Synergism and rules of the new combination drug Yiqijiedu formulae (YQJD) on ischemic stroke based on amino acids (AAs) metabolism

Abstract

The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.

Figure 1. Effects of YQJD on the infarct volume of MCAO rats monitored by T2 weighted MRI.

(A) Illustrative MRI images of coronal sections showing the infarct volumes of the cerebral hemisphere as a distinct, bright pale area in the rats subjected to ischemia and the attenuation of the infarct volume by treatment with YQJD; (B) The effects of YQJD on the infarct volume of rats induced by MCAO. YQJD was administered i.g. 15 min prior to MCAO. The values are expressed as the mean ± SD (n = 8), and the data were analyzed with one-way ANOVA. #p < 0.01 versus the sham group; *p < 0.01 versus the vehicle control.

Figure 2. Total ion chromatogram (TIC).

Full chromatograms of 12 AAs standard solutions (100 ng·mL⁻¹, a) and a sample from the vehicle group (b).

Figure 3. RRLC-MS/MS MRM chromatogram of the 12 AAs and IS.

1, GABA; 2, Gly; 3, Glu; 4, Met; 5, Naa; 6, I.S.; 7, Phe; 8, Trp; 9, Ala; 10, Ser; 11, Asp; 12, Hcy; 13, Tyr.

Figure 4. Effect of YQJD on the neurological deficits induced by MCAO.

The neurological examination scores (behavior test). In the neurological examination, a higher score means a worse pathological condition. The score of each experimental group came from the average of ten subjects' behavioral performances. The values are expressed as the mean ± SD (n = 9), and the data were analyzed using one-way ANOVA; ** p < 0.01 versus the sham group, ^# p < 0.05 versus vehicle group. Drugs A, B and C represent ginsenosides, berberine and jasminoidin, respectively.

Figure 5. PLS-DA score plots derived from sham and vehicle group at 12 h after MCAO by SIMCA-P12.0.

In the score plots, the spots of vehicle group were clearly separated from the sham group.

Figure 6. Effect of YQJD on the levels of 7 amino acids (ng·mL⁻¹).

(a) GABA, (b) Ala, (c) Gly, (d) Asp, (e) Ser, (f) Glu, (g) Naa; the values were analyzed using one-way ANOVA and expressed as the mean ± SD (n = 9). **p < 0.01,*p < 0.05 versus the sham group; ^##p < 0.01, ^#p < 0.05 versus the vehicle group. Drugs A, B and C represent ginsenosides, berberine and jasminoidin, respectively.

Figure 7. The scree plots at the dosages of 5 mg·kg⁻¹ (A) and 25 mg·kg⁻¹ (B).

The rate of accumulation of the previous two principal components reached 86% at the dosages of 5 mg·kg⁻¹ and 25 mg·kg⁻¹.

Figure 8. The association between the values of each PC and the various drugs combinations (a: the PCs at the dosage of 25 mg·kg⁻¹; b: the PCs at the dosage of 5 mg·kg⁻¹).

At the dosage of 25 mg·kg⁻¹, the ABC group had the best performance in PC1 and PC2 and was close to the level of the sham group (Figure 8 a). At the dosage of 5 mg·kg⁻¹, the ABC- and only A-treated groups performed relatively well in the first two principal components (PC 1 and PC 2), respectively (Figure 8 b). Drugs A, B and C represent ginsenosides, berberine and jasminoidin, respectively.