*Article* **Neurotherapy of Yi-Gan-San, a Traditional Herbal Medicine, in an Alzheimer's Disease Model of** *Drosophila melanogaster* **by Alleviating A**β**<sup>42</sup> Expression**

**Ming-Tsan Su <sup>1</sup> , Yong-Sin Jheng <sup>1</sup> , Chen-Wen Lu <sup>1</sup> , Wen-Jhen Wu <sup>1</sup> , Shieh-Yueh Yang <sup>2</sup> , Wu-Chang Chuang <sup>3</sup> , Ming-Chung Lee <sup>4</sup> and Chung-Hsin Wu 1,\***


**Abstract:** Alzheimer's disease (AD), a main cause of dementia, is the most common neurodegenerative disease that is related to the abnormal accumulation of amyloid β (Aβ) proteins. Yi-Gan-San (YGS), a traditional herbal medicine, has been used for the management of neurodegenerative disorders and for the treatment of neurosis, insomnia and dementia. The aim of this study was to examine antioxidant capacity and cytotoxicity of YGS treatment by using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in vitro. We explored neuroprotective effects of YGS treatment in alleviating Aβ neurotoxicity of Drosophila melanogaster in vivo by comparing survival rate, climbing index, and Aβ expressions through retinal green fluorescent protein (GFP) expression, highly sensitive immunomagnetic reduction (IMR) and Western blotting assays. In the in vitro study, our results showed that scavenging activities of free radical and SH-SY5Y nerve cell viability were increased significantly (*p* < 0.01–0.05). In the in vivo study, Aβ42-expressing flies (Aβ42-GFP flies) and their WT flies (mCD8-GFP flies) were used as an animal model to examine the neurotherapeutic effects of YGS treatment. Our results showed that, in comparison with those Aβ<sup>42</sup> flies under sham treatments, Aβ<sup>42</sup> flies under YGS treatments showed a greater survival rate, better climbing speed, and lower Aβ<sup>42</sup> aggregation in *Drosophila* brain tissue (*p* < 0.01). Our findings suggest that YGS should have a beneficial alternative therapy for AD and dementia via alleviating Aβ neurotoxicity in the brain tissue.

**Keywords:** Alzheimer's disease; amyloid β; immunomagnetic reduction; *Drosophila melanogaster*

### **1. Introduction**

Dementia is the most frequent age-related neurocognitive disorder. Patients with dementia are known to frequently experience disturbing behavioral and psychological symptoms, such as excitement, aggression, hallucinations, insomnia, anxiety, wandering, and depression [1–3]. Alzheimer's disease (AD), a main cause of dementia, is the most common neurodegenerative disease that is related to the abnormal accumulation of amyloid β (Aβ) proteins [4]. Pathological indicators of AD include the presence of Aβ plaques, which damage neurons, particularly those surrounding the hippocampus [5]. Aβ plaques are neuropathological biomarkers for AD. The challenge with assaying AD biomarkers is ascribed to the ultralow concentrations of Aβ<sup>42</sup> proteins in the cerebrospinal fluid and the blood [6].

Yi-Gan-San (YGS, Shun-Ning-Yi OTC medicine), a traditional Chinese (Kampo) herbal medicine, is composed of Atractylodes, Poria, Chuanxiong, Angelica, Bupleurum, Licorice and Uncaria. Since ancient times, YGS has been used to treat patients who have symptoms,

**Citation:** Su, M.-T.; Jheng, Y.-S.; Lu, C.-W.; Wu, W.-J.; Yang, S.-Y.; Chuang, W.-C.; Lee, M.-C.; Wu, C.-H. Neurotherapy of Yi-Gan-San, a Traditional Herbal Medicine, in an Alzheimer's Disease Model of *Drosophila melanogaster* by Alleviating Aβ<sup>42</sup> Expression. *Plants* **2022**, *11*, 572. https://doi.org/10.3390/plants 11040572

Academic Editors: Juei-Tang Cheng, I-Min Liu and Szu-Chuan Shen

Received: 26 January 2022 Accepted: 19 February 2022 Published: 21 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

such as nervousness, short temper, irritability and sleeplessness. Recently, several studies have shown that the administration of YGS is effective for treating the behavioral and psychological symptoms of dementia (BPSD) [7,8]. In Japan, YGS is also called yokukansan and served as a remedy for restlessness and agitation in children [9]. Yokukansan has been approved by the Japanese Ministry of Health, Labor and Welfare as a remedy for neurosis, insomnia, irritability and night crying in children. YGS is the extract of multiple crude drugs containing a large number of ingredients. YGS can protect against the cytotoxic effect of a low concentration of corticosterone on hippocampal neurons [10]. To date, over 70 basic research articles have been published on the pharmacological efficacy and mechanisms of YGS about the pharmacokinetics, metabolism and brain distribution of their active ingredients. Among these research articles, YGS has been confirmed as a new potential therapeutic agent for the management of neurodegenerative disorders such as AD, and for the treatment of neurosis, insomnia and dementia [7,8,11–14]. Although molecular complexity is the main obstacle to studying the mechanism of YGS, it is an advantage for various pharmacological effects.

The argument that recapitulation of human AD by using transgenic animal models has improved the understanding of its pathological mechanisms is indisputable. The *Drosophila melanogaster* model has made its mark as an effective tool for the study of human AD [15–17], including its cellular aspects and associated physiological and behavioral traits, through the use of both conventional and innovative genetic tools. It is undeniable that human genetics research has improved the understanding of genes related to neurodegenerative diseases. However, the inspection of human subjects is hampered by moral and technical limitations. Therefore, many studies turn to AD animal models, such as the fruit fly (*Drosophila melanogaster*), mouse (*Mus musculus*), zebrafish (*Danio rerio*), and nematode (*Caenorhabditis elegans*); with each mirroring differing aspects of AD to generalize human diseases. Among these animal models, the *Drosophila melanogaster* AD model has been selected as an ideal tool to study AD disorders because targeted expression of Aβ proteins in adult *Drosophila* can result in changes in the appearance of the structure, including a reduction in external eye size, and a loss of ommatidia organization. Thus, this study selected *Drosophila* as an AD model as a drug discovery tool for AD. Pathological evidence of AD includes the presence of Aβ protein in the brain tissue [18–20]. It is very important to find suitable analytical methods that can detect Aβ expression in *Drosophila* brain tissue. The novel techniques that have been successfully developed to detect Aβ in cerebrospinal fluid (CSF) are clearly suitable for detecting Aβ expression in *Drosophila* brain tissue [21,22]. For example, immunomagnetic reduction (IMR) can detect ultralow concentrations of Aβ protein in human CSF and blood for early diagnosis of AD through the use of antibodyfunctionalized magnetic nanoparticles dispersed in an aqueous solution [23–26].

In this study, we shed light on how *Drosophila melanogaster* became an animal model for AD, as well as its contribution as a tool for discovering therapeutic drugs for AD. We used highly sensitive IMR assay technology that is capable of detecting ultralow concentrations of Aβ in *Drosophila* brain tissue. Our results demonstrated that YGS treatments had better antioxidant capacity, and low cytotoxicity for SH-SY5Y nerve cells for the in vitro study, and had a greater survival rate, better climbing speed, and lower Aβ<sup>42</sup> aggregation in the brain tissue. On the basis of our data, YGS treatment may be a beneficial therapy for alleviating neurodegenerative disorders in AD.

### **2. Results**

### *2.1. Chromatographic Fingerprints of YGS*

YGS is widely used as a traditional herbal medicine that is composed of seven dried medicinal herbs: *Uncis ramulus*, *Cnidii rhizoma*, *Bupleuri radix*, *Atratylodis Lanceae rhizoma*, Poria, *Angelicae radix* and *Glycyrrhizae radix* in specific ratios. Each plant material was identified by external morphology, and the marker compound of the plant specimen according to the Taiwan Pharmacopoeia standard. In Figure 1, we used 3D high-performance liquid chromatography (HPLC) and UV detection methods to analyze individual active substances

and confirm which medicinal material corresponds to the chromatographic peak of YGS. We adopted chlorogenic acid, ferulic acid, liquiritin, glycyrrhizic acid, atractylenolide III and ligustilide, and other standard products to prepare a standard solution, and analyzed and compared the standard and sample solution with the same analytical method. Our 3D HPLC data showed the bioactive substances of YGS were chlorogenic acid, ferulic acid, liquiritin, glycyrrhizin, atractylenolide III, ligustilide, and were determined qualitatively within 60 min. The possible function of these bioactive substances was described in detail separately in the discussion section.

**Figure 1.** Chromatographic fingerprints of YGS from 3D HPLC. The bioactive marker compounds, namely chlorogenic acid, ferulic acid, liquiritin, glycyrrhizin, atractylenolide III, and ligustilide, were determined qualitatively within 60 min under the selected HPLC condition. Abbreviations: YGS, Yi-Gan-San; AU, arbitrary perfusion units; 3D, three-dimension; HPLC, high-performance liquid chromatography.

### *2.2. YGS Treatment Shows Better DPPH Free Radical Scavenging Activity*

Figure 2A shows quantified DPPH free radical scavenging activities of YGS extracts at varying concentrations. By using the radical scavenging activity of L-ascorbic acid as the reference standard, we measured 56.4–91.7% of radical scavenging activity under 0.1–100 mg/mL YGS extract treatments. Quantified DPPH radical scavenging activities of YGS extract treatments at 0.1 and 1.0 mg/mL had similar free radical scavenging activity (56.4–61.2%), while there were significant differences (*p* < 0.01, Figure 2A) for those YGS extract treatments at 10–100 mg/mL (78.3–91.7%) from YGS extract treatments at 0.1 and 1.0 mg/mL.

**Figure 2.** Antioxidant capacity and cytotoxicity of YGS treatment. (**A**) Quantified scavenging activities of free radicals are significantly greater with concentrations of YGS treatments by DPPH assay (*N* = 3 for each group). (**B**) Quantified relative SH-SY5Y cell viability was significantly greater with concentrations of YGS treatments by MTT assay (*N* = 3 for each group). Values are mean ± SEM (\*\* *p* < 0.01, \* *p* < 0.05, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test). Abbreviations: YGS, Yi-Gan-San; AU, DPPH, 1,1-diphenyl-2-picrylhydrazyl; L-AA, L-Ascorbic acid; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SEM, standard error of the mean; ANOVA, analysis of variance.

### *2.3. YGS Treatment Shows Higher Cell Viability of SH-SY5Y Cells*

Figure 2B shows the quantified cell viability of human neuroblastoma SH-SY5Y cells at different concentrations of YGS extracts treatment. Significant cell viability of SH-SY5Y cells was observed under YGS treatment at 0.5–10 mg/mL. When compared to sham treatment, the cell viability of SH-SY5Y cells was significantly enhanced from 120.9–140.8% under YGS treatment at 0.5–10 mg/mL (*p* < 0.01–0.05, Figure 2B). However, the cell viability of SH-SY5Y cells under YGS treatment at 20 mg/mL has no significant difference from those with sham treatment (*p* > 0.05, Figure 2B). Our results reveal that SH-SY5Y cells show better cell viability only under YGS treatment from 0.5–10 mg/mL.
