*2.8. YGS Treatment Shows Reduced Aβ Expression for GFP-Aβ<sup>42</sup> Flies by Western Blotting Assay*

For further verification, we used Western blotting to examine the Aβ<sup>42</sup> expressions of mCD8 and Aβ<sup>42</sup> flies under sham and YGS treatments, shown in Figure 7. Our results showed that Aβ<sup>42</sup> expressions of mCD8 flies under sham and YGS treatments are quite low, while the Aβ<sup>42</sup> expression of Aβ<sup>42</sup> flies under sham treatment was greater than those of GFP-Aβ<sup>42</sup> flies under YGS treatment (Figure 7A). We quantified and then compared that Aβ<sup>42</sup> concentrations of Aβ<sup>42</sup> concentrations of Aβ<sup>42</sup> flies under sham treatment are significantly greater than those of GFP-Aβ<sup>42</sup> flies under YGS treatment (sham, 0.96 vs. YGS, 0.61, *p* < 0.01, Figure 7B). The results further confirmed the down-regulation of Aβ<sup>42</sup> expression for Aβ<sup>42</sup> flies under YGS treatment.

**Figure 7.** Western blotting analysis of Aβ<sup>42</sup> expressions of Aβ42-GFP flies under sham and YGS treatments. (**A**) An example of Western blotting of Aβ<sup>42</sup> expressions of Aβ42-GFP flies under sham and YGS treatments. (**B**) Quantified Aβ<sup>42</sup> expressions of the Aβ42-GFP flies under YGS treatment were significantly weaker than those of Aβ42-GFP flies under sham treatment (*N* = 100 for each group). Values are mean ± SEM (\*\* *p* < 0.01, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test). Abbreviations: YGS, Yi-Gan-San; Aβ, amyloid-beta, GFP, green fluorescent protein; kDa, kilodaltons; WT, wild-type; SEM, standard error of the mean; ANOVA, analysis of variance.

### **3. Discussion**

Oxidative stress has been implicated in the progression of a number of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) [27]. These diseases are characterized by extensive oxidative damage to lipids, proteins and DNA. Oxidative stress is the result of an imbalance in the pro-oxidant/antioxidant homeostasis, leading to a generation of toxic reactive oxygen species (ROS). Aβ can generate H2O<sup>2</sup> with the further generation of ROS through Fenton chemistry. To prove YGS has a neuroprotective function, we first examine whether YGS has the antioxidant ability by using the DPPH assay. In this study, we found that YGS has a good antioxidant ability because YGS extracts show significant DPPH radical scavenging activity at concentrations of 0.1–100 mg/mL. Bioactive markers of YGS were chlorogenic acid, ferulic acid, liquiritin, glycyrrhizin, atractylenolide III, and ligustilide that were determined qualitatively. It has been reported that YGS has neuroprotective effects and rescues neurons, possibly via the PI3K/Akt pathway [28]. Chlorogenic acid, an active compound of Uncaria, could protect against neurodegeneration with supplementation via inhibiting oxidative stress in the brain [29–32]. In addition, chlorogenic acid treatment can protect Aβ-induced injury in SH-SY5Y neurons and alleviate cognitive impairments in AD transgenic mice via enhancing the activation of the mTOR/TFEB signaling pathway [33]. Ferulic acid, an active compound of Angelica, can delay Aβ-induced pathological symptoms [34]. Liquiritin, an active compound of Licorice, can attenuate rheumatoid arthritis via reducing inflammation, inhibiting the MAPK signal pathway [35], and ameliorating Aβ-induced spatial learning and memory impairment by inhibiting oxidative stress and neural apoptosis [36]. Glycyrrhizin, another active compound of Licorice, can ameliorate inflammatory pain via blockage of the HMGB1-TLR4- NF-kB pathway [37], and prevent cognitive impairment in aged mice by reducing neuroinflammation and AD-related pathology [38]. Atractylenolide III, an active compound of Atractylodes, has anti-inflammatory and neuroprotective effects that may serve as a therapeutic agent in the treatment of depression [39]. Ligustilide, an active compound of Chuanxiong, can improve aging-induced memory deficit by regulating mitochondrial related inflammation and inhibiting oxidative stress in SAMP8 mice [40].

In this study, we modeled GFP fluorescence in Aβ42-expressing flies that were more sensitive and suitable for analyzing Aβ<sup>42</sup> toxicity and identified relevant therapeutic compounds. *Drosophila* Aβ models can help us to approach AD studies in uncovering crucial mechanisms and pathways. In addition, *Drosophila* models can be developed as an excellent tool for various drug testing purposes. As suggested from our results, YGS treatment could significantly reduce GFP fluorescence in the external eyes. These results should provide supporting evidence for the neurotherapy of YGS in an Alzheimer's disease model of *Drosophila melanogaster* by alleviating Aβ<sup>42</sup> expression. Although *Drosophila* was an excellent tool for studying AD mechanisms and pathways, there are still risks when using it as a disease model because the pathology may be unique to vertebrates and cannot be transformed into the invertebrate *Drosophila*.

Since the expression of Aβ in the *Drosophila* brain tissue is extremely low, it is very important to find a suitable analytical method to detect the Aβ change. We found that IMR, an ultra-high-sensitivity technology, should be very suitable for detecting ultralow concentrations of Aβ protein in the *Drosophila* brain tissue for diagnosis of AD through the use of antibody-functionalized magnetic nanoparticles dispersed in aqueous solution and the superconducting-quantum-interference-device (SQUID) [23–25]. Most studies utilizing SQUID IMR have focused on exploring the relationship between Aβ protein in plasma and in CSF [25,26]. We have achieved promising results in terms of the feasibility of detecting AD in *Drosophila* brain tissue. Our Western blotting data confirmed the results that down-regulation of Aβ<sup>42</sup> expression for Aβ<sup>42</sup> flies under YGS treatment by IMR assay is credible. As far as we know, this study should be the first application of IMR technology to detect Aβ protein expressions in the *Drosophila* brain tissue for the diagnosis of AD.

The exact mechanism by which YGS treatment alleviates Aβ<sup>42</sup> expression remains unclear. Therefore, further clinical trials are warranted to verify the benefits of YGS treatment in AD patients.

### **4. Materials and Methods**

### *4.1. Yi-Gan-San (YGS) Preparation*

The YGS, an over-the-counter drug called Shun-Ning-Yi, in Taiwan, was manufactured by the Sun-Ten Pharmaceutical company, New Taipei City, Taiwan. The Ministry of Health, Labor and Welfare of Japan has approved this as a remedy for neurosis, insomnia and irritability in children. YGS is a traditional herbal medicine consisting of seven herbs: *Uncis ramulus*, *Cnidii rhizoma*, *Bupleuri radix*, *Atratylodis Lanceae rhizoma*, Poria, *Angelicae radix* and *Glycyrrhizae radix* in specific ratios. As suggested by the Sun-Ten Pharmaceutical company, YGS is a Chinese herbal formula and its mass fraction of constituents was composed of Bupleurum 2 g, Licorice 2 g, Chuanxiong 3.2 g, Angelica 4 g, Atractylodes 4 g, Poria 4 g, and Uncaria 4 g per 100 g of the final product. In Taiwan, YGS was traditionally used as powders. In the prescription of YGS powder, such as *Angelica sinensis* and Chuanxiong, it is rich in volatile oil, which can retain a high content of volatile oil when used in powder, while the volatile oil can be easily volatilized when used in soup. YGS follows the traditional concept of using powder as medicine, mixing crude drug powder according to the proportion of traditional Chinese medicine formula, and then adding a small amount of starch for granulation, with the proportion of starch being 3.3%.

### *4.2. Phytochemical Screening*

Before the phytochemical screening, precisely weigh 0.5 g of the YGS powder, ultrasonically shake with 20 mL of 70% methanol at room temperature for 15 min, then shake at 160 rpm for 20 min in a 40 ◦C water bath, and then centrifuge to take the supernatant. Another 20 mL of 70% methanol was added to the precipitate after centrifugation, ultrasonically shaken for 15 min at room temperature, and then centrifuged again at 160 rpm for 20 min in a 40 ◦C water bath, and the supernatant was taken. The two supernatants were combined and made up to 50 mL with 70% methanol, mixed evenly, and filtered through a 0.45 µm filter to obtain the test solution. The chromatographic fingerprint analysis of YGS was conducted using 3D HPLC (Burdick & Jackson, Gyeonggi-do, Korea). To confirm which bioactive substances of YGS were in the chromatographic peaks, we used

chlorogenic acid, ferulic acid, liquiritin, glycyrrhizic acid, atractylenolide III and ligustilide and other standard products to prepare a standard solution and analyze and compare the standard and sample solution with the same analytical method. HPLC analysis systems regarding column type and identification are described: the model of controller and pump pressurized system was a Waters 600, the degasser was a Waters In-Line Degasser AF, the autosampler was a Waters 717 plus, the photodiode array detector was a Waters 2996, the pre-column was a Lichrospher RP-18 endcapped (5 µm, ID × L = 4.0 × 10 mm, Merck), and the analytical column was a Cosmosil 5C18-MS-II (5 µm, ID × L = 4.6 × 250 mm, Nacalai tesque). HPLC analysis conditions regarding mobile phase, flow rate and detector time are described: column temperature was 35 ◦C, the flow rate was 1.0 mL/min, and analysis time was 65 min.

### *4.3. DPPH Assay*

Antioxidant activities of the YGS treatment were assessed by DPPH assay. YGS extracts that were diluted in distilled water in a concentration range of 0.1 to 100 mg/mL were mixed with 100 µL of 1.5mM/mL DPPH (D9132, Sigma-Aldrich Co., St. Louis, MO, USA) in methanol in a 96-well plate. After 30 min at room temperature, the absorbance of the samples was recorded. The color changes were recorded spectrophotometrically at 517 nm using a microplate spectrophotometer (µQuant™, BioTek Intruments, Inc., Winooski, VT, USA). Appropriate blanks (methanol) and standards (L-ascorbic acid in water, L-AA; A5960, Sigma-Aldrich Co., St. Louis, MO, USA) were recorded simultaneously. Each assay was carried out in triplicate. The DPPH scavenging was calculated by using the following expression:

DPPH scavenging (%) = 100 × [(absorbance of sample + DPPH) − (absorbance of sample blank)]/[(absorbance of DPPH) − (absorbance of methanol)]

Concentrations of YGS that cause 50% scavenging (IC50) were calculated from the graph in which scavenging activity was plotted against the corresponding YGS concentration.

### *4.4. MTT Assay under YGS Treatment*

Triplicate cultures of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> SH-SY5Y cells per well for each 24-well plate. After the YGS or sham treatment, we added 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, M5655, Sigma-Aldrich Co., St. Louis, MO, USA) to the culture media. SH-SY5Y cells were incubated for 1 h at 37 ◦C in a humidified atmosphere (95% air and 5% CO2), and then the MTT solution was discarded, and 100 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich Co., St. Louis, MO, USA) was added. The absorbance was read at an optical density (OD) of 570 nm with a microplate spectrophotometer (µQuant™, BioTek Intruments, Inc., Winooski, VT, USA).

### *4.5. Animals and Experimental Design*

Aβ42-expressing flies were generated by the Goldstein Laboratory (stock No. 32038). In this study, Aβ42-expressing flies (Aβ42-GFP flies) and their WT (mCD8-GFP flies) were used to examine the effect of YGS treatment by comparing retinal GFP expression without the need for histological assessment. The stronger Aβ<sup>42</sup> expressions in GFP-Aβ<sup>42</sup> flies and the weaker retinal GFP expression was monitored [15]. In this study, unless otherwise specified, both GFP-Aβ<sup>42</sup> and their WT flies were cultured and maintained on a standard cornmeal-yeast-agar medium at 25 ◦C and 60% humidity, in which the standard medium of GFP-Aβ<sup>42</sup> was uniformly mixed with 1% and 0.1% YGS by weight of the medium.

### *4.6. Survival Rate and Behavior Analysis in Drosophila*

Aβ<sup>42</sup> flies and mCD8 flies (*N* = 300 for each group) were exposed to sham and 1.0% YGS treatment. The flies were observed daily for the incidence of mortality, and the survival rate was determined by counting the number of dead flies for 50 days. The data were

subsequently analyzed and plotted as cumulative mortality and percentage survival after the treatment period.

The negative geotaxis assay was used to evaluate the locomotor performance of flies (*N* = 30 for each group). In brief, after the treatment period of 5 days, the flies from each group were briefly immobilized in ice and transferred into a clean tube (11 cm in length, 3.5 cm in diameter) and labeled accordingly. The flies were initially allowed to recover from immobilization for 10 min and thereafter were tapped at the bottom of the tubes. Observations were made for the total number of flies that crossed the 6 cm line within a period of 6 s and recorded. The results were expressed as a percentage of flies that escaped beyond a minimum distance of 6 cm in 6 s during three independent experiments.

### *4.7. Retinal GFP Expression Assay in Drosophila*

We took an image of the outer Drosophila eye with an Olympus-BH2 microscope. Tissue sections and fluorescence were imaged using a Leica DM IRB fluorescence microscope. For examination with fluorescence microscopy, we removed the Drosophila head with spring scissors between the head and the thorax that was arranged in pairs so that individual experimental flies could be imaged and compared directly to individual flies from their respective control groups. Fluorescence was quantified by using publicly available NIH Image J software. The mean retinal fluorescence from Aβ<sup>42</sup> flies was normalized to the mean fluorescence from mCD8 flies (30 files for each group).

### *4.8. IMR Assay in Drosophila*

*Drosophila* (100 files for each group) was homogenized and mixed with a reagent, which consisted of magnetic nanoparticles that were functionalized with monoclonal antibodies against a target protein, and then dispersed in PBS of pH 7.2 (MagQu Co., Ltd.) at room temperature. The magnetic nanoparticles used were dextran-coated Fe3O<sup>4</sup> particles (MF-DEX-0060, MagQu Co., Ltd.). For each sample at each target protein concentration, IMR signal measurements were performed in duplicate. The signals were converted to biomarker concentrations using standard curves. All plasma samples were blinded for IMR measurements. The tau reagent (MF-TAU-0060, MagQu Co., Ltd.) contained magnetic nanoparticles immobilized with a monoclonal antibody (T9450, Sigma) against human tau protein. The Aβ1–42 reagent (MF-AB2-0060, MagQu Co., Ltd.) contained magnetic nanoparticles coated with a monoclonal antibody against human Aβ1–42 protein. These reagents were superparamagnetic, with a saturated magnetization of 0.3 emu/g. A SQUIDbased AC magnetic susceptometer (XacPro-S, MagQu Co., Ltd.) was used to determine the time-dependent AC magnetic susceptibility, which approximates the association between magnetic nanoparticles and target protein molecules in the plasma [24] of each mixture. The IMR signal, which refers to the reduction in magnetic susceptibility caused by the association between magnetic nanoparticles and the target protein molecule, as detected by the magnetic susceptometer, represents the concentration of the target protein.

### *4.9. Western Blotting in Drosophila*

Total proteins were extracted from the head tissue of *Drosophila* following the treatment described (100 files for each group). The removed tissue was homogenized in a buffer solution that was placed on ice for one hour and then centrifuged at 4 ◦C for 13,000 rpm for another 20 min. The separated solution was quantified by using a BCA protein assay kit (Thermo Fisher Scientific Inc. Waltham, MA, USA). Proteins were separated on 12.5% or 15% SDS polyacrylamide gels (Bionovas Pharmaceuticals Inc., Washington, DC, USA), and proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Barrington, IL, USA). The antibodies used in this study were anti-Histone H3.3B (Thermo Fisher Scientific Inc.), and anti-amyloid-beta (anti-Aβ) (Covance Cat#SIG-39220, BioLegend, Dedham, MA, USA). Antibodies were detected by suitable horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc.), and then proteins' immunoreactive bands were visualized by the enhanced chemiluminescence

(ECL) substrate (Millipore, Billerica, MA, USA), and the band intensities were quantified with the Image J analysis software (version 1.48t, Wayne Rasnabd, USA).

### *4.10. Statistical Analysis*

All data are presented as means ± standard errors of the mean. One-way or two-way analysis of variance was performed, followed by the Student–Newman–Keuls post hoc test. The *p*-values of at least < 0.05 were considered significant. All data are obtained in at least three independent experiments.

### **5. Conclusions**

Transgenic Drosophila Aβ models have successfully provided valuable information for studying AD mechanisms and pathways. In addition, Drosophila provided valuable drug testing in vivo experiments for YGS treatment. For in vitro experiments, our results showed that YGS treatment has a good antioxidant ability and low cytotoxicity. For in vivo experiments, our results showed that YGS treatment can reduce Aβ and Tau expressions in Drosophila melanogaster by IMR assay and Western blotting that were quite consistent with the change in appearance traits. To the best of our knowledge, this study was the first to conduct an evidence-based investigation of the effectiveness of alternative therapy with the traditional Chinese medicine YGS treatment in alleviating Aβ neurotoxicity of *Drosophila melanogaster* assessed by highly sensitive IMR assay and evaluated from appearance traits in GFP expression of external eyes.

**Author Contributions:** Conceived and designed the experiments: C.-H.W. and M.-T.S.; analyzed the data, S.-Y.Y., C.-W.L. and W.-J.W.; contributed reagents/materials/analysis tools, S.-Y.Y., M.-C.L. and W.-C.C.; wrote the paper, C.-H.W. and M.-T.S.; surgical procedures, Y.-S.J.; behavioral tests, Y.-S.J. and M.-T.S.; Western blotting assay, Y.-S.J. and W.-J.W.; immunohistochemistry, Y.-S.J. and M.-T.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants from the Ministry of Science and Technology of Taiwan under grant numbers MOST 107-2321-B-003-001-, MOST 108-2321-B-003-001-, MOST 109-2321-B-003- 001-, and MOST 107-2320-B-003-003-MY3.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Care and Use of Laboratory Animals of the National Institutes of Health.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is confidential.

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

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