CP seeds were kindly provided by Queen Sirikit Botanic Garden, Botanical Garden Organization of the Ministry of Natural Resources and Environment (Chiang Mai, Thailand). Dried plant material was ground into powder using a mill. The extraction process was conducted according to the maceration method, where CP seeds (300 g) were macerated with 95% ethanol at room temperature for 72 h. Thereafter, the filtrate was separated from the residue. This was repeated three times. The filtrate was combined and evaporated for 16 h under reduced pressure at 50˚C to obtain the crude ethanolic extract.

A part of the crude extract was dissolved in methanol and water (1:4 v/v). The extract solution was inserted into a separating funnel and n-hexane (H) solvent was then added with a volume ratio of 1:1 and shaken until the two solutions were mixed into one phase. When A and H phase were separated, and the H fraction was placed in a glass beaker. This process was repeated three times until the H fraction was almost clear. Fractionation was continued via the addition of ethyl acetate (EA) into the A fraction using a separating funnel (100 ml or in a 1:1 ratio). The same procedure was performed as in the H fraction. Then, each fraction was evaporated until dry using a rotary evaporator to obtain the H, EA and A fractions (Fig. 1). All extracts were refrigerated at 4˚C until further use.

The total phenolic content of extract was determined according to the method reported by Khongkaew and Chaemsawang (15). The phenolic component was obtained from CPSE: 25 µl Folin-Ciocalteu reagent was added to cause a reaction with the phenolic compound and then incubated at room temperature in a dark room for 6 min. Then, 100 µl 7.5% sodium carbonate was added to each well and incubated at room temperature for 90 min in the dark. Light absorption was analyzed at 765 nm using a microplate reader to calculate the total phenolic compound content in the extract and to compare it with the standard gallic acid. All samples were subtracted from the absorbance of the blank sample. The result was reported as µg gallic acid equivalent/mg extract.

The antioxidant activity in terms of half-maximal inhibitory concentration (IC50), CPSE antioxidant capacity was measured on the basis of DPPH• scavenging. A 0.1 mM DPPH solution was prepared using 1.97 mg DPPH in 50 ml methanol The CPSE (100 µl) was added into 96-well plates with DPPH and incubated at room temperature in the dark for 30 min, after which, the wavelength was determined at 520 nm using a microplate reader. If the extract contains antioxidants, the purple color will change to yellow. Inhibition was calculated using the following equation: Inhibition (%)=[(absorbance of control-absorbance of sample)/absorbance of control) x100.

ABTS solution was mixed with potassium persulfate together in a 1:1 ratio and then incubated in the dark for 16 h at room temperature. Then, the crude extract, hexane fraction, ethyl acetate fraction and water fraction were taken and dissolved in methanol to a concentration of 1,000, 500, 300, 100 µg/ml. Subsequently, a volume of 100 µl/well was pipetted into a 96-well plate and 100 µl ABTS solution was added, and then incubated in the dark for 6 min at room temperature. The absorbance was then calculated at a wavelength of 734 nm. Inhibition was calculated using the following equation: Inhibition (%)=[(absorbance of control-absorbance of sample)/absorbance of control) x100.

The SH-SY5Y neuroblastoma cell line was purchased from American Type Culture Collection. The cells were maintained in a 1:1 mixture of modified Eagle medium and Ham's F-12 nutrient mixture with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37˚C in a humidified environment containing 5% CO₂. The culture medium was changed every 3-4 days. SH-SY5Y cells were grown in a 6-well plate. CPSE was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 µg/ml as a stock solution. The stock solution was diluted with complete medium before use. For pre-treatment, CPSE (10 µg/ml) was added to SH-SY5Y cells. Then, cells were maintained in a humidified 5% CO₂ atmosphere at 37˚C for 24 h. After washing with 1X PBS, 2 mM MPP⁺ was added into the culture plate and incubated at room temperature for 3 h. In the CPSE post-treatment group, MPP⁺ was added 3 h before CPSE treatment.

Cell viability was assessed by MTT assay, which is based on the cleavage of tetrazolium salts by mitochondrial succinate reductase in viable cells to form formazan dye. SH-SY5Y cells were cultured in 96-well plates at a density of 1x10⁴ cell/ml for 24 h. Following the aforementioned treatments, MTT (0.5 mg/ml) was added to each well and incubated for 3 h at 37˚C. The formazan crystals formed were then dissolved in DMSO. The absorbance was measured at 540 nm using a microplate reader and cell viability was expressed as the percentage of control.

LDH activities were measured spectrophotometrically by a CyQUANT™ LDH Cytotoxicity Assay (cat. no. C20300, Invitrogen™), using pyruvate as substrate, at 340 nm. Following the aforementioned treatments, an aliquot of the culture medium was taken to determine the activity of LDH that leaked through the cell membranes. Data are presented as the percentage of LDH leakage relative to the control. Four independent assays were conducted in triplicate.

Following 24 h treatment with all CP extract, cell vitality was measured quantitatively using the reagent WST-1. Cells were incubated with the reagent for 4 h at 37˚C. The absorption of the color product was measured at a 450/620 nm wavelength with a microplate reader. Each condition was tested in duplicate.

SH-SY5Y cells were harvested following the aforementioned treatments and lysed using Pierce^® RIPA Buffer (Thermo Fisher Scientific, Inc.). For measuring the concentrations of proteins, Bradford protein assay is used. Subsequently, equal amounts of protein (30 µg) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis on 10% gels and transferred to nitrocellulose membranes. The membrane were blocked with 5% skim milk in TBST for 1 h at room temperature. The membranes were incubated with primary antibodies at a dilution of 1:1,000 as follows: Mouse anti-p-GSK-3β (Ser9; cat. No. 05-643), anti-GSK-3β (cat. No. G7914), anti-Bcl-2 (Cat. No. SAB4500003) and anti-β-actin (Cat. No. A2228) antibodies (all Sigma-Aldrich) for 24 h at 4˚C. Membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cat. No. sc-2357; Santa Cruz Biotechnology, Inc.) at a dilution of 1:5,000 for 3 h at 4˚C and detected with Thermo Scientific SuperSignal^® West Pico Substrate (Thermo Fisher Scientific, Inc.). Densitometric analysis was performed using the ImageJ analysis software version 1.48p (ImageJ, NIH, Bethesda, Maryland, USA) and results were normalized to β-actin.

All experiments were performed in triplicate. Data are presented as the mean ± SD. Statistical significance was analyzed with one-way analysis of variance followed by Tukey's post hoc test. All data were analyzed by GraphPad Prism version 7.0 (GraphPad Software Inc.; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

The present study determined the effect of different concentrations of CPSE and its fractions including H, EA, and A on SH-SY5Y cell viability. Cell viability was significantly increased following treatment with 10 µg/ml EA and A fractions but not in response to the crude extract or H fraction (Fig. 2A). In addition, treatment with EA and A fractions for 24 h significantly increased the viability of SH-SY5Y cells (Fig. 2B).

High concentrations of all CP fractions (200 µg/ml) and long-term CP treatment (48 h) did not affect cell viability. Viability was highest following treatment with all fractions 10 µg/ml for 24 h; therefore, this treatment condition was selected for further experiments.

The total phenolic content of CPSE and its fractions was quantified (Table I). The A fraction presented the highest levels of individual phenolic compounds (30.20 µg/100 g), followed by EA (21.40 µg/100 g) and H fractions (8.83 µg/100 g). The crude extract had a low phenolic content (3.01 µg/100 g).

The antioxidative mechanisms of CPSE and its fractions were assessed using ABTS and DPPH assays (Table II). In the EA fraction, half-maximal inhibitory concentration (IC50) values tested by ABTS and DPPH were 492.11±43.92 and 1462.84±233.20 µg/ml, respectively; in the A fraction, the IC50 values were 393.35±32.53 and 719.17±107.50 µg/ml, respectively. In the crude extract and H fraction, higher IC50 values indicated low antioxidant capacity.

Oxidative stress and activation of the apoptotic cascade serve central roles in the pathogenesis of PD (1). Thus, to investigate the potential effect of CPSE and its fractions on MPP⁺-induced SH-SY5Y cell injury, SH-SY5Y cells treated with crude extract, H, EA or A fractions were stimulated with 2 mM MPP⁺ for 3 h before or after CPSE treatment, followed by the MTT assay (Fig. 3A and B). Treatment with 2 mM MPP⁺ resulted in decreased viability of SH-SY5Y cells, whereas treatments with all types of CPSE did not significantly affect the viability of SH-SY5Y cells compared with control. Both CPSE pre- and post-treatment reversed the MPP⁺-induced reduction of SH-SY5Y cell viability, especially H, EA or A fractions.

The balance between apoptosis and proliferation is key to maintaining normal cell activity. Cell proliferation was evaluated by WST-1 assay. Similar to the MTT assay, pre-treatment with H, EA and A fractions significantly increased the proliferation of MPP⁺-treated cells. (Fig. 4A). Post-treatment with all CPSE fractions increased the proliferation of MPP⁺-treated cells (Fig. 4B). This confirmed the significant neuroprotective effect of CPSE when administered either before or after MPP⁺ treatment.

To explore the neuroprotective effect of CPSE on MPP⁺-induced cell injury, SH-SY5Y cells were treated with CPSE 24 h before MPP⁺ treatment or after 3 h of MPP⁺ treatment. LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged, a key feature of cells undergoing apoptosis (14). MPP⁺ treatment significantly increased cytoplasmic LDH release; this was partially restored by CPSE and its fractions (Fig. 5). Therefore, both pre- and post-treatment of CPSE alleviated MPP⁺-induced SH-SY5Y cell damage. However, post-treatment with CPSE and H fraction did not significantly affect LDH levels (Fig. 5B).

The expression levels of anti-apoptotic proteins following MPP⁺ treatment with CPSE post-treatment were examined by western blot analysis. SH-SY5Y cells treated with MPP⁺ had decreased levels of the anti-apoptotic protein Bcl-2 (63.86±4.2%) compared with control; however, the protein expression levels of Bcl-2 were significantly increased in the EA- and A-treated groups (Fig. 6). The expression levels of Bcl-2 indicate the susceptibility of cells to apoptosis (4,16). These data suggested that MPP⁺ can induce the apoptosis of SH-SY5Y cells and CPSE treatment can protect SH-SY5Y cells by upregulating Bcl-2 expression.

Since a previous study reported that GSK-3β is involved in MPP⁺-induced SH-SY5Y cells, the present study investigated the expression levels of this protein (5). The results showed that MPP⁺ treatment reduced expression (but not but not statistically significant) of p-GSK-3β (Ser9), whereas post-treatment with crude extract, H, EA and A fractions significantly increased the expression levels of p-GSK-3β (Ser9) compared with those in the MPP⁺-treated group (Fig. 7). This phosphoserine exerts an inhibitory effect on the kinase by blocking the catalytic site; therefore, low p-GSK-3β (Ser9) levels can indicate increased kinase activity (6).