Polyphenols Derived from Lychee Seed Suppress Aβ (1-42)-Induced Neuroinflammation

Amyloid-β (Aβ) is commonly recognized as the most important factor that results in neuronal cell death and accelerates the progression of Alzheimer’s disease (AD). Increasing evidence suggests that microglia activated by Aβ release an amount of neurotoxic inflammatory cytokines that contribute to neuron death and aggravate AD pathology. In our previous studies, we found that lychee seed fraction (LSF), an active fraction derived from the lychee seed, could significantly improve the cognitive function of AD rats and inhibit Aβ-induced neuroinflammation in vitro, and decrease neuronal injuries in vivo and in vitro. In the current study, we aimed to isolate and identify the specific components in LSF that were responsible for the anti-neuroinflammation effect using preparative high performance liquid chromatography (pre-HPLC), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) methods. To this end, we confirmed two polyphenols including catechin and procyanidin A2 that could improve the morphological status of BV-2 cells and suppress the release, mRNA levels, and protein expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) through downregulating the nuclear factor-κB (NF-κB) signaling pathway using ELISA, RT-PCR, and Western blotting methods. Furthermore, catechin and procyanidin A2 could inhibit Aβ-induced apoptosis in BV-2 cells by upregulating Bcl-2 and downregulating Bax protein expression. Therefore, the current study illustrated the active substances in lychee seed, and first reported that catechin and procyanidin A2 could suppress neuroinflammation in Aβ-induced BV-2 cells, which provides detailed insights into the molecular mechanism of catechin and procyanidin A2 in the neuroprotective effect, and their further validations of anti-neuroinflammation in vivo is also essential in future research.


Introduction
Alzheimer's disease (AD) is the most common type of late-onset dementia, which is characterized by dysfunction in cognition and behavior. Senile plaques (SPs) and neurofibrillary tangles (NFTs) are

Isolation, Purification, Identification, and Elucidation of Catechin and Procyanidin A2 in LSF
LSF was prepared according to the previous study [19,20] and first analyzed using RP-HPLC monitored at 280 nm. A total of 12 main peaks were collected using AKTA protein purification system ( Figure S1A,B). The purity of 12 collected peaks were measured by using RP-HPLC ( Figure S1C). Through the preliminary evaluation of the anti-neuroinflammation effect of these 12 fractions using Wright-Giemsa staining and ELISA methods, Fractions 3 and 9 were found to improve the morphological status, decrease the ratio of activated BV-2 cells, increase cell density, and decrease the release of pro-inflammatory cytokines in Aβ(1-42)-induced BV-2 cells ( Figures S2 and S3), suggesting that the compounds in Fraction 3 and 9 could be the major bioactive components that are responsible for the anti-inflammation effect. Therefore, we applied pre-HPLC to further purify the compounds in Fractions 3 and 9, and obtained compounds 1 and 2 from Fractions 3 and 9, respectively. Through the elucidation using ultra-high-performance liquid chromatography with diode array detector coupled with electrospray ionization-tandem mass spectrometry (UHPLC-DAD-ESI-/MS/MS) and NMR instruments ( Figures S4-S6), Compounds 1 and 2 were confirmed to be catechin and procyanidin A2, respectively. Their structures, DAD chromatograms, and MS spectrums are displayed in Figure 1 and Figure S7.
LSF was prepared according to the previous study [19,20] and first analyzed using RP-HPLC monitored at 280 nm. A total of 12 main peaks were collected using AKTA protein purification system ( Figure S1A,B). The purity of 12 collected peaks were measured by using RP-HPLC ( Figure S1C). Through the preliminary evaluation of the anti-neuroinflammation effect of these 12 fractions using Wright-Giemsa staining and ELISA methods, Fractions 3 and 9 were found to improve the morphological status, decrease the ratio of activated BV-2 cells, increase cell density, and decrease the release of pro-inflammatory cytokines in Aβ(1-42)-induced BV-2 cells ( Figures S2 and S3), suggesting that the compounds in Fraction 3 and 9 could be the major bioactive components that are responsible for the anti-inflammation effect. Therefore, we applied pre-HPLC to further purify the compounds in Fractions 3 and 9, and obtained Compounds 1 and 2 from Fractions 3 and 9, respectively. Through the elucidation using ultra-high-performance liquid chromatography with diode array detector coupled with electrospray ionization-tandem mass spectrometry (UHPLC-DAD-ESI-/MS/MS) and NMR instruments ( Figures S4-S6), Compounds 1 and 2 were confirmed to be catechin and procyanidin A2, respectively. Their structures, DAD chromatograms, and MS spectrums are displayed in Figure 1 and Figure S7.  The above data were consistent with the reported papers and identified as catechin [21,22].  30.06 (C-4 ), 29.43 (C-4). The above data were consistent with the reported papers and identified as procyanidin A2 [23].

Catechin and Procyanidin A2 Improve the Morphology of BV-2 Cells
The cellular morphology of BV-2 cells can reflect the inflammatory response [24]. In this part, we first examined the cytotoxicity of catechin and procyanidin A2 against BV-2 cells using a CCK-8 kit, and there no cytotoxicity was observed under 80 µM (Figure 2A). Figure 2B showed the morphologic changes of BV-2 cells treated with Aβ(1-42) alone and Aβ(1-42) co-treated with LSF, catechin, and procyanidin A2, which were detected by the Wright-Giemsa staining method. As shown in Figure 2B-D, LSF, catechin and procyanidin A2 could reduce the ratio of activated BV-2 cells that displayed ameboid shape, fusiform, more synapse and obvious nucleus fragmentations, and increase cell density in Aβ(1-42)-induced BV-2 cells, suggesting that LSF and its derived compounds could suppress Aβ(1-42) induced neuroinflammation. Therefore, catechin and procyanidin A2 could be the bioactive components that are responsible for the anti-neuroinflammation effect.  The density of BV-2 cells, which was quantified by counting the number of all the BV-2 cells, and a minimum of 150 cells from three randomly selected fields were scored. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 μM Aβ(1-42) alone were set as the Aβ group. ### p < 0.001 vs. Control group; ** p < 0.005, *** p < 0.001 vs. Aβ group. The red arrows indicate the activated BV-2 cells with ameboid shape. Magnification: ×100; Scale bar: 50 µm. (C) The ratio of activated BV-2 cells, which was calculated by counting the number of activated BV-2 cells in all of the BV-2 cells views; (D) The density of BV-2 cells, which was quantified by counting the number of all the BV-2 cells, and a minimum of 150 cells from three randomly selected fields were scored. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ group. ### p < 0.001 vs. Control group; ** p < 0.005, *** p < 0.001 vs. Aβ group.

Catechin and Procyanidin A2 Inhibit Pro-Inflammatory Cytokines in Aβ(1-42)-Induced BV-2 Cells
Our previous study proved that LSF could inhibit the release, mRNA levels, and protein expressions of TNF-α, IL-1β, COX-2, and iNOS. Based on the preliminary screen result of the anti-neuroinflammation effect of the 12 isolated fractions, we further validated the anti-neuroinflammation effect of catechin and procyanidin A2 in Fractions 3 and 9 in the current experiment. As shown in Figure 3, LSF, catechin and procyanidin A2 could significantly decrease the release of TNF-α, IL-1β, and IL-6 levels in cell culture supernatants using an ELISA kit in Aβ(1-42)-induced BV-2 cells. Additionally, the mRNA levels of TNF-α, IL-1β, and iNOS were measured using RT-PCR. Figure 4 shows that LSF, catechin, and procyanidin A2 could significantly reduce the mRNA levels in Aβ(1-42)-induced BV-2 cells. Correspondingly, the protein expressions of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2 were detected by Western blotting. As shown in Figure 5, LSF, catechin, and procyanidin A2 dramatically reduced the protein content of TNF-α, IL-1β, and iNOS. In summary, catechin and procyanidin A2 could inhibit the inflammatory response in Aβ(1-42)-induced BV-2 cells, suggesting that catechin and procyanidin A2 could be the key active components in LSF. Our previous study proved that LSF could inhibit the release, mRNA levels, and protein expressions of TNF-α, IL-1β, COX-2, and iNOS. Based on the preliminary screen result of the anti-neuroinflammation effect of the 12 isolated fractions, we further validated the anti-neuroinflammation effect of catechin and procyanidin A2 in Fractions 3 and 9 in the current experiment. As shown in Figure 3, LSF, catechin and procyanidin A2 could significantly decrease the release of TNF-α, IL-1β, and IL-6 levels in cell culture supernatants using an ELISA kit in Aβ(1-42)-induced BV-2 cells. Additionally, the mRNA levels of TNF-α, IL-1β, and iNOS were measured using RT-PCR. Figure 4 shows that LSF, catechin, and procyanidin A2 could significantly reduce the mRNA levels in Aβ(1-42)-induced BV-2 cells. Correspondingly, the protein expressions of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2 were detected by Western blotting. As shown in Figure 5, LSF, catechin, and procyanidin A2 dramatically reduced the protein content of TNF-α, IL-1β, and iNOS. In summary, catechin and procyanidin A2 could inhibit the inflammatory response in Aβ(1-42)-induced BV-2 cells, suggesting that catechin and procyanidin A2 could be the key active components in LSF.    Our previous study proved that LSF could inhibit the release, mRNA levels, and protein expressions of TNF-α, IL-1β, COX-2, and iNOS. Based on the preliminary screen result of the anti-neuroinflammation effect of the 12 isolated fractions, we further validated the anti-neuroinflammation effect of catechin and procyanidin A2 in Fractions 3 and 9 in the current experiment. As shown in Figure 3, LSF, catechin and procyanidin A2 could significantly decrease the release of TNF-α, IL-1β, and IL-6 levels in cell culture supernatants using an ELISA kit in Aβ(1-42)-induced BV-2 cells. Additionally, the mRNA levels of TNF-α, IL-1β, and iNOS were measured using RT-PCR. Figure 4 shows that LSF, catechin, and procyanidin A2 could significantly reduce the mRNA levels in Aβ(1-42)-induced BV-2 cells. Correspondingly, the protein expressions of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2 were detected by Western blotting. As shown in Figure 5, LSF, catechin, and procyanidin A2 dramatically reduced the protein content of TNF-α, IL-1β, and iNOS. In summary, catechin and procyanidin A2 could inhibit the inflammatory response in Aβ(1-42)-induced BV-2 cells, suggesting that catechin and procyanidin A2 could be the key active components in LSF.

Catechin and Procyanidin A2 Inhibit the Activation of NF-κB Signaling Pathway
Emerging evidence implies that the NF-κB signaling pathway is activated in AD pathogenesis. This pathway can regulate the release and expression of pro-inflammatory cytokines, and its function is related to its inhibitory factor, IκBα. Aβ can activate the NF-κB signaling pathway then release the pro-inflammatory cytokines. In this study, we investigated the inhibition effect of LSF and the isolated components, catechin and procyanidin A2 in neuroinflammation in Aβ(1-42)-induced BV-2 cells. As shown in Figure 6, LSF, catechin, and procyanidin A2 could significantly suppress the protein expression of NF-κB and the ratio of p-IκBα/IκBα, suggesting that LSF, catechin, and procyanidin A2 suppressed the neuroinflammation via inhibiting the NF-κB signaling pathway in Aβ(1-42)-induced BV-2 cells.

Catechin and Procyanidin A2 Inhibit the Activation of NF-κB Signaling Pathway
Emerging evidence implies that the NF-κB signaling pathway is activated in AD pathogenesis. This pathway can regulate the release and expression of pro-inflammatory cytokines, and its function is related to its inhibitory factor, IκBα. Aβ can activate the NF-κB signaling pathway then release the pro-inflammatory cytokines. In this study, we investigated the inhibition effect of LSF and the isolated components, catechin and procyanidin A2 in neuroinflammation in Aβ(1-42)-induced BV-2 cells. As shown in Figure 6, LSF, catechin, and procyanidin A2 could significantly suppress the protein expression of NF-κB and the ratio of p-IκBα/IκBα, suggesting that LSF, catechin, and procyanidin A2 suppressed the neuroinflammation via inhibiting the NF-κB signaling pathway in Aβ(1-42)-induced BV-2 cells.

Catechin and Procyanidin A2 Inhibit Cell Apoptosis in Aβ(1-42)-Induced BV-2 Cells.
As is known to us, over-expression of pro-inflammatory cytokines can induce the microglial activation and lead to neuronal death. Our previous study have proved that LSF could inhibit cell apoptosis in Aβ(1-42)-induced BV-2 cells. In this current experiment, we also investigated the suppression effect of cell apoptosis in Aβ(1-42)-induced BV-2 cells under the treatments of LSF and the isolated components, catechin and procyanidin A2. As shown in Figure 7, LSF, catechin and procyanidin A2 could inhibit Aβ(1-42)-induced BV-2 cells apoptosis by reducing Bax/Bcl-2 ratio and attenuating the proportion of cleaved-PARP in total PARP. These results suggested that the decreased cell apoptosis could relate to the inhibition of inflammatory response in Aβ(1-42)-induced BV-2 cells with the treatments of catechin and procyanidins A2.

Catechin and Procyanidin A2 Inhibit Cell Apoptosis in Aβ(1-42)-Induced BV-2 Cells
As is known to us, over-expression of pro-inflammatory cytokines can induce the microglial activation and lead to neuronal death. Our previous study have proved that LSF could inhibit cell apoptosis in Aβ(1-42)-induced BV-2 cells. In this current experiment, we also investigated the suppression effect of cell apoptosis in Aβ(1-42)-induced BV-2 cells under the treatments of LSF and the isolated components, catechin and procyanidin A2. As shown in Figure 7, LSF, catechin and procyanidin A2 could inhibit Aβ(1-42)-induced BV-2 cells apoptosis by reducing Bax/Bcl-2 ratio and attenuating the proportion of cleaved-PARP in total PARP. These results suggested that the decreased cell apoptosis could relate to the inhibition of inflammatory response in Aβ(1-42)-induced BV-2 cells with the treatments of catechin and procyanidins A2.

Discussion
AD is the most common type of dementia worldwide. Its pathological hallmarks are recognized as the accumulated Aβ in SPs and the hyperphosphorylation of tau in NFTs [25,26]. The inactivated microglia can improve Aβ clearance and promote anti-inflammatory effects, and also protect the CNS from injury and invading pathogens [27]. However, it has recently been shown that microglial depletion for 2-4 weeks in the mouse model at advanced stage cannot reduce Aβ plaque, suggesting that the aged microglia is inactivated in clearing plaques in the AD brain [28]. It has been reported that immune activation early in life could trigger the onset of AD, while exposing microglia to Aβ could induce sustained chronic neuroinflammation, tau hyperphosphorylation, and a loss of synapses and neurons [7,[29][30][31]. Therefore, the timing of Aβ pathogenesis is recognized to be critical for microglia to form a physical barrier to limit neurotoxicity [32], and a potent inhibitor of microglia activation is essential for inhibiting neuroinflammation. Lychee seed, a commonly used TCM, has been reported to exert many biological activities such as anti-oxidation, anti-virus, and improving insulin resistance (IR) [33][34][35][36]. In our previous study, LSF, a bioactive fraction from lychee seed, was first proven to improve the cognitive function of AD rats, protect PC12 cells against Aβ-induced injury, and suppress neuroinflammation in Aβ(1-42)-induced BV-2 cells [19,20]. However, the

Discussion
AD is the most common type of dementia worldwide. Its pathological hallmarks are recognized as the accumulated Aβ in SPs and the hyperphosphorylation of tau in NFTs [25,26]. The inactivated microglia can improve Aβ clearance and promote anti-inflammatory effects, and also protect the CNS from injury and invading pathogens [27]. However, it has recently been shown that microglial depletion for 2-4 weeks in the mouse model at advanced stage cannot reduce Aβ plaque, suggesting that the aged microglia is inactivated in clearing plaques in the AD brain [28]. It has been reported that immune activation early in life could trigger the onset of AD, while exposing microglia to Aβ could induce sustained chronic neuroinflammation, tau hyperphosphorylation, and a loss of synapses and neurons [7,[29][30][31]. Therefore, the timing of Aβ pathogenesis is recognized to be critical for microglia to form a physical barrier to limit neurotoxicity [32], and a potent inhibitor of microglia activation is essential for inhibiting neuroinflammation. Lychee seed, a commonly used TCM, has been reported to exert many biological activities such as anti-oxidation, anti-virus, and improving insulin resistance (IR) [33][34][35][36]. In our previous study, LSF, a bioactive fraction from lychee seed, was first proven to improve the cognitive function of AD rats, protect PC12 cells against Aβ-induced injury, and suppress neuroinflammation in Aβ(1-42)-induced BV-2 cells [19,20]. However, the specific components being responsible for this effect are still unknown. In this experiment, we isolated and identified two bioactive components from LSF that are responsible for these neuroprotective effects.
It has been reported that SP is surrounded by a cluster of reactive microglia as phagocytic cells. These microglia can remove Aβ in brain and protect neurons [29,37,38]. On the other hand, studies have shown that the content of Aβ in microglia was remarkably increased in AD patients and model animals' brains, suggesting that the microglia are over activated and then contribute to the occurrence of AD [39,40]. Therefore, the over-activated microglia induced by accumulated Aβ or tau will induce neuroinflammation and accelerate AD progression. Based on this evidence, we used Aβ(1-42)-induced BV-2 cells to simulate the over activated microglia in AD patients or rats. With persistent activation of BV-2 cells by Aβ(1-42), many cytotoxic factors including TNF-α, IL-1β, IL-6, nitric oxide (NO), and superoxide are released from the over-activated microglia [29,[41][42][43]. The released toxic inflammatory cytokines induce microglial activation and lead to neuronal death, and ultimately dementia [44]. In the present study, we symmetrically isolated and identified two polyphenols including catechin and procyanidin A2 from LSF using column chromatography, pre-HPLC, and NMR technologies. The bioactive validation proved that catechin and procyanidin A2 could suppress Aβ-induced neuroinflammation in Aβ(1-42)-induced BV-2 cells by inhibiting the release, mRNA levels, and protein expressions of TNF-α, IL-1β, IL-6, and iNOS.
Polyphenols widely exist in most natural plants such as Radix et Rhizoma Rhei, Radix Scutellariae, Rhizoma et Radix Polygoni Cuspidati, etc. As is known to us, polyphenols exert a potent anti-oxidative effect and protect cells or tissue against oxidative damage, which results in improved immunity, anti-cancer, and anti-aging effects. Recently, many polyphenols such as resveratrol from Rhizoma et Radix Polygoni Cuspidati [45]; epigallocatechin gallate (EGCG) from green tea [46]; curcumin from the spice turmeric [47]; and the flavonoids-including baicalin, baicalein, wogonoside, wogonin, oroxylin A, etc.-from Radix Scutellariae [48] have been proved to inhibit Aβ aggregation. Catechin and procyanidin A2 widely exist in fruit seed and skin including grape seed, apple seed, and the skin from Hylocereus undatus 'Foo-Lon'. Catechin has been reported to stimulate antioxidant activity by scavenging free radicals, inhibiting pro-oxidant enzymes, and stimulating antioxidant enzymes. In this study, our data suggested that catechin inhibited neuroinflammation, which is consistent with previous studies that claimed that catechin possessed a neuroprotective effect [49]. Procyanidins are members of the proanthocyanidin (or condensed tannins) class of flavonoids. They are oligomeric compounds composed of catechin and epicatechin. It has been reported that procyanidins have an extremely strong antioxidant and free radical clearing effect. At the same time, catechin and procyanidins have also been proved to inhibit Aβ-induced apoptosis in PC12 cells and inhibit Aβ aggregation, respectively [50,51]. However, up to now, there have been no reports on the inhibition effect of catechin and procyanidin A2 in Aβ-induced neuroinflammation.
In this study, we identified the key active components of LSF to inhibit Aβ(1-42)-mediated neuroinflammation. In general, morphological appearance is often related to functional properties, and we found the appearance of BV-2 cells treated with Aβ(1-42) were ameboid shaped, indicating a fully activated state [52,53]. Furthermore, the proportion of ameboid-shaped cells in Aβ(1-42) pre-treated BV-2 cells was decreased after the treatment of LSF, catechin, or procyanidin A2, suggesting that LSF, catechin, and procyanidin A2 can inhibit BV-2 cells activation.
NF-κB is a protein complex that regulates the transcription of DNA, pro-inflammatory cytokine release, and cell survival [54][55][56][57][58]. Pro-inflammatory mediators such as TNF-α, IL-1, IL-6, iNOS, and cyclooxygenase-2 (COX-2) are released from cells as NF-κB is activated [59]. NF-κB usually exists in the cytoplasm in the form of homo-and/or heterodimers, and forms an inactive complex with its inhibitory factor IκBα [60,61]. IκBα is phosphorylated by IκB kinase (IKK) and releases the NF-κB dimer when the pathway is activated. Our previous studies proved that LSF could inhibit neuronal and BV-2 cells apoptosis, which could be associated with the inhibition of the NF-κB pathways [20]. In the current study, catechin and procyanidin A2 in LSF were found to inhibit the NF-κB pathway by downregulating the p-IκBα/IκBα and NF-κB expressions.
Aβ also induces microglial apoptosis and further aggravates the inflammatory response. The Bcl-2 family plays an important role in the regulation of apoptosis. It consists of anti-apoptotic proteins such as Bcl-2, Bcl-xl, Bcl-w, and Mcl-1, and pro-apoptotic proteins including Bax, Bak, Bad, Bid, and Bim. In the present study, we found that catechin and procyanidin A2 could decrease the Bax/Bcl-2 ratio in Aβ(1-42)-induced BV-2 cells. Additionally, another apoptosis related protein, the poly (ADP-ribose) polymerase (PARP) protein family, is associated with many cellular processes such as DNA repair, genomic stability, and programmed cell death. The cleaved-PARP is generally considered to be an important indicator of apoptosis and caspase 3 activation [62,63]. Similarly, catechin and procyanidin A2 could reduce the proportion of cleaved-PARP in total PRAP in Aβ(1-42)-induced BV-2 cells. Taken together, all of the above results indicated that catechin and procyanidin A2 could suppress apoptosis in Aβ-induced BV-2 cells.
The blood-brain barrier (BBB) permeability of the drugs acting on CNS is becoming an important issue to which we should pay attention. The permeability of dietary polyphenols across the blood-brain barrier (BBB) is selective, has poor absorption, and rapid metabolism, which limits their bioavailability and neuroprotective effect. Most polyphenols have been proven to have a neuroprotective effect in vitro and vivo. Recently, a study found that the bioavailable polyphenol metabolites could transport across the BBB endothelium and protect brain endothelial cells and neuronal cells, and reduce neuroinflammation [64]. Although the polyphenols intracellular concentrations in neurons and microglial cells are limited, such concentrations are in fact beneficial [65]. Catechin, the compound identified from LSF in the current study, has previously been reported to pass through BBB after intravenous (iv) administration [66]. In addition, nanosystems could be a promising strategy for delivering resveratrol, a polyphenol, into the brain, which can protect it from degradation in the blood stream [67]. Therefore, a rational administration or formulation could avoid the first pass effect, protect degradation, and improve the BBB permeability of the polyphenols.
Collectively, our study confirmed that the two key active substances of LSF, catechin and procyanidin A2, could inhibit Aβ-induced neuroinflammation. In future study, we will try to investigate the concentration of catechin and procyanidin A2 passed through BBB by selecting the optimal administration route and formulation, which are expected to be developed as novel drugs for AD.

Reagent and Instrument
LSF was prepared according to our previous method [19,20]. HPLC-grade acetonitrile and methanol, sodium dodecyl sulfate (SDS), and Aβ

Instrument and Chromatograph Condition for the Separation of the Components in LSF
The separation and purification of the components in LSF was performed using AKAT protein purification system equipped with a UV-vis detector at 280 nm on an amethyst C 18 -H column (21.2 × 250 mm, 5 µm) which was purchased from Sepax Technologies Inc. (Suzhou, China) at a flow rate of 10 mL/min. The parameters of the gradient elution program were applied as follows: the ratio of mobile phase B (acetonitrile) to mobile phase A (0.1% formic acid in water) changed from 15% to 35% in 45 min. The peaks displaying in the chromatograms were collected according to their retention time. All the collected solutions were concentrated by rotary evaporator.

Cell Culture
BV-2 cells, an immortalized mouse microglia cell line, was purchased from the Kunming Institute of Zoology, Chinese Academy of Science (Kunming, China). It was cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin at 37 • C with CO 2 (5%) [8].

Cell Viability
Twelve isolated fractions were dissolved with dimethyl sulfoxide (DMSO) to make a final concentration of 80 g/L. Cell viability of Fractions 1-12 against BV-2 cells was measured with a CCK-8 kit according to the manufacture's protocols. In brief, BV-2 cells were plated on 96-well plates (100 µL, 1 × 10 5 cells/well) one day before LSF treatment under the indicated concentrations. After 48 h treatment, 10 µL of CCK-8 solution was added into each well and incubated for another 2 h. Colorimetric reading of the solute mixture was then determined at OD 450 nm using a standard plate-reader (DG5032, Hua Dong, Nanjing, China). The percentage of cell viability was calculated using the following formula: Cell viability (%) = Cells number treated /Cells number DMSO control × 100. Data were obtained from three independent experiments.

Identification of the Components in LSF
Reverse phase ultra-high-performance liquid chromatography (RP-UHPLC) (Shimadzu Corporation, Hadano, Japan) equipped with DGU-20A5R degasser, two LC-20ADXR pumps, a SIL-20ACXR autosampler, a CTO-20AC column oven, a SPD-M20A diode array detector, and a Shimadzu CBM-20A system controller was used for analysis in the current study. All the samples dissolved in methanol were separated by a C18 column (4.6 × 250 mm, 5.0 µm particle size) (GL Science, Torrance, CA, USA) at a flow rate of 1 mL/min. The gradient elution system consisting of mobile phase A (acetonitrile) and mobile phase B (0.1% acetic acid, v/v) was set as follows: 0-10 min Ultra-high-performance liquid chromatography (UHPLC) (Shimadzu Corporation, Hadano, Japan) equipped with DGU-30A5R degasser, two LC-30ADXR pumps, a SIL-30ACXR autosampler, a CTO-30AC column oven, a SPD-M30A diode array detector, a Shimadzu CBM-30A system controller, and a 8045 electrospray ionization tandem mass spectroscopic (ESI-MS/MS) was used to identify the isolated components from LSF in negative and positive modes. Data were acquired in scan mode from m/z 100 to 1000 Da with 2.0 spectra/s. Data analysis was carried out using Shimadzu LabSolutions software B.01.03 (Shimadzu Corporation, Hadano, Japan).

Wright-Giemsa Staining
BV-2 cells were seeded on coverslips in a 6-well plate a day before treatments. The cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of the test fractions or compounds for another 12 h. After treatments, these coverslips were air-dried and stained with Giemsa solution as described previously [68]. In brief, slides stained in the diluted Giemsa solution (1:20) in PBS for 15-20 min were washed with PBS to remove the excess stain and air-dried at room temperature. The air-dried slides were mounted with FluorSave TM mounting media (Calbiochem, San Diego, CA, USA), and the optical microscope images were observed and taken with an optical microscope (Leica DM750 optical microscope, Leica, Germany). The ratio of activated BC-2 cells was calculated by counting the number of the activated BV-2 cells in all of the BV-2 cells. In addition, the density of BV-2 cells was quantified by counting the number of all of the BV-2 cells. A minimum of 150 cells from three randomly selected fields were scored.

Cytokines ELISA
BV-2 cells were plated on a 96-well plate (100 µL, 1 × 10 5 cells/well) a day before treatments. The cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by an incubation of the fractions or compounds for another 12 h. The cell-free supernatants were subsequently employed for TNF-α, IL-1β, and IL-6 assays using the ELISA kits according to the manufacturer's instructions.

Quantitative Reverse Transcription PCR (qRT-PCR)
The total RNA of BV-2 cells was extracted by TRIZOL reagent, and its reverse transcription into cDNA were performed according to the manufacture's protocol for the Prime Script RT reagent kit

Western Blotting
After treatment, cells were washed with pre-cooled PBS and lysed by RIPA lysis buffer (Beyotime, Shanghai, China) containing 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 140 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSE). Protein concentrations were measured by BCA kit (Beyotime, Shanghai, China) as per the previous report [20]. Equal amounts of each protein (50 µg/well) were loaded onto SDS-PAGE and the separated proteins on the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) for 1 h. The membrane was then washed with TBST three times and incubated with the primary antibodies including β-actin, TNF-α, IL-1β, iNOS, NF-κB, IκBα, p-IκBα, PARP, cleaved-PARP, Bax, and Bcl-2 overnight at 4 • C. After the membrane was washed with TBST three times, it was followed with a further incubation for 1 h at room temperature with horseradish peroxidase (HRP) conjugated secondary antibodies. The protein expression bands were revealed by the ultra ECL Detection Reagent ECL (4A Biotech Co., Ltd., Beijing, China) and detected by the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Band intensity was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA) and the ratio of the interest proteins to β-actin was calculated.

Statistical Analysis
All data were presented as the mean ± standard deviation (S.D.) and were analyzed using GraphPad Prism 5.0 statistical analytical software (GraphPad Software, San Diego, CA, USA). The data were considered to have significant difference as p < 0.05. One-way ANOVA followed by the post-Tukey test was applied for statistical analysis to compare all the different groups in the current study.

Conclusions
In the current study, two polyphenols-catechin and procyanidin A2-were isolated and identified in LSF derived from lychee seed; they were proven to be the bioactive components responsible for the inhibition effect of neuroinflammation in Aβ(1-42)-induced BV-2 cells by modulating the NF-κB signaling pathway. Furthermore, catechin and procyanidin A2 could also inhibit cell apoptosis in Aβ(1-42)-induced BV-2 cells. This study illuminated the action substances and molecular mechanism of LSF in anti-neuroinflammation. Further studies on brain pharmacokinetics and the pharmacological activity in vivo are also essential.

Conflicts of Interest:
The authors declare no conflict of interest.