**4. Conclusions**

We developed two in vitro models of AD, namely the Aβ-induced axonal growth cone collapse model and the Aβ-induced axonal atrophy model. Using these models, we found several drugs derived from natural medicines, which prevented growth cone collapse or enhanced axonal growth. These agents prevented or induced recovery from memory deficits in AD model mice, and some of them were observed to be effective in humans. These findings indicate that axonal growth is possibly an important target for AD therapy and our models have high predictive validity for anti-AD activity.

Clarifying molecular mechanisms of natural medicines is complicated. Natural medicines contain thousands of constituents, and each of them shows various pharmacological effects. It is very complicated to identify active compounds and molecular targets of their compounds. These have made it difficult to analyze natural medicines scientifically. We advocated to resolve these problems by a combination of in vivo screening and the DARTS method. Our strategies could comprehensively identify active compounds in natural medicines and their target molecules, allowing to gain a foothold for usages of the natural medicines based on scientific evidence.

Several target molecules of anti-AD agents were identified in our studies. Most of these molecules had not been reported as AD-related molecules. Further detailed analysis of the signaling cascade of these molecules might reveal novel and effective pathways for AD prevention and cure. Natural medicines are clinically used for thousands of years. Safety of natural medicines has already been established. Studies of natural medicine might be a shortcut for the development of novel anti-AD drugs.

**Author Contributions:** All authors wrote the manuscript and drew the figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially funded by JSPS KAKENHI, Grant Numbers 18K07389 (T.K.) and 19K16288 (X.Y.), and research grants from the Takeda Science Foundation, Japan.

**Acknowledgments:** We would like to express our gratitude to all members in Division of Neuromedical Science, Institute of Natural Medicine, University of Toyama.

**Conflicts of Interest:** T.K. and C.T. declare that they received research fund from Kobayashi Pharmaceutical Co., Ltd., Japan. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**

