Identification and screening of active components from Ziziphora clinopodioides Lam. in regulating autophagy

Abstract This study investigated the flavonoid constituents of a traditional Chinese medical plant Ziziphora clinopodioides Lam. by using ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry and screened the active components in regulating autophagy.Normal rat kidney (NRK) cells transfected with green fluorescent protein- microtubule-associated protein 1 light Chain 3(GFP-LC3) were treated with Z. clinopodioides flavonoids and its chemical compositions. After 4 h of treatment, the auto-phagy spot aggregation in NRK cells was photographed and observed by laser scanning confocal microscopy. The following 10 flavonoid components of Z. clinopodioides were identified: baicalein(1), quercetin(2), hyperoside(3), quercetin3-O-β-d-glucopyranoside(4), apigenin(5), kaempferol(6), chrysin(7), diosimin(8), linarin(9) and rutin(10). Among these flavonoids, chrysin, apigenin and quercetin were identified as the active principles in activating autophagy. This research may provide a reference for further developing and utilizing Z. clinopodioides.


Introduction
Ziziphora clinopodioides Lam. is a traditional Chinese medicinal plant that is widely distributed in Xinjiang (China) and used to treat hypertension, coronary heart disease and other cardiovascular diseases (Tian et al. 2011). This plant has many biological activities, such as antibacterial, antimicrobial, anti-inflammatory and antioxidant properties; a relaxing effect on the vascular system; and an immunity-boosting effect on laying hens (Nobakht et al. 2012) People mainly focused on the essential oil of Z. clinopodioides (Amiri 2009); however, information on its flavonoid components is limited.
Autophagy refers to 'self-eating' where cells degrade their cellular constituents to maintain cellular homeostasis as a normal response to stresses, such as hypoxia, oxidative stress, starvation, and toxic reactions (Mizushima et al. 2008). Some studies support that autophagy is involved in the occurrence and development of atherosclerosis (AS), which is the main pathological basis of cardiovascular and cerebrovascular diseases (Shao et al. 2016). Normal rat kidney (NRK) cells stably transfected with a fusion protein between green fluorescent protein and light chain 3 (GFP-LC3) were used to investigate autophagy (Mi et al. 2015). Drug screening with autophagy as the target has become a new research focus (Shu et al. 2012). Therefore, this study investigated the identification of the flavonoid composition of Z. clinopodioides and screening of the active components in regulating autophagy.

Effects of Z. clinopodioides flavonoids on the viability of NRK cells
The cytotoxicity of Z. clinopodioides flavonoids was evaluated by MTT assay to ensure the suitable concentrations of Z. clinopodioides flavonoids. The results revealed that Z. clinopodioides flavonoids at concentrations of ≤ 100 μg/mL did not affect the viability of NRK cells, as shown in Figure 2 (IC50 = 300 μg/mL).

Effects of Z. clinopodioides flavonoids on NRK cells with green fluorescent protein-microtubule-associated protein 1 light Chain 3 (GFP-LC3)
Z. clinopodioides flavonoids induced GFP-LC3 puncta. The control group showed few points of autophagy (Figure 3(A)). The number of positive LC3 puncta increased with the increase of Z. clinopodioides flavonoid dose. The 100 μg/mL group of Z. clinopodioides flavonoids exhibited the most significant effect among the drug groups (Figure 3(E)).

Effects of flavonoid components of Z. clinopodioides Lam on NRK cells with GFP-LC3
After intervention with different flavonoid components of Z. clinopodioides Lam. on NRK cells with GFP-LC3, three of these components, including chrysin, apigenin and quercetin, can significantly activate autophagy, whereas the others cannot ( Figure 4). Quercetin showed the most significant effect among the drug groups (Figure 4(G)), which was consistent with previous results (Liu et al. 2015). The activation of autophagy in Z. clinopodioides flavonoids and its active ingredients may provide a possible strategy for AS prevention and treatment.