**3. Discussion**

In this study, the purity of EGCG extracted from green tea was detected to be 90.5%, and the free radical scavenging activity of EGCG was more extensive than 80% within 3 min. It was increasing with a higher concentration of EGCG or longer reaction time. The particle size of PC-EGCG-VE- and PS-EGCG-VE-liposomes were 161.5 and 142.9 nm, which were smaller than that of EGCG loaded in PLGA microspheres [28] with additional β-cyclodextrin (ranging from 1–14 μm). The encapsulation efficiency of PC-EGCG-VEand PS-EGCG-VE-liposomes were 60.2% and 76.8%, respectively. These results showed that PS-containing liposomes were smaller, more stable, and had higher encapsulation efficiency due to the charge on PS and resulted in a repulsive force between liposomes to avoid aggregation.

Expression of TNF-α in cells pretreated with PS-EGCG- and PS-EGCG-VE-liposomes and then induced by LPS had statistically significant differences compared with that of cells induced with LPS, and similar results were observed in the pretreatment of EGCG on LPS-induced TNF-α expression in BV-2 cells [25] and human macrophages [26]. In summary, the cell morphology and expression of TNF-α of the group treated with EGCG showed an inhibition effect of the inflammation induced by LPS.

In the present study, EGCG pretreatment can reduce the release of NO. Suppression of NO production from LPS-induced BV-2 cells by pretreatment of EGCG-loaded PLGA microspheres was also investigated in our previous study [28].

In the present study, Parkinsonian syndrome in rats is created by the damage to the unilateral substantia nigra region induced by LPS. Another important finding is that, through statistical quantitative analysis, EGCG-loaded liposomes can alleviate the syndrome due to damage to the unilateral midbrain nigrosome region of the rat brain induced by LPS in the rotational test, and production of neuroinflammatory factors TNF-α in the substantia nigra area of the rat brain can also be reduced by EGCG-loaded liposomes. This study indicates that improvement of limb coordination and reduction of neuroinflammation in LPS-induced Parkinsonian syndrome is caused by locally administering EGCG-loaded liposomes but not by increasing expression of BDNF. However, the antineuroinflammation results are necessary for a neuroprotective effect [35]. BV-2 activation releases pro-inflammatory factors, which are neurotoxic and lead to cell damage. By preventing BV-2 activation, EGCG provides a neuroprotective effect. Some post-treatment of EGCG for improving proliferation, survival rate, and neuronal differentiation of adult neural stem cells in dentate gyrus induced by LPS indicates that EGCG may be a potential therapeutic agent for neuroinflammatory diseases [27].

Previous pharmacokinetic studies showed that exogenous PS can cross the blood-brain barrier (BBB), in which it appears to have an affinity for the hypothalamus [6], and the oral administration results in peak levels in 1–4 hours. In addition, it was found that PScontaining liposomes mimic apoptotic cells to promote the secretion of anti-inflammatory mediators, such as transforming growth factor-β1 (TGF-β1) (to downregulate NO produced from macrophages) [36] and prostaglandin E2 (PGE2) by macrophages and microglia cells in vitro [6,37], and also promote the alleviation of inflammation in vivo [38]. Accordingly, PS-containing EGCG-loaded liposomes demonstrated in this study have the advantage of smaller particle size, higher encapsulation efficiency, and inhibiting activation of Parkinsonian syndrome both in microglia cells and in the in vivo rat model, showing an improving anti-inflammatory function and neuroprotection.

The limitation is that our study was carried out with some missing analyses, such as in the case of the neuroprotective effect, and the NeuN staining was not investigated. We also analyzed only BDNF as a neurotrophic factor. FGF2 and IGF2 are also neurotrophic factors that should be considered [39].
