Zhong Guo Zhong Yao Za Zhi 2013; 38 (5): 748-752. Guo ZL, Zhu Y, Su XT, et al. Danhong injection dose-dependently varies amino acid metabolites and metabolic pathways in the treatment of rats with cerebral ischemia. Acta Pharmacol Sin 2015; 36(6): 748-757. Panatier A, Theodosis DT, Mothet JP, et al. Glia derived d-serine controls NMDA receptor activity and synaptic memory. Cell 2006; 125 (4): 775-784. Gustafson EC, Stevens ER, Wolosker H, et al. Endogenous d-serine contributes to NMDA-receptor-mediated light-evoked responses in the vertebrate retina. J Neurophysiol 2007; 98 (1): 122-130. Wolosker H, Dumin E, Balan L, et al. D-Amino acids in the brain: D-serine in neurotransmission and neurodegeneration. FEBS J 2008; 275 (14): 3514-3526. Yang RS, Wu X, Ma WK. Effect of L-Arginine-NO pathway in myocardial ischemia and reperfusion in isolated langendorff rat heart model. Jiangsu Yi Yao Za Zhi 2002; 28 (9): 668-670. Ralph DM, Robinson SR, Campbell MS, et al. Histidine, cystine, glutamine, and threonine collectively protect astrocytes from the toxicity of zinc. Free Radical Biol Med 2010; 49(4): 649-657.
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