Electronic Supplementary Materials

Acute psychoactive and toxic effects of D. metel on mice explained by 1H NMR based metabolomics approach

Yonghong Fu a, Zhihong Si b, Pumin Li a, Minghui Li a, He Zhao a, Lei Jiang a, Yuexiao Xing a, Wei Hong a, Lingyu Ruan a and Jun-Song Wang* a

aCenter for Molecular Metabolism, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China

bCancer Hospital, Chinese Academy of Sciences, 350 Shu Shan Hu Road, Hefei 230031, PR China

*To whom correspondence should be addressed.

Jun-Song Wang,

Tel: +86 25 8430 3216; E-mail:

Fig. S1 Typical 1H NMR (500 MHz ZGPR) spectra of mice brain and liver tissue extracts. Labeled metabolites: 1 Lactate, 2 Alanine, 3 Acetate, 4 4-Aminobutyrate (GABA), 5 N-acetyl- glycine, 6 Acetylcholine (Ach), 7 Glutamate, 8 Succinate, 9 Glutamine, 10 Glutathione, 11 N-acetyl-aspartate (NAA), 12 Malate, 13 Aspartate, 14 Creatine, 15 Choline, 16 Betaine, 17 Taurine, 18 myo-inositol, 19 Ascorbate, 20 Theronine, 21 ATP, 22 AMP, 23 Valine, 24 Leucine, 25 Iso-leucine, 26 3-Hydroxybutyrate, 27 Alanine, 28 Lysine, 29 Ethanolamine, 30 O-phosphocholine, 31 Glucose, 32 Inosine, 33 Lactate, 34 Fumarate, 35 Tyrosine, 36 Histidine, 37 Phenylalanine, 38 Nicotinurate, 39 NAD+, 40 2-Phosphtoglycerate, 41 Formate, 42 Glycine.

Fig. S2 Typical 1H NMR (500 MHz ZGPR) spectra of mice serum. Labeled metabolites: 1 Valine, 2 Leucine, 3 Iso-leucine, 4 Lactate, 5 Alanine, 6 Glutamine, 7 Succinate, 8 Citrate, 9 Choline, 10 Arginine, 11 Glucose, 12 Creatinine, 13 Tyrosine, 14 fumarate, 15 Histidine, 16 Phenyalanine.

Fig. S3 PCA Score plots of 1H NMR data of aqueous tissue extracts of mice brain and liver. (a) The brain samples; (c) the liver samples. Two PCs explained 40.56% and 46.93% of total variances in the brain and the liver samples respectively. The ellipses represent the 95% confidence interval for each group.

Fig. S4 PCA Score plots of 1H NMR data of serum. Two PCs explained 42.73% and 19.43% of total variances in serum. The ellipses represent the 95% confidence interval for each group.

Fig. S5 OSC-PLS-DA analysis of NMR data from serum of D. metel dosed mice. (a) Scores plot. Two components explained 59.8% of total variances in serum samples. (b) S-plot. (c) Color-coded loading plot. The color bar corresponds to the weight of the corresponding variable in the discrimination of statistically significant (Red) or no statistically significant (Blue). Positive and negative peaks indicate a relatively decreased and increased metabolite level in the D. metel dosed groups.

Fig. S6 OSC-PLS-DA scatter plots of statistical validation obtained by 2000 times permutation test for the brain (a) and liver (b). With R2 and Q2 values in the vertical axis, the correlation coefficients (between the permuted and true class) in the horizontal axis, and the ordinary least squares (OLS) line for the regression of R2 and Q2 on the correlation coefficients. Intercepts: R2 = (0.0, 0.574), and Q2 = (0.0, -0.528) for brain (a); R2 = (0.0, 0.597), and Q2 = (0.0, -0.496) for liver (b).

Fig. S7 OSC-PLS-DA model validation of serum. (a) OSC-PLS-DA scatter plots of statistical validation obtained by 2000 times permutation test for serum. With R2 and Q2 values in the vertical axis, the correlation coefficients (between the permuted and true class) in the horizontal axis, and the ordinary least squares (OLS) line for the regression of R2 and Q2 on the correlation coefficients. (b)Histograms for permutation test scores of OSC-PLS-DA models of serum. Permutation test was on the basis of 2000 times permutations: the red arrow indicating the performance based on the original labels, significant for a P-value less than 0.05.

Fig. S8 Histograms for permutation test scores of OSC-PLS-DA models of brain and liver. (a) The brain samples, (b) the liver samples. Permutation test was on the basis of 2000 times permutations: the red arrow indicating the performance based on the original labels, significant for a P-value less than 0.05.

Table. S1 Metabolites identified from the serum, and their fold change values versus control group

Metabolites / Chemical shift (ppm) / LDM vs. CON / HDM vs. CON
log2(FC) / p value / log2(FC) / p value
Lactate / 1.35(d), 4.13(m) / 0.44 / * / 0.64 / ***
Arginine / 3.24(m) / -0.25 / -0.40 / *
Choline / 3.22(m) / 0.16 / 0.47 / **
Glucose / 3.48(m), 3.92(m) / -0.56 / *** / -0.68 / ***
Tyrosine / 6.92(d), 7.20(d) / -0.07 / -0.15
Glutamine / 2.45(m) / -0.12 / * / -0.17
Leucine / 0.99(d), 1.01(d) / 0.10 / 0.13
Isoleucine / 0.93(d),0.97(d) / 0.02 / -0.01
Valine / 0.98(d), 1.03(d) / 0.01 / 0.07
Alanine / 1.49(t) / 0.24 / 0.45 / *
Pyruvate / 2.40(s) / 0.33 / 0.39 / *
Fumarate / 6.51(q) / 0.01 / 0.03
Citrate / 2.59(d), 2.71(d) / -0.14 / -0.15 / *
creatinine / 4.01(q) / -0.09 / 0.24 / *
Histidine / 7.13(q), 7.90(q) / -0.02 / -0.19
Phenylalanine / 7.40(t) / 0.02 / 0.01

Multiplicity: s, singlet; d, double; t, triplet; q, quartets; m, multiplet. Fold Change (FC) = D. metel dosed samples / control samples; Color coded according to the log 2 (FC) using color bar , red the increased and blue the decreased in D. metel dosed groups. P values corrected by BH (Benjamini Hochberg) methods were calculated based on a parametric Student’s t-test or a nonparametric Mann-Whitney test. * P < 0.05, ** P < 0.01, *** P < 0.001.

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