Gami–Chunggan Formula Prevents Motor Dysfunction in MPTP/p-Induced and A53T α-Synuclein Overexpressed Parkinson’s Disease Mouse Model Though DJ-1 and BDNF Expression

The Gami–Chunggan formula (GCF) is a modification of the Chunggan (CG) decoction, which has been used to treat movement disorders such as Parkinson’s disease (PD) in Traditional East Asian Medicine. To evaluate the neuroprotective effects of GCF in chronic PD animal models, we used either a 5-week treatment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with probenecid (MPTP/p) or the α-synuclein A53T overexpressed PD mouse model. C57BL/6 mice were treated with MPTP, in combination with probenecid, for 5 weeks. GCF was administered simultaneously with MPTP injection for 38 days. The A53T α-synuclein overexpressed mice were also fed with GCF for 60 days. Using behavioral readouts and western blot analyses, it was observed that GCF prevents motor dysfunction in the MPTP/p-induced and A53T α-synuclein overexpressed mice. Moreover, GCF inhibited the reduction of dopaminergic neurons in the substantia nigra (SN) and fibers in the striatum (ST) against MPTP/p challenge. The expression of DJ-1 was increased but that of α-synuclein was decreased in the SN of PD-like brains by GCF administration. In vitro experiments also showed that GCF inhibited 6-OHDA-induced neurotoxicity in SH-SY5Y neuroblastoma cell lines and that it did so to a greater degree than CG. Furthermore, GCF induced BDNF expression through phosphorylation of Akt, ERK, CREB, and AMPK in the SN of PD-like brains. Therefore, use of the herbal medicine GCF offers a potential remedy for neurodegenerative disorders, including Parkinson’s disease.


INTRODUCTION
Parkinson's disease (PD) is a chronic neurodegenerative disorder that is predominantly characterized by three representative motor features: akinesia, stiffness, and tremors (Conley and Kirchner, 1999;Kalia and Lang, 2015). Although the exact pathogenic mechanism of PD remains unknown, one study with PD patients suggested that oxidative stress and inflammatory pathways together induce apoptosis of dopaminergic neurons, eventually leading to manifestation of the disease (Hartmann, 2004). In addition, several genes have been correlated with the development of PD (de Silva et al., 2000). One among them is the presynaptic protein α-synuclein, a fibrillar factor of Lewy bodies that is linked to neuropathological features of PD; several different missense mutations of α-synuclein such as A53T, A30P, and E46K have been linked to early onset PD (Polymeropoulos et al., 1997;Krüger et al., 1998). Overexpression of wild-type or mutant A53T human α-synuclein in mice causes human PD-like symptoms such as neuronal degeneration and movement impairments (van der Putten et al., 2000).
Several toxins or reagents that mimic Parkinsonism both in vitro and in vivo have been reported in recent times, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), and rotenone (Beal, 2001). MPTP specifically targets dopaminergic neurons and causes severe and irreversible PD-like syndrome in non-human primates and humans. These subjects display biochemical and pathological hallmarks of PD (Przedborski et al., 2000) such as the obvious loss of dopaminergic neurons, astrogliosis, and activated microglia in the substantia nigra pars compacta (SNpc) (Beal, 2001). A second reagent, probenecid, can accelerate the mitochondrial toxicity of MPTP by interfering with ATP metabolism (Alvarez-Fischer et al., 2013). In the chronic MPTP/probenecid (MPTP/p) model, approximately 40-45% of dopaminergic neurons in the SNpc are lost within 3 weeks of treatment while 25% are lost in subchronic models without probenecid (Petroske et al., 2001;Meredith et al., 2008). In either case, death of dopaminergic neurons continues for at least 6 months, unlike in the subchronic and acute MPTP models (Petroske et al., 2001;Meredith et al., 2008). 6-OHDA, on the other hand, mimics symptoms of PD by generating free radicals after it is transported by the dopamine transporter, resulting in the cell death of dopaminergic neurons.
Treatment for PD typically comprises L-3,4-dihydroxyphenylalanine (L-dopa), a dopamine precursor and/or a dopamine agonist. Although this can reduce symptoms of PD, long-term use of the drug reduces its effectiveness and does not in fact stop disease progression (Kostic et al., 1991).
Chunggan (CG) extract has been used for the treatment of motor-related disorders, such as PD, in traditional oriental medicine. It includes six herbs: Paeonia lactiflora root, Angelica gigas root, Bupleurum falcatum Linne root, Ligusticum chuanxiong root, Gardenia jasminoides Ellis fruit, and Paeonia suffruticosa Andrews root bark. We have previously presented evidence for the pharmaceutical effects and mechanism of action of modified CG extract and its combination with L-dopa in the MPTP-induced PD model (Ahn et al., 2017;Chang et al., 2018). Although we already demonstrated the pharmacological properties of CG on acute PD symptoms, we did not examine its effects in a chronic disease model. Therefore, in this study, MPTP/p or α-synuclein A53T overexpression was used to establish a chronic PD mouse model. To improve the treatment efficacy of CG, a modified formula named Gami-Chunggan formula (GCF) was prepared, consisting of CG plus the Syzygium aromaticum bud and the Agastache rugosa O. Kuntze herb that has strong radical scavenging activities (data not shown). We aim to demonstrate the effect and mechanisms of action of GCF on PD-like phenotypes such as motor symptoms and neuroprotection in the chronic MPTP/p-induced or α-synuclein A53T overexpression induced PD mice models.

Apparatus, Chemicals, and Reference Compounds
All analytical experiments were conducted with the Shimadzu LC-20AD XR High Performance Liquid Chromatography (HPLC) system and an SPD-M20A Photo Diode Array (PDA) detector (Kyoto, Japan). Acetonitrile and ethanol were obtained from J.T. Baker (PA, United States), and 18.2 M distilled water was purified using Younglin's Aqua Max Ultra 370 (Anyang, South Korea) series. Geniposide, paeoniflorin, tilianin, paeonol, eugenol, saikosaponin A, ligustilide and decursin reference compounds were used for HPLC analysis and all were purchased from ChemFaces (Hubei, China).

Preparation of GCF and CG Extract
All packages of P. lactiflora root, L. chuanxiong root, A. gigas root, B. falcatum Linne root, G. jasminoides Ellis fruit, S. aromaticum bud, P. suffruticosa Andrews root bark, and A. rugosa O. Kuntze herb were purchased from the Tae-won-dang herb supplier (Daegu, South Korea). The origins of all plant batches were confirmed and deposited at Dongkwang Pharmaceutical Research and Development Center for extraction and HPLC analysis. For extraction of GCF, air-dried P. lactiflora root (60 g), L. chuanxiong root (40 g), A. gigas root (40 g), B. falcatum Linne root (32 g), G. jasminoides Ellis fruit (16 g), S. aromaticum bud (60 g), P. suffruticosa Andrews root bark (16 g), and A. rugosa O. Kuntze herb (40 g) were uniformly mixed and 30% ethanol (3.24 L) added to make a 30% ethanol mixture (10% w/v). For preparation of CG extract, S. aromaticum bud and A. rugosa O. Kuntze herb were excluded from GCF. After heating was initialized under reflux and the temperature reached 95 • C, the 30% ethanol mixture was extracted for a further 4 h. The 30% ethanol extract was cooled down for 30 min and filtered with Whatman #2 filter paper. The filtered extract was freeze dried to obtain GCF dried extract powder. The extraction was repeated 10 times.

Preparation of Standards and Samples
For standardization of GCF extract, The Korean Pharmacopoeia and scientific papers were reviewed and its major components identified (Yun et al., 2008;Tuan et al., 2012;Korean Food and Drug Administration [KFDA], 2015;Baek et al., 2016). One reference compound from each plant component of GCF was selected as a standard; these were geniposide, paeoniflorin, paeonol, eugenol, saikosaponin A, and decursin. For HPLC analysis, each reference compound was dissolved and mixed thoroughly to make stock solution. Individual stock solutions were added in uniform amounts to make working standard mixtures, which were used for the simultaneous separation and determination of compounds. For HPLC analysis, 1 g of freeze-dried GCF extract powder was weighed and added into a 10 mL volumetric flask with HPLC-grade 70% ethanol as solvent. The GCF extract powder was further extracted using an ultra-sonicator for 1 h. After sonication, the extract was filtered and used as GCF extract sample for HPLC analysis.

HPLC Analysis of GCF
All experiments were conducted with the Shimadzu LC-20AD XR HPLC system. GCF extract samples were analyzed under the developed HPLC method and the reference compounds in GCF extract samples were quantified using Shimadzu's Lab Solutions software. Chromatographic separation was accomplished by using a YMC Pack Pro C18 (250 × 4.6 mm, 5 µm) column (YMC Company, Japan) with a flow rate of 1.0 mL/min at 30 • C. To optimize detection, the entire UV spectrum of each reference compound was reviewed at different wavelengths. For optimum analysis, we selected 210 nm for saikosaponin A and ligustilide, and 230 nm for geniposide, paeoniflorin, tilianin, paeonol, eugenol and decursin.

Transgenic Mice
83Vle mice (Prnp-SNCA * A53T) with a B6C3H background (Jackson Laboratory, Bar Harbor, Maine, United States) were bred at the Dongguk University and animal protocols followed previously described methods (Lee et al., 2017. A53T hemizygous mice (n = 22) at 13-14 months of age were divided into three groups: (1) Control (intraorally saline-treated group, n = 7) (2) GCF 100 (intraorally 100 mg/kg of GCF -treated group, n = 7) (3) GCF 300 (intraorally 300 mg/kg of GCF-treated group, n = 8) Saline or GCF was orally administered every day for 60 days. Behavior experiments were done on day 60 and the mice were then sacrificed. The feed efficiency ratio (FER) was calculated as total increased weight divived by the total amount of food consumption.

Biochemical Analysis of Blood
For biochemichal blood analysis, total blood was collected with heparin-syringe tubes and centrifuged at 3000 rpm for 15 min at 4 • C. Plasma was collected and kept at −70 • C. Glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol, GOT (Glutamate Oxaloacetate Transaminase)/GPT (Glutamate Pyruvate Transaminase), and triglyceride (TG) were examined with analysis kits (Asan Pharmaceutical, Seoul, South Korea).

Akinesia
Akinesia was measured as the latency in time taken to move four limbs. The test was administered as previously described (Ahn et al., 2017). The test was repeated four times for each animal.

Catalepsy
Catalepsy was recorded as the time period for which animals retained their front legs, once placed, on a bar suspended above the floor of the test apparatus (Ahn et al., 2017). The time point at which the mice lifted their front paws from the bar marked the end of the time period. This experiment was repeated four times for each animal and mean value was calculated.

Rotarod Test
The rotarod test was used to assess neurological impairment such as motor coordination and balance. The experimental procedure followed was as previously reported (Ahn et al., 2017).

Pole Test
We performed a pole test 60 days after GCF administration, using an instrument 55 cm in height and 1.3 cm in diameter. The mice were held by their tails, with their heads positioned upward near the top of the pole and their forepaw on top of the pole. The time taken by the mouse to fall fully head down (orient down time) and the time taken to reach the bottom (transverse down time) were recorded. Mice were adjusted to the task by performing five trials per day for 3 days before the behavior test.

Brain Immunohistochemistry
Brain tissue preparation and immunohistochemistry methods were performed as per a previous report (Ahn et al., 2017). Briefly, sectioned slices were incubated with rabbit antityrosine hydroxylase (1:1000; Santa-Cruz Biotechnology, TX, United States) overnight at room temperature. They were then stained using ABC methods (Vectastain Elite ABC kit; Vector Laboratories, Inc., CA, United States) and developed with diaminobenzidine (Sigma, MO, United States). The sections were mounted, coverslipped, and imaged using a light microscope (BX51; Olympus Japan Co., Tokyo, Japan).

Cell Culture
The human neuroblastoma SH-SY5Y cell line was purchased from the American Type Culture Collection (Rockville, MD, United States) and maintained in DMEM containing 10% FBS and 1% antibiotics (Hyclone Laboratories Inc., UT, United States). Serum-deprived cells were treated with GCF for 30 min and then stressed with 200 µM of 6-OHDA (Biosource International, CA, United States) for 24 h. To examine cell viability, 100X of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma) was added to the media and the cells were incubated for 3 h at 37 • C in a CO 2 incubator. The media was then removed and remaining dye in the cells solubilized with DMSO. The optical density of each well was calculated using a spectrophotometer (Versamax microplate reader, Molecular Device, CA, United States) at a wavelength of 570 nm.

Statistical Analyses
GraphPad Prism (ver. 5; GraphPad Software, Inc., CA, United States) was used to perform all statistical analyses. One-way ANOVA was used to analyse the data excluding AIMs. Two-way ANOVA was used in AIMs data considering time and group as factors. All data are expressed as means ± SEM. p < 0.05 were considered as statistically significant.

Development of the Chemical Profile of GCF and Identification of Major Components
Plants contain hundreds of constituents, some of which are present at very low concentrations. Compositions may vary within different batches of plant material due to factors like freshness, temperature, light, water, time of collection, drying, and storage methods. To minimize this variation, the origins and suppliers of plant materials and the chemical compositions of GCF extract powder were controlled and standardized. One reference compound from each plant source used in GCF was selected for standardization based a thorough literature scan ( Figure 1A; Yun et al., 2008;Tuan et al., 2012;Korean Food and Drug Administration [KFDA], 2015;Baek et al., 2016). Eight reference compounds were analyzed using the developed HPLC method; all of them were well-resolved and detected in the chromatograms (Figure 1B). The retention times of these compounds were used for identification of those from the GCF extract samples. When the GCF extract samples were analyzed, their chromatograms showed eight peaks matching those of the eight reference compounds ( Figure 1B). The composition of the eight compounds in GCF extract powder are geniposide (13.97 ± 0.03 mg/g), ligustilide (0.8 ± 0.12 mg/g), tilianin (6.23 ± 0.03 mg/g), eugenol (12.13 ± 0.03 mg/g), saikosaponin A (1.1 ± 0.04 mg/g), paeoniflorin (19.26 ± 0.27 mg/g), paeonol (0.97 ± 0.08 mg/g), and decursin (11.20 ± 0.14 mg/g). No significant differences were found between batches.

GCF Inhibits MPTP-Induced Movement Impairments
To verify the protective effect of GCF in a PD animal model, GCF or L-dopa was administered to MPTP-treated mice for 14 days. MPTP induced severe motor impairments in the pole and rotarod tests when compared to the control group. Administration of GCF, however, significantly reduced the orient down time (F (5,35) = 8.88, p < 0.0001), traverse down time (F (5,34) = 6.81, p < 0.0002) and increased the time taken to fall from the rod (F (5,35) = 3.04, p < 0.224). L-dopa-treated mice were used as positive controls and they also recovered movement impairments, but not to the extent of the GCF-treated group (Figures 3A-C).

Suppressive Effect of GCF on the Level of α-Synuclein and TNF-α in the MPTP/p PD Model
The levels of α-synuclein, a Lewy Body marker, were assessed by immunoblotting. Chronic MPTP/p treat m ent significantly enhanced α-synuclein expression in the SN and ST (SN: F (4,15) = 8666.00, p < 0.0001; ST: F (4,15) = 3672.00, p < 0.0001) when compared to vehicle treatment (p < 0.001, respectively) (Figures 5A,B). GCF treatment, on the other hand, significantly and dose-dependently suppressed the MPTP/pinduced α-synuclein expression in both the SN and ST (MPTP/p vs. GCF 100, 200 and 300, p < 0.001 in all cases) (Figures 5A,B). We also examined the levels of tumor necrosis factor α (TNF-α), a cytokine involved in the inflammatory response and regulation of immune cells, in the SN of chronic MTPT/p mice. The expression level of TNF-α (F (4,15) = 19.80, p < 0.0001) was significantly increased in the chronic MPTP/p group when compared to the control group (p < 0.001). However, treatment with GCF significantly reduced the levels of TNF-α (MPTP vs. GCF 100, p < 0.01; GCF 200 and 300, p < 0.001 each) ( Figure 5C).
To assess the efficacy of GCF on motor symptoms, we conducted behavior tests on A53T α-synuclein Tg mice. In the pole test, GCF showed a dose-dependent reduction of the orient down time (F (2,7) = 3.27, p < 0.0857) and traverse down time (F (2,7) = 3.00, p < 0.1043) when compared to control Tg mice (each p < 0.001) (Figures 8A,B). Additionally, in the rotarod test we observed significant improvement of the latency time (F (2,7) = 4.54, p < 0.0431) in GCF 300 treated mice in comparison to the control mice (p < 0.01) (Figure 8C).
A53T α-synuclein Tg mice showed no differences in TH expression in the SN as compared to control mice (Supplementary Figure S1). This is in agreement with previous reports showing that, in transgenic mice expressing A53T α-synuclein under the tyrosine hydroxylase promoter, the number of nigral neurons and levels of striatal dopamine are unchanged relative to wild-type mice for up to 1 year (Matsuoka et al., 2001). It has also been shown that the total number of SN neurons is not changed between the Tg and wild-type mice (Jia et al., 2018), indicating that dopaminergic cell loss has not yet occurred in the Tg mice. Despite lacking these markers of PD, the A53T mutation was identified in familial cases leading to early onset of parkinsonian symptoms (Athanassiadou et al., 1999), as well as in cases of motor impairment in mice (Uchihara and Giasson, 2016).

DISCUSSION
In this study, we demonstrated that administration of GCF significantly improves behavioral impairments in A53T α-synuclein overexpressed mice and blocks the loss of doparminergic neurons in MPTP/p-induced mice, both of which are models of chronic Parkinson's disease. In addition, GCF induces BDNF and DJ-1 expression in the SN and downregulates α-synuclein in PD models.
The CG decoction has been used to improve motor function of PD patients in traditional oriental medicine. GCF is a modification of the CG decoction, consisting of some herbal components in addition to the standard CG mixture. Among the GCF components, paeoniflorin shows the most prominent anti-PD activities (Li et al., 2013). Paeoniflorin comes from the P. lactiflora root, which has been used to treat neurodegenerative disorders like PD in traditional medicine clinics. Paeoniflorin has been known to rescue MPP + and acidic damage-induced PC12 cell apoptosis through the autophagic pathway (Cao et al., 2010). This treatment also alleviates neurological deficits associated with unilateral striatal 6-OHDA lesion PD models (Liu et al., 2007). It attenuates neuroinflammation and dopaminergic neurodegeneration in PD mouse models by activation of the adenosine A1 receptor (Liu et al., 2006). Geniposide, an active element of G. jasminoides Ellis, has tranquilizing effects and is an important herb used in Traditional Chinese Medicine for dementia (Su et al., 2016). It shows neuroprotective effects by suppressing α-synuclein expression (Su et al., 2016), induces growth factors and reduces apoptosis in PD models (Chen et al., 2015). Eugenol, a phenol extracted from cloves, is an antioxidant (Ito et al., 2005), monoamine oxidase (MAO) FIGURE 6 | Mechanisms of protective effects of GCF in MPTP/p-induced PD mouse model. Immunoblots of SN extracts probed using the following antibodies: (A) pAkt/Akt/β-actin, (B) pERK/ERK/β-actin, (C) pCREB/CREB/β-actin, (D) BDNF/β-actin, (E) pAMPK/AMPK/β-actin, (F) DJ-1/β-actin, (G) pSynapsin-1/Synapsin-1/β-actin, and (H) BAX/BCL-2/β-actin. The intensities of protein bands were quantitated by densitometry and the bands indicating phosphorylated protein were normalized against either the total protein or β-actin. Data are expressed as mean ± SEM ( * p < 0.05, * * p < 0.01, * * * p < 0.001 compared to control group; ## p < 0.01, ### p < 0.001 compared to MPTP/p group; n = 4).
In the present study, we examined six active components (geniposide, paeoniflorin, tilianin, eugenol, saikosaponin A, and decursin) from the GCF mixture using in vitro assays, and for the first time demonstrated that tilianin has anti-PD activity in SH-SY5Y neuroblastoma cells. In addition, we showed that GCF has a stronger neuroprotective effect on the neuroblastoma cells than CG or its individual components (Figure 2C), suggesting that GCF might be a good formulation to treat PD patients. One possible caveat of this study is that we treated the cells first with GCF and then with 6-OHDA. The antioxidant properties of GCF may have inhibited the oxidation of 6-OHDA, thus weakening its toxicity toward the cells. However, GCF administered at a high concentration of 200 ug/ml did not show cytotoxicity and inhibited the cell death induced by 6-OHDA. This indicated that it most likely did not suppress the oxidation of 6-OHDA, but had a genuine protective effect on the cells. cAMP response element binding is a transcription factor stimulated by Ser-133 phosphorylation, and it has numerous downstream effecters: protein kinase C (PKC), protein kinase A (PKA), ERK1/2, and several PI3K/Akt/GSK-3β pathways (Adams and Sweatt, 2002;Carlezon et al., 2005). In this study, we found that GCF treatment increased the levels of CREB phosphorylation in the SN of MPTP/p-treated and A53T a-synuclein Tg mice. An upstream activator of CREB, Akt, was phosphorylated by GCF treatment as well. The observed ERK phosphorylation, however, may be attributed not just to GCF treatment, but to increased proinflammatory cytokine release, which could be neurotoxic in itself (Hua et al., 2002). In addition to these elements, administration of GCF was shown to suppress TNF-α expression ( Figure 5C). These alterations in protein expression indicate that GCF activates Akt, which in turn enhances CREB activation and exerts neuroprotective effects in the PD model mice.
DJ-1, a sensor of oxidative stress (Canet-Avilés et al., 2004), can decrease the accumulation and toxicity of α-synuclein in PD models (Shendelman et al., 2004;Zhou et al., 2011;Sun et al., 2012;Zondler et al., 2014;Lee et al., 2017). It is also known to activate ERK and Akt pathways to induce cell proliferation and survival (Oh and Mouradian, 2017). In this study, we demonstrated that GCF activates Akt, which might in turn be due to the induction of DJ-1 expression. The final cellular output of these signaling pathways is the reduction of α-synuclein accumulation, relieving the symptoms of PD in animal models.
MPTP-induced oxidative stress induces apoptosis through the activation of BCL-2 family proteins, including anti-apoptotic BCL-2 and pro-apoptotic BAX (Yang et al., 1997;Crompton, 2000). It has been reported that DJ-1 translocates to the mitochondria and binds to BCL-X L in response to UV-B irradiation, inhibiting both rapid degradation of BCL-X L and mitochondrial apoptosis (Ren et al., 2011). Specifically, BCL-X L interacts with BAX to block its oligomerization in the mitochondrial membrane, thereby protecting cells from BAX-induced mitochondrial membrane permeabilization (Yin et al., 1994). In the present study, it was shown that GCF induces DJ-1, which may suppress BCL-2 degradation and BAX expression, resulting in the inhibition of MPTP-induced apoptosis in PD models.
Autophagy is the process of degradation and elimination of aggregated proteins. Inhibition of this process induces neuronal degeneration in the central nervous system (Hara et al., 2006;Komatsu et al., 2006). AMPK activation is linked to the maintenance of autophagy (Mihaylova and Shaw, 2011) and neurogenesis (Dagon et al., 2005). AMPK activity in dopaminergic neurons has also been shown to be necessary for neuroprotection in a mouse model of PD (Bayliss et al., 2016). In addition, neuronal AMPK is part performance were measured in each group. The lysates of SN were electrophoresed and immunoblotted with each antibodies against α-synuclein, DJ-1, BDNF, and the phosphorylated forms of Akt, CREB, and ERK. The intensity of each band was normalized to that of β-actin (D) or total form and β-actin (E) and presented in bar graphs. The data are expressed as mean ± SEM ( * p < 0.05, * * p < 0.01 compared to control group; # p < 0.05 compared to GCF 100 mg/kg treated group; n = 7).
of an important signaling pathway that regulates BDNF, an essential mediator of neurogenesis (Kim and Leem, 2016;Liu et al., 2014). In this context, we demonstrated that treatment with GCF activates AMPK and induces BDNF expression in MPTP/p-induced PD mice models. Thus, this study provides evidence for the neuroprotective effects of GCF through AMPK activation in the SN.
We conducted both in vitro and in vivo efficacy tests of GCF in SH-SY5Y cells and mouse models. However, we primarily focused on the in vivo test to determine the effective doses of GCF in humans. We found that the effective doses in mice were 200 mg/kg and 300 mg/kg and the Human Equivalent Doses (HED) (Reagan-Shaw et al., 2008) were the following: 972.9730 mg/day (200 mg/kg × (3/37) × 60 kg) and 1459.4595 mg/day (300 mg/kg × (3/37) × 60 kg], respectively. In line with this observation, we conducted a 13-week oral toxicity study in Sprague-Dawley (SD) rats to determine the No Observed Adverse Effect Level (NOAEL). Based on this oral toxicity study, the NOAEL was assessed to be 2000 mg/kg/day by the KFDA-certified Good Laboratory Practice (GLP) Contract Research Organization (CRO), ChemOn (Yongin-Si, Gyeonggi, South Korea). The concentrations of GCF that were used in mice are therefore safe for therapeutic usage (data not shown).
Generally, when animals are tested for therapeutic effects of new compounds, the drug is administered simultaneously with disease induction. Therefore in this experiment, medication in the form of GCF was administered at the same time as MPTP, implying that the protective effects of GCF in the MPTP/p study are due to blockage of MPTP metabolism to MPP + . However, we also examined the neuroprotective effects of GCF after PD induction in the MPTP PD models (data not shown) and in the A53T α-synuclein Tg animal model, and found similar effects to those seen with simultaneous GCF administration. Hence, it can be inferred that GCF is an effective reagent for neuroprotection and regeneration when administered to PD patients.
In this study, we compared the anti-PD effects of GCF with L-dopa in the MPTP PD model (Figure 3). Although L-dopa did not recover the loss of dopaminergic cells, it did improve behavioral impairment, which is in concert with previous reports that L-dopa improves abnormal behavior in Parkinson's patients by supplementing deficient dopamine. However, longterm use of L-dopa may induce certain complications such as eventual loss of symptom control, leading to dyskinesia (Hauser, 2009). We found that the effect of GCF was more significant than that of L-dopa in terms of motor control (Figure 3), suggesting that GCF may normalize the function of dopaminergic cells in MPTPtreated mice. However, further studies are essential to determine the levels of dopamine and its metabolites in order to fully illustrate the protective effects of GCF.
In summary, the present findings demonstrate the neuroprotective effect of GCF against MPTP-or MPTP/ p-induced motor deficits and dopaminergic cell death. In addition, GCF administration diminishes behavioral impairments in α-synuclein A53T overexpressed mice. GCF activates elements of cell survival pathways such as Akt, ERK, and CREB in PD models and induced DJ-1 and BDNF expression. Moreover, GCF decreases α-synuclein expression and proapoptotic BAX expression through DJ-1 induction in chronic PD models. Therefore, the use of GCF, a herbal medicine, could be a potential remedy for neurodegenerative disorders such as Parkinson's disease.

ETHICS STATEMENT
The experimental processes were approved from the Institutional Animal Treatment Ethical Committee, Dongguk University Campus (Nos. 2017(Nos. -0992, 2017(Nos. -025, and 2017 and followed the NIH guidelines.

AUTHOR CONTRIBUTIONS
All authors were responsible for the study concept and design. SA, J-HJ, and JP carried out the immunoblotting of animal experiments in MPTP/p PD model. QL and HJ carried out the cell study, MPTP PD model, and α-synuclein PD animal experiments. YK, DK, GJ, and SO participated in the extraction of herbal medicine and analytical experiments. S-UP and S-YC organized the oriental medicine prescription. H-JP and SJ conceived the study and wrote the draft manuscript.