Original Article
Tissue metabolic changes in rats after
administration of Aidi injection by GC-MS
Zhennan Zhang1*, Liming Hu2*, Qianqian Wang1, Yanwen Dong1, Miaomiao Zhang1, Xianqin Wang1, Shuanghu Wang3
1Analytical and Testing Centre, School of Pharmaceutical Sciences, Wenzhou Medical University, Xueyuan Road No.
268, Wenzhou, China; 2Department of Pharmacy, The First People’s Hospital of Wenling, South Chuanan Road No.
327, Wenling, China; 3Laboratory of Clinical Pharmacy, People’s Hospital of Lishui, Dazhong Street No. 15, Lishui,
China. *Equal contributors.
Received October 4, 2017; Accepted October 7, 2018; Epub April 15, 2019; Published April 30, 2019
Abstract: Aidi injection has various pharmacological effects, including tumor angiogenesis inhibition, induction of apoptosis in tumor cells, enhancement of immunity, and relief of chemotherapy-related side effects. In this study, we developed a tissue (liver, kidney and heart) metabolomics method using gas chromatography-mass spectrom-etry (GC-MS) to evaluate the effect of Aidi injection on rats. The Sprague-Dawley rats were divided into three groups, namely the control (8 mL/kg/day normal saline) group, Low-dose (5 mL/kg/day) Aidi injection-treated group and High-dose (10 mL/kg/day) Aidi injection-treated group, 7 rats per group. Rats were given intraperitoneal injection for 7 days. Tissue (liver, kidney, heart) samples were collected from rats of the 3 groups after 7 day. The tissue samples were protein precipitated with acetonitrile and derivatized for GC-MS analysis. Partial least squares-discriminant analysis (PLS-DA) revealed that Aidi injection induced metabolic perturbations. Compared to the control group, the metabolites (urea, glycerol, hexadecanoic acid, [9, 12] octadecadienoic acid, arachidonic acid, d-ribose, cholesterol,
galactose, glucitol, 9h-purine, threitol, tetradecanoic acid) were changed in the Aidi group. The changes of
metabo-lites indicated that Aidi injection-treated rats exhibited induced fatty acid metabolism, energy metabolism, amino
acid metabolism, nucleotide metabolism, and urea cycle perturbations. This is the first report to characterize the
tissue metabolic changes in rats treated with Aidi injection.
Keywords: Metabolomics, liver, kidney, heart, PLS-DA, Aidi
Introduction
Chinese herbal medicine is widely used to treat cancer, and it has become one of the main ways for cancer comprehensive treatment pro-gram. Strengthening the body resistance to eliminate pathogenic factors is a basic principle of Chinese herbal medicine in the treatment of cancer [1]. Aidi injection, a traditional Chinese medicine, has various pharmacological effects, including tumor angiogenesis inhibition, induc-tion of apoptosis in tumor cells, enhancement of immunity and relief of chemotherapy-related side effects [2, 3]. Aidi injection is made from an extraction of Renshen (Radix Ginseng, Araliaceae), Huangqi (Astragalus
membrana-ceus (Fisch.) Bunge, Papilionaceae), Ciwujia
(Radix et Caulis Acanthopanacis Santicosi,
Araliaceae) and Banmao (Lytta Vesicatoria,
Meloidae) [4, 5]. Its main components include cantharidin, ginsenoside, astrogaloside and acanthopanax senticosus polysaccharide [6, 7].
To more comprehensively investigate the effect of Aidi injection on metabolic pathways, we
examined the metabolic profiles of rats that
received Aidi injections. To the best of our
knowledge, this study is the first to report the
tissue metabolic changes in rats after Aidi injections.
Material and methods
Chemicals
Tissue metabolic changes after Aidi injection
purity ≥ 99.0%) and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, purity ≥ 98.5%)
were purchased from Sigma-Aldrich Corp. (Shanghai, China). HPLC-grade acetonitrile and
n-hexane were purchased from Tedia Reagent Company (Shanghai, China). Methylhydroxyla- mine hydrochloride and pyridine were pur-chased from Aladdin Industrial, Inc. (Shanghai, China).
Instrumentation and conditions
The Agilent 6890N-5975B GC-MS and HP-5MS
(0.25 mm × 30 m × 0.25 μm) were obtained
from Agilent Technologies Inc. (Santa Clara, CA,
USA). Mass detection was conducted first in EI
mode with electron energy of 70 eV, then in
full-scan mode with m/z 50-550, and finally, by
the splitless mode injection [8, 9]. The GC oven was initially set at 80°C and was kept at this temperature for 5 min. The temperature was then gradually increased to 260°C at a rate of 10°C/min, and then kept at 260°C for 10 min.
Sample preparation
A volume of 250 µL of acetonitrile was added to 100 mg of tissue (liver, kidney, heart), the sus-pension was kept in an ice-bath for 15 min and then centrifuged at 13000 g for 10 min at 4°C. A volume of 150 µL of the supernatant was transferred to a GC vial and evaporated to dry-ness under a stream of nitrogen gas. Methyl oximation was carried out at 70°C for 24 h after 50 µL of methylhydroxylamine hydrochloride (15 mg/mL in pyridine) was added. Then, a 50
µL volume of MSTFA (with 1% TMCS as the cat -alyst) was added and kept at 70°C for another hour, and then vortexed after adding 150 µL
n-hexane [9].
Metabolomics study
Sprague-Dawley rats (male, 220 ± 20 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). Rats were housed under natural light-dark cycle condi-tions with controlled temperature (22°C). All rats were housed at the Laboratory Animal Research Center of Wenzhou Medical Univer- sity. All experimental protocols were ethically approved by the Administration Committee of experimental animals of Wenzhou Medical University.
Twenty-one Sprague-Dawley rats were divided into three groups, namely the control group, Low-dose Aidi injection-treated group, High-dose Aidi injection-treated group, 7 rats per group. In the Low-dose Aidi injection-treated group, 5 mL/kg/day of Aidi was given to rats by intraperitoneal injection for 7 days. In the High-dose Aidi injection-treated group, 10 mL/kg/ day of Aidi was given to rats by intraperitoneal injection for 7 days. In the control group, 8 mL/ kg/day of normal saline was given to rats by intraperitoneal injection for 7 days.
Tissue (liver, kidney, heart) samples were sepa-rately collected from the 3 groups of rats at 10:00 am on the eighth day. The tissue sam-ples were stored at -80°C until measurement.
Main ingredient in Aidi injection
A volume of 100 µL Aidi injection was added in the eppendorf tube, vortexed after adding methanol 900 µL, and then centrifuged at 13000 g for 10 min. The supernatant was used for UPLC-MS/MS analysis.
UPLC-MS/MS with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrome-ter (Waspectrome-ters Corp., Milford, MA, USA) equipped with electrospray ionization (ESI) interface and
BEH C18 column (2.1 mm × 50 mm, 1.7 μm) were used in our work. Methanol and 0.1% for
-mic acid were used as mobile phases; the flow
rate was 0.4 mL/min. A gradient elution pro-gram was conducted for chromatographic
sep-aration with mobile phase A (0.1% formic acid),
and mobile phase B (methanol) as follows:
0-0.2 min (10-10% B), 0.2-1.5 min (10-80% B), 1.5-3.0 min (80-80% B), 3.0-3.5 min (80-10% B), 3.5-5.0 min (10-10% B). Mass spectrometry
parameter multiple reaction monitoring (MRM) settings was according to the literature (Liu et al., 2016). The main chemical compositions of Aidi injection acquired through UPLC-MS/MS technique are presented in Figure 1 and Table 1.
Data analysis
Tissue metabolic changes after Aidi injection
ples acquired through GC-MS technique are presented in Figure 2. A total of 50 endogen-
ous metabolites were identified in the tissue
samples using the NIST 2005 mass spectrom-etry database.
In order to evaluate the metabolic profile chang -es in rats caused by different dosag-es (5 and 10 mL/kg/day, for the Low and High groups, respectively) of Aidi, we compared the PCA of the Aidi injection groups (Low and High) with the spectrum from rats in the control group, as illustrated for different tissues in the following
figures: Figure 3A (liver), Figure 3C (kidney),
Figure 3E (heart); the corresponding load dia-gram is shown in Figure 3B (liver), Figure 3D
(kidney), Figure 3F (heart). We compared the GC-MS spectra from the PLS-DA of the rats in the Aidi injection groups (Low and High) with those of rats in the control group (Figure 4A,
4D, 4G); the corresponding load diagram is shown in Figure 4B, 4E, 4H. Additionally, the PLS-3D results are shown in Figure 4C, 4F, 4I. The PLS-3D score charts in Figure 4 showed
that the first principal components of the rats in
the Aidi injection groups (Low and High) were distinguished from the rats in the control group. Actually, the results of PLS-3D were better than those of the PCA.
Changes in metabolite
The identification of endogenous compounds
that can be used as metabolic biomarkers of Aidi injection poisoning would represent an
alternative approach of significant importance
to detect hidden effects. In this study, the changes of metabolites between the Aidi
injec-Table 1.MRM parameters for the main ingredient in Aidi injec-tion
NO. Compound name Parent (m/z) Daughter (m/z) Cone (V) Collision (V)
1 Isofraxidin 223.1 208.1 25 15
2 Syringin B 395.2 232.2 30 20
3 Calycosin-7-O-β-d-glucoside 469.2 307.1 30 23
4 Syringin E 765.4 603.4 45 40
5 Astragaloside IV 807.3 627.2 30 25 6 Ginsenoside Rg1 823.6 643.8 25 15 7 Astragaloside II 849.6 669.2 30 25 8 Ginsenoside Re 969.7 789.7 45 20 9 Ginsenoside Rd 969.9 789.7 40 25 10 Ginsenoside Rc 1101.7 789.4 50 20 11 Ginsenoside Rb1 1131.7 789.6 40 25
Figure 2. Typical GC-MS total ion chromatogram of rat tissue after Aidi injection.
Statistical analysis
Statistical analysis was conducted using the SPSS software (Version 18.0, SPSS, IBM Corp., Armonk, NY, USA). Independent samples
t-test was applied in order to de-
tect significant differences in all
metabolites between two groups.
P < 0.05 was considered
statisti-cally significant.
Results
Metabolomics study
The typical metabolic profiles of
sam-Figure 3. PCA score results of the rat liver, kidney and heart samples (A, C, E) after Aidi injection (0.5, 1.0 g/kg, Low,
High), Low (Class 1), High (Class 2), Control (Class 3); the corresponding load diagram (B, D, F).
tion groups and their corresponding control group are shown in Tables 1-3.
Discussion
Metabolomics is a newly emerging omics ap- proach to the investigation of metabolic
[image:5.612.90.525.72.591.2]Tissue metabolic changes after Aidi injection
Figure 4. PLS-DA score results of the rat liver, kidney and heart samples (A, D, G) after Aidi injection (0.5, 1.0 g/kg, Low, High), low (Class 1), high (Class 2), control
Table 2. Relative levels of metabolites in rat liver after Aidi injection
NO. Retention time/min VIP Metabolite Control Low Pa High Pb Pc
1 8.94 1.215 Urea 0.712 1.061* 0.011 0.969* 0.025 0.216
2 12.89 1.113 Glycerol 0.736 0.427** 0.007 0.395** 0.001 0.517
3 15.19 4.614 d-Glucose 3.772 6.074 0.093 12.339**,## 0.005 0.009
4 15.37 2.803 Galactose 13.464 15.272* 0.048 16.391**,# 0.006 0.038
5 17.51 2.176 Hexadecanoic acid 4.023 2.895** 0.005 2.553**,# 0.002 0.024
6 19.41 2.580 9,12-Octadecadienoic acid 4.128 2.386** 0.007 2.052** 0.002 0.458
7 20.97 1.454 Arachidonic acid 2.271 1.575* 0.027 1.382* 0.019 0.332
8 21.83 1.635 Tetradecanoic acid 6.540 7.637 0.172 8.024* 0.037 0.657
9 22.72 1.666 d-Ribose 1.661 1.140** 0.009 0.764**,## 0.005 0.008
10 26.59 1.322 Cholesterol 1.807 1.334* 0.032 1.145** 0.008 0.084
Note: Variable importance in the projection (VIP) was acquired from the PLS-DA model with a threshold of 1.0. Compared with control group, Pa (Low-Control), Pb (High-Control), *P < 0.05 and **P < 0.01; Compared with Low-dose Aidi injection treated group, Pc (High-Low), #P < 0.05 and ##P < 0.01.
Compared to the control group, urea, glyce- rol, hexadecanoic acid, 9,12-octadecadienoic acid, arachidonic acid, d-ribose and cholesterol were decreased in the Low and High Aidi injec-tion groups, while galactose was increased (Table 2). In addition, compared to the control group, glycerol, glucitol, 9h-purine were decre- ased in the Low and High Aidi injection groups (Table 3). Also, compared to the control group, threitol, hexadecanoic acid, 9,12-octadecadie-noic acid, tetradeca9,12-octadecadie-noic acid, d-ribose, choles-terol were increased in the Low and High Aidi injection groups (Table 4).
Glycerol is generally obtained from plant and animal sources where it occurs as triglycerides. Triglycerides are esters of glycerol with long-chain carboxylic acids. Hexadecanoic acid is
the first fatty acid produced during fatty acid
synthesis and is the precursor to longer fatty acids. Arachidonic acid is a polyunsaturated fatty acid present in the phospholipids of mem-branes of the cells of the body, and is abundant in the brain, muscles, and liver. Cholesterol,
given that it composes about 30% of all animal
cell membranes, is required to build and main-tain cell membranes and modulates membrane
fluidity over the range of physiological
tem-peratures.
The changes of metabolites (urea, glycerol, hexadecanoic acid, 9,12-octadecadienoic acid, arachidonic acid, d-ribose, cholesterol, galac-tose, glucitol, 9h-purine, threitol, tetradecanoic acid), increased or decreased, indicate that Aidi injection-treated rats had induced fatty acid metabolism, energy metabolism, amino acid metabolism, nucleotide metabolism, urea cycle
perturbations. These findings may be useful for
evaluating new evidence in Aidi injection stu- dies.
Conclusion
In this study, we developed a tissue metabolo-mic approach by GC-MS to evaluate the effect of Aidi injection in rats. PLS-DA revealed that Aidi injection induced metabolic perturbations. We demonstrated that metabolomic methods based on GC-MS could provide a useful tool for Table 3. Relative levels of metabolites in rat kidney after Aidi injection
NO. Retention time/min VIP Metabolite Control Low Pa High Pb Pc
1 12.89 1.276 Glycerol 1.439 1.166* 0.048 1.179* 0.031 0.715
2 13.05 1.047 2-aminohexanoic acid 1.083 1.118 0.534 0.823* 0.026 0.351
3 13.22 1.887 l-Proline 1.778 1.744 0.112 1.294**,## 0.007 0.009
4 14.82 1.013 Glucitol 0.433 0.370* 0.043 0.300**,## 0.002 0.005
5 17.04 2.336 9h-Purine 1.870 1.499** 0.007 1.161**,## 0.001 0.005
6 17.45 1.544 Hexadecanoic acid 2.877 2.937 0.079 3.368* 0.032 0.275
Note: VIP was acquired from the PLS-DA model with a threshold of 1.0. Compared with control group, Pa (Low-Control), Pb (High-Control), *P < 0.05 and **P < 0.01; Compared with Low-dose Aidi injection treated group, P
[image:7.612.91.524.286.378.2]Tissue metabolic changes after Aidi injection
exploring biomarkers to elucidate the effects of Aidi injection and its mechanism of action.
Acknowledgements
This study was supported by grants from the Wenling City Science and Technology Project (2015C311029, 2015C312060, 2016C311- 111 and 2016C311113) and Wenzhou Science and Technology Bureau (Y20160532).
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Xianqin Wang, Analytical and Testing Centre, School of Pharma- ceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China. Tel: (86) 57786699156; E-mail: lankywang@foxmail.com; Dr. Shuanghu Wang, Laboratory of Clinical Pharmacy, People’s Hospital of Lishui, Lishui 323000, China. Tel: (86) 18957091837; E-mail: wangshuangfux@163.com
References
[1] Jiancheng W, Long G, Ye Z, Jinlong L, Pan Z, Lei M and Kehu Y. Effect of Aidi injection plus che-motherapy on gastric carcinoma: a meta-anal-ysis of randomized controlled trials. J Tradit Chin Med 2015; 35: 361-74.
[2] Xu HX, Huang XE, Li Y, Li CG and Tang JH. A
clinical study on safety and efficacy of Aidi in -jection combined with chemotherapy. Asian Pac J Cancer Prev 2011; 12: 2233-6.
[3] Sun XF, Pei YT, Yin QW, Wu MS and Yang GT.
Application of Aidi injection in the bronchial artery infused neo-adjuvant chemotherapy for stage III A non-small cell lung cancer before surgical operation. Chin J Integr Med 2010; 16: 537-41.
[4] Zhang H, Zhou QM, Lu YY, Du J and Su SB. Aidi
injection alters the expression profiles of mi -croRNAs in human breast cancer cells. J Tradit Chin Med 2011; 31: 10-16.
[5] Zhang H, Jiang H, Hu X and Jia Z. Aidi injection combined with radiation in the treatment of non-small cell lung cancer: a meta-analysis
evaluation the efficacy and side effects. J Can -cer Res Ther 2015; 11 Suppl 1: C118-121. [6] Wang T, Nan H, Zhang C, Wang Y, Zhang X, Li Y;
Mulati. Aidi injection combined with FOLFOX4
chemotherapy regimen in the treatment of advanced colorectal carcinoma. J Cancer Res Ther 2014; 10 Suppl 1: 52-5.
[7] Xu XT, Song Y, Qin S, Wang LL and Zhou JY. Radio-sensitization of SHG44 glioma cells by Aidi injection in vitro. Mol Med Rep 2012; 5: 1415-8.
[8] Wang X, Zhang M, Ma J, Zhang Y, Hong G, Sun
F, Lin G and Hu L. Metabolic changes in para -quat poisoned patients and support vector ma-chine model of discrimination. Biol Pharm Bull 2015; 38: 470-5.
[9] Zhang M, Deng M, Ma J and Wang X. An
evalu-ation of acute hydrogen sulfide poisoning
in rats through serum metabolomics based on gas chromatography-mass spectrometry. Chem Pharm Bull (Tokyo) 2014; 62: 505-7. [10] Zhang Q, Wu H, Wen C, Sun F, Yang X and Hu L.
Metabolic changes in rats after intragastric administration of MGCD0103 (Mocetinostat), a hdac class I inhibitor. Int J Clin Exp Pathol 2015; 8: 9320-5.
[11] Li Y, Lin GT, Chen BB, Zhang J, Wang LT, Li ZX, Cao YG, Wen CC, Yang XZ, Cao GZ, Wang XQ and Cao GQ. Effect of alprazolam on rat serum
metabolic profiles. Biomed Chromatogr 2017;
31: e3956.
[12] Geng PW, Cai JZ, Zhang LJ, Xu MZ, Liu ZZ, Zhang J, Wang ZY, Wang XQ, Wen CC and Ma JS. Liver tissue metabonomics in rat after acute paraquat poisoning gas chromatogra-phy-mass spectrometry. Int J Clin Exp Med 2017; 10: 937-43.
[13] Bao SH, Zhang J, Lin ZX, Su K, Mo JJ, Hong L,
Qian SY, Chen LG, Sun F, Wen CC, Wu Q, Hu LF,
[image:8.612.90.523.85.186.2]Lin GY and Wang XQ. Serum metabolic chang-es in rats after intragastric administration of dextromethorphan. Biomed Chromatogr 2017; 31: e3814.
Table 4. Relative levels of metabolites in rat heart after Aidi injection
NO. Retention time/min VIP Metabolite Control Low Pa High Pb Pc
1 9.02 1.233 Urea 2.350 2.765 0.291 3.868** 0.040 0.445
2 12.82 1.066 Threitol 0.028 1.112** 0.003 1.048** 0.001 0.176
3 17.48 1.712 Hexadecanoic acid 7.134 8.224* 0.023 8.859** 0.007 0.209
4 19.38 2.977 9,12-Octadecadienoic acid 7.729 9.995* 0.036 7.903# 0.102 0.047
5 21.87 1.227 Tetradecanoic acid 1.594 1.885* 0.019 2.261**,# 0.008 0.035
6 22.77 3.190 d-Ribose 15.312 20.574* 0.045 21.053* 0.034 0.185
7 26.58 2.113 Cholesterol 6.806 8.754* 0.028 9.572** 0.006 0.142
Note: VIP was acquired from the PLS-DA model with a threshold of 1.0. Compared with control group, Pa(Low-Control), Pb(High-Control), *P < 0.05 and **P < 0.01; Compared with Low-dose Aidi injection treated group, P
[14] Wang SH, Zhang LJ, Wang XQ, Wang ZY, Wen
CC, Ma JS, Gao ZM and Hu LF. Metabolic
changes in rat lung after acute paraquat poi-soning by gas chromatography-mass spec-trometry. Int J Clin Exp Med 2016; 9: 21514-20.
[15] Wu Q, Hu QP, Sun F, Dong YW, Wang QQ, Ma JS, Wang XQ and Zhou YF. Brain metabolic chang -es in rats after dextromethorphan. Lat Am J Pharm 2017; 36: 1882-6.
[16] Ma JS, Sun F, Chen BH, Tu XT, Peng XF, Wen CC, Hu LF and Wang XQ. Tissue metabolic changes