**1. Introduction**

Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. Recent statistics showed CRC caused 832,000 deaths and nearly 1.6 million new cases were diagnosed [1]. Among the various CRCs, Kirsten rat sarcoma viral oncogene homolog (KRAS) genetic mutation driven-CRC are known to be di fficult to target, and prone to drug-induced side e ffects [2,3]. Mutated KRAS proteins cause persistent activation of GTP, which endlessly send signals to the RAS/extracellular signal–regulated kinases (RAS/ERK) or protein kinase B/mammalian target of rapamycin (AKT/mTOR) cell regulation pathways [2]. ERK or mTOR proteins activate transcription factors involved in cell death, cell cycle progression, and transcription [2,4]. To inhibit RAS activation, antibody therapy was developed to target EGFR, such as Cetuximab, but various drug-resistance issues were documented [5–7]. Therefore,

novel medicinal substances with e ffective anti-cancer properties are needed and plant-derived foods or medicinal plants can o ffer potential means.

Several natural products have exhibited anti-CRC activity. For example, Danshen (*Salvia miltiorrhiza*) improved the survival rate of patients with colorectal cancer [8]. Triterpenoids from *Rhus chinensis* Mill [9], protopine from *Nandina domestica* [10], Paris saponin VII from *Trillium tschonoskii* [11] and protein hydrolysates from Fenugreek (*Trigonella foenum graecum)* [12] suppressed the proliferation and induced apoptosis of CRC cells. Additionally, rosemary (*Rosmarinus o*ffi*cinalis*) inhibited CRC growth by inducing necrosis [13]. However, limited studies have examined the e ffects of natural extracts targeting mutated KRAS-driven CRC and none of these studies tested the e fficacy of these extracts on muscle function. In our previous study, *Cordyceps militaris* germinated soybeans suppressed mutated KRAS-driven CRC via the RAS/ERK pathway [14]. *Phellinus linteus* grown on germinated brown rice increased the sensitivity of Cetuximab to inhibit KRAS-CRC progression both in vitro and in vivo [15]. Although the two natural compounds have anti-CRC activities, more medicinal substances are needed to target the notorious and drug-resistant nature of mutated KRAS-driven CRC.

Morning glory (*Pharbitis nil*) is a well-known ornamental plant, and its seeds have been used in traditional medicine to treat various cancers and inflammatory diseases in Korea, China, and Japan [16]. Recent studies have demonstrated its suppressive e ffects on breast cancer [17], lung cancer [18] and gastric cancer cells [19]. In addition, the seed of *Pharbitis nil* is the main constitute of the approved drug, DA-9701 (Motilitone) [20]. DA-9701 is a botanical drug for treating functional dyspepsia and composed of the seed of *Pharbitis nil* and *Corydalis* tuber. Therefore, we anticipated its safety on human and can be adapted for therapies in a relatively short timeframe if proven its e fficiency. In view of its anticancer e ffects, we considered the active compound of *Pharbitis* semen (PN) may inhibit the proliferation and progression of mutated KRAS-driven CRC without inducing serious side e ffects.

In addition to the suppressive e ffect of PN on CRCs, we evaluated the e ffect of compound PN on muscle cell function. During the course of CRC, cachexia, a complex syndrome showing severe muscle weight loss, is often accompanied with CRC progression and worsened treatment therapies [21–23]. Therefore, treating cachexia becomes an important combinational therapy in the treatment of CRCs. We mimicked cancer-associated environment with conditioned media and evaluated the e ffect of PN on muscle cells.

In the present study, the anticancer activity of PN was investigated by examining its anti-proliferation and clonogenic features in five di fferent CRC cell models. Cell cycle and apoptotic analyses were performed, and the changes in RAS/ERK and AKT/mTOR pathways were investigated. Finally, we assessed the e ffect of the compound PN on muscle cell proliferation and function.
