Colorectal cancer (CRC) is an emerging public health problem and the second leading cause of death worldwide, with a significant socioeconomic impact in several countries. The 5-year survival rate is only 12% due to the lack of early diagnosis and resistance to available treatments, and the canonical Wnt signaling pathway is involved in this process. This review underlines the importance of understanding the fundamental roles of this pathway in physiological and pathological contexts and analyzes the use of naturally occurring small molecules that inhibits the Wnt/β-catenin pathway in experimental models of CRC. We also discuss the progress and challenges of moving these small molecules off the laboratory bench into the clinical platform.Colorectal cancer (CRC) ranks second in the number of cancer deaths worldwide, mainly due to late diagnoses, which restrict treatment in the potentially curable stages and decrease patient survival. The treatment of CRC involves surgery to remove the tumor tissue, in addition to radiotherapy and systemic chemotherapy sessions. However, almost half of patients are resistant to these treatments, especially in metastatic cases, where the 5-year survival rate is only 12%. This factor may be related to the intratumoral heterogeneity, tumor microenvironment (TME), and the presence of cancer stem cells (CSCs), which is impossible to resolve with the standard approaches currently available in clinical practice. CSCs are APC-deficient, and the search for alternative therapeutic agents such as small molecules from natural sources is a promising strategy, as these substances have several antitumor properties. Many of those interfere with the regulation of signaling pathways at the central core of CRC development, such as the Wnt/β-catenin, which plays a crucial role in the cell proliferation and stemness in the tumor. This review will discuss the use of naturally occurring small molecules inhibiting the Wnt/β-catenin pathway in experimental CRC models over the past decade, highlighting the molecular targets in the Wnt/β-catenin pathway and the mechanisms through which these molecules perform their antitumor activities. According to the World Health Organization (WHO) definition, cancer is a general term for a group of diseases characterized by changes in cell proliferation that can affect any part of the body, invade other tissues, and cause death . More thColorectal cancer (CRC) has a high incidence and mortality rate and is the second leading cause of cancer deaths worldwide ,4. EnvirEarly diagnosis allows family follow-up and the adoption of appropriate medical conduct for better chances of cure . HoweverCSCs share some regulatory mechanisms of normal intestinal stem cells (ISC), which generate one of the main therapeutic challenges because CSC-targeted therapies may inadvertently harm ISC homeostasis or stem cells in bystander organs ,18. The The cell signaling by the Wnt family of secreted glycoproteins is one of the fundamental mechanisms that regulate cell proliferation, polarity, and cell fate ,28,29. TThe canonical Wnt signaling pathway relies on the fine balance of β-catenin protein level ,31. In tDrosophila), all Wnt proteins are palmitoylated in the endoplasmic reticulum (ER) [Wnt proteins are crucial in the canonical signaling pathway as well as in polarized epithelial cells . Excludilum (ER) ,45,46 bylum (ER) , leadinglum (ER) ,49. Oncelum (ER) ,51. Thislum (ER) ,52,53,54lum (ER) . Howeverlum (ER) ,32,56. Tlum (ER) , which cAdditionally, negative feedback mediated by the E3 ubiquitin-protein ligases ZNRF3 and Ring Finger Protein 43 (RNF43) promote the regulation of β-catenin through the degradation of LRP5/6 and Frizzled receptors, reducing the availability of binding sites for the Wnt protein, resulting in subsequent downstream β-catenin degradation ,59. HoweThe intestine is one of the most dynamic organs in the human body ,19. The As the Wnt pathway is crucial for promoting homeostasis in the ISC niche, it is not surprising that changes in its components trigger pathological events ,20,60. AAccording to The Cancer Genome Atlas (TCGA), changes in Wnt/β-catenin signaling members such as APC are frequently the classic driver event in more than 90% of CRC cases . The APCThe increase of genomic instability and the successive mutations in genes such as KRAS and SMADs trigger the formation of dysplastic tubular adenomas ,74,75. AThe constitutive activation of Wnt signaling triggered by the loss of APC confers mutant ISCs a clonal advantage, thereby enhancing the critical role of the Wnt signaling pathway in the development and progression of CRC ,80,81. IThese discoveries reinforce the importance of understanding the molecular mechanisms that implicate the Wnt pathway in the promotion and progression of CRC by the scientific community and draw attention to its potential for targeted therapies.Treatment of CRC depends on the staging and may include laparoscopic removal of polyps or surgical removal of damaged areas of the intestine ,84,85,86Although combination drugs for cancer treatment have improved overall survival compared to monotherapy, CRC recurrence remains the reality for approximately 40% of the patients after primary treated tumors . The steSeveral FDA-approved anticancer drugs are based on natural sources such as natural compounds. The alkaloids vincristine and vinblastine are used as first-line therapy for leukemia and lymphomas ,92, whilGenistein is one of the isoflavones present in soybean, and it inhibits the Wnt/β-catenin pathway through epigenetic modifications that positively regulate the expression of several Wnt antagonists. It has been demonstrated that treatment with genistein reduced methylation in CpG islands of the Wnt5a and sFRP2 genes, attenuating the nuclear translocation of β-catenin ,102. AddTussilago farfara is an inhibitor of the Wnt/β-catenin signaling pathway in a range between 10 to 30 µM, downregulating both cytoplasmic and nuclear β-catenin levels in HEK293T reporter cells stimulated by Wnt3a or a GSK3-β inhibitor. TSL promotes proteasome-mediated degradation of the β-catenin since the proteasome inhibitor MG132 recovers the TSL-mediated reduction of the cytosolic level of β-catenin. Additionally, TSL suppressed the proliferation of colon cancer cells via the downregulation of Wnt target genes, such as CCND1 (cyclin D1) and MYC (c-myc) and decreased the cell viability in the MTT assay [The natural compound tussilagone (TSL) isolated from the flower buds of TT assay .Lonicera japonica inhibits the Wnt/β-catenin signaling pathway in a dose-dependent manner at concentrations between 5 and 40 µM. Hydnocarpin promotes the upregulation of Axin1/2 and enhances the β-catenin phosphorylation at Ser33/37/Thr41 residues, leading to its subsequent ubiquitin-dependent proteasomal degradation. In addition, it inhibits the proliferation of human colon cancer cells SW480 in the sulforhodamine B (SRB) assay [Anemone raddeana that inhibits the Wnt/β-catenin signaling pathway by the activation of GSK3-β in a concentration less than 5 µM, thereby initiating β-catenin phosphorylation at Ser33/37/Thr41 residues and subsequently, its ubiquitin-dependent proteasomal degradation. Furthermore, raddeanin A reduces cell viability, inhibits cell proliferation, and induces apoptosis in SW480 and LoVo cells in a concentration-dependent manner at concentrations between 0.6 and 1.2 µM. Moreover, raddeanin A efficiently inhibited tumor growth in a xenograft mouse model by reducing the expression of the β-catenin protein and the Wnt-target genes CCND1 (Cyclin D1) and MYC (c-myc) with 2.5 and 5 mg/kg [The flavonolignan hydnocarpin isolated from B) assay . Raddean 5 mg/kg .Min/+ mice by modulating the localization of β-catenin in intestinal adenomatous cells and negatively regulates the expression of PCNA and the Wnt-target gene cyclin D1, reducing the number of intestinal polyps, besides promoting apoptosis by regulating pro-apoptotic proteins and inflammatory cytokines [Myricetin is a flavonoid found in citrus fruits, wild fruits, and grapes . Myricetytokines .Oroxylum indicum and medicinal plants of the genus Scutellaria [The flavone Oroxylin A is present in the tree tellaria . Oroxylitellaria .Luteolin inhibits the canonical Wnt pathway through GKS3 activity, promotes phosphorylation and the subsequent proteasomal degradation of β-catenin dose-dependently in a range between 25 to 100 µM ,111. TreOcimum sanctum. Yang et al. [Vicenin-2 is the 6,8-di-C-glucoside of apigenin that is widespread in the medicinal plant g et al.  showed tg et al. .Coptis and Berberis [CCND1 (cyclin D1) and MYC (c-myc) [Min/+ mice treated with 330 mg/kg three times per day. Berberine reduced the expression of Wnt-target genes Cyclin D1 and c-Myc in mice, in addition to decreasing the recurrence of polyps in seven patients with familial adenomatous polyposis by reducing Cyclin D1 expression [Berberine is an isoquinoline alkaloid of medicinal plants of the genus Berberis . Berberi (c-myc) ,118. Berpression ,118.Rhizoma copidis and has a similar chemical structure to berberine [Jatrorrhizine is a protoberberine alkaloid that is one of the main components of erberine ,159. Jaterberine .Sanguisorba officinalis, a popular Chinese herb called DiYu. TGG significantly inhibits the Wnt/β-catenin signaling pathway by downregulating β-catenin protein levels in a concentration range between 20 to 70 µg/mL. Moreover, TGG induces apoptosis by increasing the protein levels of Caspase-3, PARP and the Bax/Bcl-2 ratio in HT-29 cells [1,4,6-Tri-O-galloyl-β-d-glucopyranose (TGG) is a polyphenol isolated from 29 cells . Another29 cells .Curcuma longa, inhibits the Wnt/β-catenin signaling pathway in tumor cell lines and mouse models in a dose-dependent manner. Curcumin treatment reduces β-catenin nuclear translocation and decreases transcription of target genes such as CCND1 (cyclin D1), MYC (c-myc), and AXIN2 (Axin2) in a range between 4 to 32 µM [AXIN2 (Axin2), a known Wnt target gene, was also downregulated in the same mice.Curcumin, a curcuminoid produced by to 32 µM ,125. Curto 32 µM . Furtherto 32 µM ,124,160.Physalis peruviana that inhibits the Wnt/β-catenin signaling pathway by promoting the phosphorylation and degradation of β-catenin in a concentration less than 5 µM, which impairs its subsequent nuclear translocation and attenuates expression of the endogenous Wnt target genes in colon cancer cells. 4βHWE reduces cell viability and proliferation, induces G0/G1 cell cycle arrest, and promotes Caspase-dependent apoptosis of colon cancer cells. Moreover, 4βHWE dramatically inhibited tumor growth in HCT116 xenografts by promoting β-catenin degradation and downregulating protein levels of Axin2, c-Myc, and Cyclin D1 in mice treated with 5 mg/kg and 10 mg/kg/day for 14 days, suggesting its potential use as an antitumor agent that targets the Wnt/β-catenin signaling pathway [4Hydroxywithanolide E (4βHWE) is a naturally occurring small molecule from  pathway .The flavonoid epigallocatechin-3-gallate (EGCG) is the main component of green tea and reduces the activity of the Wnt/β-catenin pathway induced by the Wnt3a conditioned medium in a range between 20 to 80 µM and promotes phosphorylation of the Ser33/37 sites of β-catenin by a GSK3-independent mechanism . In addiAtractylodes macrocephala that suppresses the Wnt/β-catenin transcriptional activity stimulated by Wnt3a or GSK3-β inhibitors in HEK293T reporter cells treated with concentrations between 5 to 20 µg/mL. Thus, atractylochromene targets the β-catenin, impairing its galectin-3-mediated nuclear accumulation and inhibiting the proliferation of SW480 colon cancer cells [Atractylochromene is a polyphenol isolated from rhizomes of er cells .Silybum marianum. Silibinin supplementation at 50 mg/kg/day for 32 weeks reduces β-catenin levels and prevents the colorectal carcinogenesis induced in Wistar rats challenged with the xenobiotic 1,2-Dimethylhydrazine (DMH). Furthermore, silibinin inhibits adenocarcinoma proliferation by downregulating protein levels of β-catenin protein, Cyclin D1 and PCNA [Silibinin is the active compound present in silymarin extracted from the medicinal plant and PCNA ,131.Broussonetia papyrifera that accelerates the intracellular turnover of β-catenin, leading to its N-terminal phosphorylation at Ser33/37/Thr41 residues and subsequent ubiquitin-dependent proteasomal degradation in a range between 5 to 20 µM. BCA decreased the intracellular β-catenin levels in colorectal cancer cells HCT116 (β-catenin mutant) and SW480 (APC mutant), and the pharmacological inhibition of GSK3-β could not abrogate the BCA-mediated degradation of β-catenin, which suggests that its mechanism of action is downstream of the destruction complex. Moreover, BCA inhibits cell viability and induces apoptosis by a Caspase-dependent mechanism [Broussochalcone A (BCA) is a prenylated chalcone isolated from echanism .Apigenin is an abundant flavone in fruits and vegetables that inhibits the reporter activity of the Wnt/β-catenin pathway downstream of the destruction complex in concentrations between 10 to 80 µM . ApigeniHumulus lupulus that promotes the β-catenin degradation and inhibits cellular proliferation by inhibiting the Wnt/β-catenin signaling pathway activity in mice orally treated with 5 mg/kg of XHA for 8 weeks. Moreover, the administration of XHA suppresses tumor proliferation by downregulating the levels c-Myc, and Cyclin D1 and Ki-67 besides inducing Caspase-3-mediated apoptosis in AOM-induced Sprague-Dawley rats [Xanthohumol (XHA) is a natural polyphenol isolated from ley rats .Rabdoternin B and maoecrystal I, are two natural compounds from a natural ent-kauranoid library identified by a dual-luciferase reporter gene assay. Both compounds inhibit the Wnt/β-catenin signaling pathway in a concentration-dependent manner in HEK293T cells with an IC50 of 13.95 ± 0.66 and 10.58 ± 0.82 µM, respectively. Nevertheless, maoecrystal I did not affect β-catenin levels in SW480 cells, indicating that it inhibits Wnt signaling downstream of β -catenin degradation events. Rabdoternin B and maoecrystal I reduced the viability and inhibited the growth of SW480, HCT116, and HT-29 tumor cells treated with 20 or 40 µM, with only weak cytotoxicity towards the normal colonic epithelial cell line CCD-841-CoN, and arrested SW480 cells at the G2/M phase .Scutellaria genus that inhibits the Wnt/β-catenin pathway in a dose-dependent manner in HCT116 cells treated with concentrations between 10 to 40 µM. Wogonin promotes the reduction of cytoplasmic and nuclear β-catenin levels and downregulates the expression of transcription factors of the TCF/LEF1 family, thereby limiting the transcriptional capacity of the TCF/LEF complex. Wogonin reduces cell viability and proliferation, induces cell cycle arrest in G1, and reduces the growth of xenographic tumors by promoting β-catenin degradation and downregulating protein levels of c-Myc and Cyclin D1 in mice treated 30 and 60 mg/kg [Wogonin is a flavone present in medicinal plants of the 60 mg/kg .xWnt8 (Xenopus Wnt8 mRNA) and the dorsalization phenotype induced by lithium chloride (LiCl) in Xenopus laevis embryos. In addition, isoquercitrin reduces the viability, proliferation, and migration of tumor cell lines HCT116, SW480, and DLD-1 without affecting the non-tumor cell line IEC-18 [Isoquercitrin, a flavonol derived from quercetin, inhibits the activity of the reporter gene for the Wnt/β-catenin pathway in a concentration-dependent manner, promoting β-catenin degradation, and preventing its translocation to the nucleus in a range between 75 to 150 µM. As a result, isoquercitrin inhibits the formation of the ectopic axis induced by e IEC-18 .Taxifolin is a flavonoid abundant in olive oil, onions, grapes, and various citrus fruits. Taxifolin interacts with β-catenin and reduces its total levels, thereby modulating the canonical Wnt signaling in a concentration-dependent manner in concentrations between 20 to 60 µM ,141. In xWnt8 DNA in embryos of X. laevis, reinforcing their inhibitor properties in a well-established canonical Wnt pathway model [The chalcones derricin and derricidin inhibit the Wnt/β-catenin signaling pathway with different mechanisms . Both inay model . Furtheray model .Antofine is an alkaloid phenanthroindolizidine present in several plants of the Asclepiadaceae family . AntofinBisleuconothine A is an alkaloid bisindole that promotes β-catenin degradation, preventing its nuclear translocation and reducing the activity of the Wnt/β-catenin signaling pathway. Bisleuconothine A reduces the transcriptional activity of the Wnt canonical pathway in a concentration-dependent manner in HCT116 and SW480 cells and induces β-catenin phosphorylation impairing its nuclear levels after treatments with concentrations between 1 to 10 µM. Furthermore, Bisleuconothine A inhibits cell proliferation by arresting the cell cycle in G0/G1 and inducing Caspase-dependent apoptosis and suppresses the growth of xenotransplanted tumors in mice, promoting the β-catenin degradation and downregulating protein levels of c-Myc and Cyclin D1 in mice treated with 2 mg/kg for 2 weeks .Glycosmis stenocarpa that inhibits Wnt/β-catenin signaling pathway according to its concentration. Murrayafoline A inhibits the pathway activated by Wnt3a conditioned medium or LiCl treatment in HEK293T cells treated with concentrations between 5 to 40 µM and promotes β-catenin degradation by a proteasome-dependent mechanism. Furthermore, murrayafoline A down-regulates the expression of c-Myc and Cyclin D1 and reduces the viability and proliferation of human colon tumor lines [Murrayafoline A is a carbazole alkaloid isolated from or lines .Stephania tetrandra roots, inhibits the Wnt/β-catenin pathway reporter gene activity in LoVo and HCT116 cells treated with concentrations between 2.5 and 10 µM [MYC (c-myc), a target gene of the Wnt canonical pathway, inhibits cell proliferation and migration, in addition to inducing Caspase-dependent apoptosis. Tetrandrine also prevents tumor growth of xenotransplanted tumors in rats treated intraperitoneally with 60 mg/kg once every 2 days [Tetrandrine, a bisbenzylisoquinoline alkaloid extracted from nd 10 µM ,164. Tety 2 days  and DMH-Magnolia obovata that suppresses the Wnt/β-catenin transcriptional activity stimulated by Wnt3a in HEK293T reporter cells. Mechanistically, magnolol inhibits the nuclear translocation of β-catenin and significantly suppresses the binding of β-catenin/TCF complexes onto their specific DNA-binding sites in the nucleus. Magnolol also effectively inhibits the proliferation of both SW480 and HCT116 cells in a concentration-dependent manner and suppresses the growth and invasion of HCT116 xenografts in a nude mouse model [Magnolol is a neolignan from the cortex of se model .Resveratrol is a flavonoid with several biological properties. It is present in peanuts and wild fruits and is abundant in grapes and their derivatives, such as red wine. Resveratrol inhibits the Wnt/β-catenin pathway in a dose-dependent manner, preventing the interaction between β-catenin/TCF4 and promoting proteasomal degradation of TCF4 in a range between 10 to 100 µM ,151. ResX. laevis embryos. Furthermore, lonchocarpin inhibits viability, proliferation, and migration in human colorectal tumor lines, in addition to reducing tumor proliferation in mice submitted to the AOM/DSS protocol [Lonchocarpin is a chalcone that inhibits the Wnt/β-catenin pathway with an IC50 of 4 µM. Lonchocarpin reduces the nuclear level of β-catenin and inhibits dnTCF4 VP16-induced activation in HEK293T cells, suggesting that its mechanism of action involves disturbing the interaction between β-catenin and TCF/LEF transcription factors. In functional assays, lonchocarpin disturbed the axial patterning and reversed the Wnt8-induced ectopic axes in protocol .Piper nigrum) and long pepper (Piper longum) that inhibits the transcriptional machinery of the Wnt/β-catenin pathway at 34 µM in SW480 β-catenin reporter gene cells and reduces the nuclear localization of β-catenin in HCT116 cells. Piperine impairs colorectal cancer progression by decreasing cell viability, proliferation, and migration in human tumor lines HCT116, SW480, and DLD-1. Still, its effects are attenuated both in RKO cells, a colorectal carcinoma lineage that lacks an overactive Wnt pathway, and HEK293T CTNNB1 (gene that encodes β-catenin) knockout cells, reinforcing its role as a selective inhibitor of the canonical Wnt pathway [Piperine is an alkaloid responsible for the spicy taste of black pepper  is a natural small-molecule derivative of coumarin identified as a potential inhibitor of the Wnt/β-catenin signaling pathway in concentrations between 20 to 80 µM. The interaction between esculetin and β-catenin inhibits the formation of the β-catenin-TCF complex, suppressing the Wnt/β-catenin transcriptional activity. Moreover, esculetin effectively decreased the cell viability and inhibited tumor growth of HCT115, HCT116, and DLD-1 cells, and impaired tumor growth in a colon cancer xenograft mouse model by reducing protein levels of Cyclin D1, c-Myc and Ki-67 .The carnosic acid from rosemary attenuates the transcriptional activity of the Wnt/β-catenin pathway in colorectal cancer cells with concentrations between 2.5 to 25 µM. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation show that the carnosic acid response requires an amino-terminal intrinsically labile α-helix (H1) next to the BCL9-binding site in β-catenin. Carnosic acid targets predominantly the oncogenic form of β-catenin for proteasomal degradation in an H1-dependent manner, providing a new strategy for developing target inhibitors of oncogenic β-catenin .Cinnamomum cassia, significantly inhibited the Wnt/β-catenin signaling pathway in HEK293T cells and HCT116 colorectal cancer cells. Mechanistically, phenylpropanoid 1 suppresses the β-catenin/TCF binding complex into the nucleus and down-regulates Wnt target genes such as MYC (c-myc) and CCND1 (cyclin D1). Furthermore, the compound inhibits tumor cells proliferation in the athymic xenograft mice model, without any apparent toxicity yet suppressing the expression of Wnt target genes associated with tumor growth, including MYC (c-myc), CCND1 (cyclin D1), and BIRC5 (survivin) [The phenylpropanoid compound 2-Hydroxycinnamaldehyde (phenylpropanoid 1) isolated from the bark of urvivin) .The Wnt canonical signaling pathway plays a prime role in regulating the levels of the β-catenin protein and is vital for the embryonic axes patterning and maintaining stem cells from different tissues ,31. It iCRC ranks second in the number of cancer deaths worldwide, mainly due to late diagnoses, restricting treatment in the potentially curable stages, and decreasing patient survival ,11,12,13Myofibroblasts, cancer-associated fibroblasts (CAFs), and CSCs secrete a range of growth factors and signaling proteins such as the agonist Wnt3a and the antagonist Notum in the TME ,165, botMYC (c-myc),AXIN2 (Axin2), and CCND1 (cyclin D1) are commonly linked to CRC development and are considered targets of the canonical Wnt pathway, in which APC is a prime regulator [The proliferative-related genes egulator . Most APegulator ,70. Theregulator . Moreoveegulator .Despite promising advances in the use of Wnt/β-catenin signaling modulators in experimental therapies against CRC, no molecules of natural or synthetic origin are clinically available . The priCombination therapy is a good strategy to overcome tumor resistance, and Wnt/β-catenin inhibitors have been shown to improve tumor sensitivity to 5-FU and oxaliplatin, which are standard drugs in regimens such as FOLFOX, used by CRC patients in stages III and IV ,25,173. In this review, we evaluated the use of small molecules of natural origin to inhibit the Wnt/β-catenin signaling pathway in experimental models of CRC and identified several molecules with high inhibitory potential. However, some papers had inconclusive information from the mechanistic perspective, which might be related to the incompatible or improper experimental design to address mechanistic questions. It is, therefore, necessary to overcome these issues with more suitable experimental models that enable a reliable characterization of their mechanisms of action and potential application in further preclinic studies.So far, we have reviewed dozens of promising naturally occurring small molecules that differently inhibit the canonical Wnt signaling pathway. Considering the mechanisms of action of each compound and the dysregulation of Wnt signaling in the different colorectal cancer types, novel pre-clinical experiments should be performed to validate and perhaps allow these small molecules to move off the laboratory bench onto the clinical platform.


Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) and semi-synthetic derivatives thereof  for their effects on Wnt/β-catenin signaling. None of these substances revealed major cytotoxicity on STF293 embryonic kidney and HCT116 colon carcinoma cells at concentrations up to 10 μM. At this concentration, HF and HM 177 showed the strongest effect on cell proliferation, whereas MC A and HM 177 most prominently inhibited anchorage-independent growth of HCT116 cells. Western blot analyses of active β-catenin and β-catenin/TCF reporter gene assays in STF293 cells revealed inhibitory activities of HF, MC A and HM 177. In line with this, the expression of endogenous Wnt target genes, Axin and Sp5, in HCT116 cells was significantly reduced. Our data suggest that the acylphloroglucinols hyperforin, myrtucommulone A and its derivative HM 177 represent potential new therapeutic agents to inhibit Wnt/β-catenin signaling in colon cancer.The therapeutic activities of natural plant extracts have been well known for centuries. Many of them, in addition to antiviral and antibiotic effects, turned out to have anti-tumor activities by targeting different signaling pathways. The canonical Wnt pathway represents a major tumorigenic pathway deregulated in numerous tumor entities, including colon cancer. Here, we investigated the acylphloroglucinols hyperforin (HF) from St. John’s wort ( Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) have attracted a lot of attention in respect to their broad spectrum of pharmacological effects. However, the molecular mechanisms behind these effects are currently not completely understood.Extracts from medical plants and herbs have a long history in traditional medicine, and a significant part of the world population relies on the therapeutic and preventive effects of plant-derived substances for health care. Up to now, many of the physiologically active compounds within plant extracts have been unknown, but interest in these compounds is constantly rising. With improvement in separation tools and analytical methods, the number of known active substances and their chemical structure is growing. Subsequent successful chemical synthesis allows for obtaining sufficient amounts of the substances of interest for detailed studies on their biological activities. Among these plant-derived substances, hyperforin (HF) from St. John’s wort (SJW)  and MC A (CAS number: 54247-21-1) represent acylphloroglucinol derivatives . Inthase-1 ,14. As anthase-1  and refe in vivo ,17,18,19 in vivo ,23,24,25 in vivo ,27, and  in vivo .Oral bioavailability of HF and MC A suggests that the intestinal epithelium is the primary site of uptake. Thus, the anti-cancer and anti-inflammatory actions of these compounds should also be active in the gastrointestinal tract. Interestingly, Satia et al. reported that continuous use of SJW extract reduces the risk of colon cancer by 65% . In lineIt is well known that the canonical Wnt signaling pathway plays a central role during colorectal carcinogenesis. In up to 80% of spontaneous colorectal cancer cases, mutations of the adenomatous polyposis coli (APC) tumor suppressor gene induce an activation of the Wnt pathway. In consequence, β-catenin accumulates in the cytosol and nucleus and activates transcription of target genes, driving tumorigenesis. In healthy cells, excess β-catenin is rapidly phosphorylated within the β-catenin destruction complex composed of APC, Axin, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β). Both kinases phosphorylate β-catenin at Ser/Thr residues within its N-terminus, marking it for ubiquitination by the β-TRCP (β-transducin repeat containing E3 ubiquitin protein ligase) complex and subsequent proteasomal degradation . This mHere, we address the question of whether HF and MC A and derivatives HM 177, HM 297 and HM 298 thereof are able to attenuate Wnt/β-catenin signaling in Wnt3a-stimulated HEK-293 cells and HCT116 colon cancer cells. MC A derivatives HM 177, HM 297 and HM 298 were chosen from our previous structure–activity relationship evaluation that identified HM 177 and HM 297 as potent MC A derivatives and as suitable tools for mechanistic studies on intrinsic mitochondrial pathways, where HM 298 served as negative control for HM 297 ,21,39. WTo define the concentration range that can be applied for subsequent functional studies of HF, MC A and its derivatives HM 177, HM 297 and HM 298  viability assays quantifying metabolically active cells, concentrations up to 3 μM of HF, MC A, HM 177 and HM 297 did not show major reduction in cell viability in STF293 cells. HF, MC A and HM 177 affected viability at 10 μM, whereas HM 297 affected viability at 30 μM. HM 298 did not exhibit any effects up to 100 µM a. Similav/v) DMSO vehicle control. At the highest tested concentration of 30 μM, all analyzed compounds  stopped cell proliferation. It cannot be excluded that cytotoxic effects contributed to this fact. At a concentration of 10 μM, the strongest reduction in cell proliferation was detected after addition of HF and HM 177. MC A revealed a clearly weaker anti-proliferative effect, and HM 297 also exhibited only minor activity  and anti-GAPDH as loading controls a. As quaanalyzed . Taken tHypericum perforatum L. (SJW) and myrtucommulone A (MC A) from Myrtus communis have attracted attention. Specifically, HF, which is considered an active ingredient of SJW for the therapy of mild and moderate depressive disorders [In ancient medicine, plant-derived extracts played an essential role to treat infections, wounds, metabolic disorders and other diseases. In recent years, more and more pharmacological active compounds of natural origin were identified, specifically from plant extracts. Among these, hyperforin (HF) isolated from isorders , was putIn this study, we used STF293 and HCT116 cells to investigate whether the anti-tumor activity of HF, as well as MC A and its derivatives HM 177 and HM 297, can be attributed to an inhibition of canonical Wnt/β-catenin signaling, an oncogenic pathway contributing to tumor formation in many different tissues, e.g., colon cancer. In most cases, the pathway is inappropriately activated by mutations of pathway components  or of modulators thereof , resulting in enhanced transcription of Wnt target genes mediated by the β-catenin/TCF transcription complex .At low concentrations of 1 and 3 μM, all tested substances did not affect the viability of both STF293, Wnt3a-treated STF293 and HCT116 cells in FDA assays. In respect to cytotoxicity, concentrations up to 10 μM did not show major effect with HM 177, revealing minor cytotoxic effects in HCT116 cells. When we analyzed growth behavior by real time impedance measurements, using the xCELLigence system, HF and HM 177 revealed a strong inhibitory effect at 10 μM, whereas MC A was less effective, and HM 297 did not show a major difference. At a concentration of 30 μM, a strong growth inhibitory effect was detectable for all four substances. Interestingly, in anchorage-independent growth assays, representing a more physiological situation, HM 177 already induced a significant reduction in colony formation at a concentration of 1 μM. MC A also showed a clear growth inhibitory activity, however weaker than HM 177. Surprisingly, HF, in this type of assay, only showed a minor effect at a concentration of 10 μM, whereas in most other assays, HF belonged to the most active compounds and already showed significant effects at lower concentrations. We currently do not know what causes this difference. We hypothesize that anchorage-independent growth is regulated by signaling pathways that may be affected by HF only in an indirect way.Axin2 and SP5. The strongest effects were obvious in response to HF and HM 177 treatment, leading to a 75% reduction in both target genes. Furthermore, the levels of active β-catenin were significantly reduced by HF, MC A and HM177, although a reduction in total β-catenin was also detectable. This, again, indicates that these substances have Wnt inhibitory activities.When testing the substances for their Wnt inhibitory activity, Wnt reporter gene assays in STF293 cells carrying a stably integrated Wnt reporter construct revealed that HF, MC A and HM 177 indeed repress expression of the luciferase reporter gene, both in unstimulated and Wnt3a-stimulated cells. This repressive activity was also detectable in HCT116 cells when we analyzed the expression of the endogenous Wnt target genes 50 value of 2.5 μM [2 synthase-1 activity [2, in binding to its receptor EP2, activates stimulatory G proteins. The activated Gαs subunits release GSK-3β from Axin within the β-catenin degradation complex, thereby abrogating β-catenin degradation. In addition, Gβγ subunits stimulate PI3K and Akt, leading to inhibitory phosphorylation of GSK-3β, again, resulting in β-catenin stabilization [2 synthase-1 by HF and MC A may contribute to a down-regulation of β-catenin signaling activity.Future studies have to unravel whether HF, MC A and its derivative HM 177 directly or indirectly modulate the canonical Wnt signaling pathway in colon cancer cells. Based on previous reports in the literature, Akt1 kinase might play a role. In this respect, Merhi et al. showed that HF directly inhibits Akt1 kinase activity in AML cells with an ICf 2.5 μM , a concef 2.5 μM . In thisf 2.5 μM , may conactivity ,19. It hlization . Thus, tTaken together, this study shows that HF, MC A and its derivative HM 177 reduce HCT116 colon cancer cell proliferation and anchorage-independent growth. Moreover, our results suggest that these effects are at least in part mediated by an inhibition of the canonical Wnt signaling pathway. Further studies are needed to define the potential crosstalk with other signaling pathways and to determine whether HF and MCs are also able to inhibit the Wnt pathway in vivo. In this respect, the encapsulation of HF into acetalated dextran polymeric nanoparticles may improve its uptake and thus bioactivity, due to reduced plasma protein binding, as shown recently for neutrophils .MC A and its derivatives HM 177, HM 297 and HM 298 were designed, synthesized and characterized by Dr. Hans Müller and Prof. Dr. Johann Jauch, Organic Chemistry II, Saarland University, Germany, as described previously ,54 and rv/v) fetal calf serum  and 1% (v/v) Pen/Strep solution  at 37 °C and 5% CO2. STF293 cells were kept under selective conditions with 0.1 mg/mL G418 . Wnt3a-conditioned medium and control medium were produced by Wnt3a-transfected and empty vector-transfected L-M-(TK-) cells, respectively, as described in [HCT116 colon carcinoma and STF293 cells  cells stably transfected with the SuperTop-Flash Wnt-reporter containing seven LEF/TCF consensus binding sites driving firefly luciferase expression)  were groribed in .4/well). After 24 h, fresh medium was added. Five hours later, cells were treated with the different compounds at different concentrations, as indicated. After 20 h, cells were washed twice with PBS and subsequently incubated with 200 µL fluorescein diacetate (FDA)  at a concentration of 10 μg/mL in PBS for 30 min at 37 °C. Released fluorescein was quantified with a Mithras LB940 microplate reader . Cytotoxicity of compounds was analyzed with the Cytotoxicity Detection KitPLUS , according to the manufacturer’s instruction, by measuring the release of lactate-dehydrogenase in response to damage of membrane integrity.HCT116 and STF293 cells were seeded in 96-well plates  as described previously . Brieflyw/v) agar in phenol red-free DMEM. After 24 h incubation at 37 °C and 5% CO2, cells were treated with the indicated concentrations of the compounds diluted in 2 mL phenol red-free DMEM. Cells were grown for 21 days in the presence of compounds until single colonies were formed. After this incubation, colonies were fixed with ice-cold methanol for 60 min at −20 °C and stained with a 0.005% (w/v) crystal violet solution in 25% (v/v) methanol at room temperature. After destaining with repeated changes in Bidest. water, colonies were quantified with the Clono-Counter software [Anchorage-independent growth of HCT116 cells was analyzed by colony formation in soft agar as described previously . Assays software . Data we4 cells/well and left for 17 h for attachment before cells were pre-treated with Wnt3a/control-conditioned medium for 5 h. After 24 h treatment with the different compounds in the indicated concentrations, with DMSO as a vehicle control, cells were lysed in PPBT-buffer  Triton X-100), and luciferase activities were measured using a Mithras LB940 microplate reader  as reported previously [STF293 cells were seeded into 96-well plates at a density of 1 × 10eviously .5 STF293 cells per well of a 12-well plate were seeded. After 24 h, medium was changed, and half of the medium volume was replaced by Wnt3a-conditioned or control-conditioned medium. Another 6 h later, cells were treated for 20 h with 3 μM HF, MC A, HM 177, HM 297 or DMSO (0.1% (v/v)) as a control. Subsequently, cells were lysed in lysis buffer  Triton X-100). After centrifugation , to remove insoluble material, cell lysates were applied to SDS-PAGE and subsequently transferred to PVDF-membranes for detection of total β-catenin with anti-β-catenin  antibody  and active β-catenin with anti-active-β-catenin  antibody . Anti-cyclinD1  antibody was obtained from Abcam , anti-c-myc  from Santa Cruz Biotechnology , and anti-GAPDH (1:4.000) antibody from Merck Millipore . HRP-labeled goat anti-mouse secondary antibodies were purchased from Dianova  and used in a dilution of 1:10.000 in TST-buffer  Tween 20). Active β-catenin signals were normalized to the GAPDH signal as a loading control. Chemiluminescence signals were detected using a G-Box imager  and quantified with AIDA software .To analyze the expression levels of total β-catenin and active β-catenin, 8 × 10Axin2 and SP5, 1 × 106 HCT116 cells/well were seeded in 6-well plates. Medium was changed 24 h after seeding, and after 5 h, compounds were added at a concentration of 3 μM for 20 h. RNA was isolated using Nucleospin RNA II isolation kit . Reverse transcription was performed using 1 μg total RNA applying the High-Capacity cDNA Reverse Transcription Kit . Quantitative PCR was performed on StepOnePlus Real-Time PCR System  using UPL probes and primers: Axin2 UPL#60  with left primer 5′-CAAGCCTGGCTCCAGAAG-3′ and right primer 5′-GCATCCTCCGGRARGGAAT-3′; SP5 UPL#36 (cat. No. 04687949001) with left primer 5′-GGGGAGACTCAGCAGACG-3′ and right primer 5′-TGGGTCCCTATGTCCGAAG-3′; GAPDH UPL#60 (cat. No. 04688589001) with left primer 5′-AGCCACATCGCTCAGACAC-3′ and right primer 5′-GCCCAATACGACCAAATCC-3′. Signals for Wnt target genes were normalized to GAPDH. Assays were performed in duplicate from 3 independent RNA isolations.For analysis of the expression of endogenous Wnt target genes t-test (unless explicitly indicated otherwise in text/legends). In the case that a data set contained at least one non-normally distributed group, all comparisons were performed by the Mann–Whitney Rank sum test, which is indicated in the accompanying figure legends. For all tests, the significance criterion p < 0.05 was used. All numbers of replications (n) in figure legends refer to biological replicates.Statistical analyses of all experiments and the creation of diagrams were performed with SigmaPlot 14.5 software . Data are presented as means ± SEM. Initially, normal distribution of values was tested by the method of Shapiro–Wilk. Normally distributed values were compared to another group by the two-tailed Student’s


To achieve this improvement, a diluent, the diglycidyl ether of bisphenol F (DGEBF or BPF), was added to TGPAP, and the blended epoxy was then cured with 4, 4′-diaminodiphenyl sulfones (DDS). A response surface methodology (RSM) was used, with the target response being to achieve a blended resin with a high glass transition temperature (Tg) and maximum pot life . Characterization through dynamic mechanical thermal analysis (DMTA) and using a rheometer indicated that the optimum formulation was obtained at 55.6 wt.% of BPF and a stoichiometric ratio of 0.60. Both values were predicted to give Tg at 180 °C and a processing window of up to 136.1 min. The predicted values were verified, with the obtained Tg and processing window (PW) being 181.2 ± 0.8 °C and 140 min, respectively, which is close to the values predicted using the RSM.The aim of this work was to improve the processability of triglycidyl- Despite showing good properties, this type of liquid epoxy resin has, however, high viscosity, which makes the liquid processing  difficult. As a consequence, blending this type of epoxy resin with another diluent is seen as an option to ease the processing and handling, for example during composite manufacturing . Thi. Thi6]. Epoxy resins cure by crosslinking via chemical reaction to form three dimensional networks. In order to crosslink, epoxy resins typically need to react with a curing agent (or hardener), most commonly a diamine . Figure Typically, the formulation of an epoxy blend is studied by the random selection of ratios between an epoxy and the diluent . In somex1 and x2 are independent factors, while y is the response that depends on both factors. The above relation can be read as the dependent factor y being a function of x1, x2, and experimental error, denoted as e. Error, e, represents the noise or any measurement error not counted in f. If the response is correlated with variables using a linear function, the function is a first-order model [A better approach in dealing with several factors (or variables) is running the experimental work through a factorial experiment. This approach is in contrast with the classical method, in that factors are varied together, instead of one at a time. Response surface methodology (RSM) is a design of experiment (DoE) approach and is a useful method that uses mathematical and statistical techniques in which a response is affected by factors (or variables). Through RSM, the interaction between factors can be analyzed and an optimization of response can be determined . The reler model ,20, someIf between these two factors, an interaction exists, that interaction can be added to the function. This will beIf the response surface produces a curvature, a higher degree of polynomial is used. This function is known as a second-order model. For two independent factors, the function can be expressed asPreviously, Guo et al.  added thg with minimum viscosity, and (2) to achieve the maximum pot life  of the epoxy blend. Despite there being a few papers reporting work related to epoxy blending, to the best of our knowledge, pot life was rarely discussed, and optimization using a design of experiment (DoE) approach for the blending of epoxy resin has also not been reported to date. The blended epoxy resin in this work is expected to offer the desired performance once cured and facilitate composite processing in later work, where it will be presented in a forthcoming paper.Herein, we aim to demonstrate the workability of TGPAP-BPF-DDS amine mixtures and how these effects are simplified by incorporating the response surface methodology (RSM) approach as the DoE. The requirements of this work were: (1) to find an epoxy blend at high Tp-aminophenol (TGPAP) and diglycidyl ether of bisphenol F (BPF). In this work, BPF is also denoted as DGEBF. The chemical structures for both resins are shown in The epoxy resins used in this study were triglycidyl-The type of hardener used to cure the blended epoxies in this work was 4,4′-diaminodiphenyl sulfone (DDS). DDS exists in the form of a white powder and is suitable for aerospace engineering applications. The chemical structure of this hardener is shown in TGPAP is the weight percentage of TGPAP, and wtBPF is the weight percentage of BPF (or DGEBF) [Blending of epoxy resin was carried out using a mechanical stirrer at 2000 rpm for 30 min in a silicone oil bath, which was heated to around 100 °C. This setup is drawn schematically in r DGEBF) .(5)Stoi® software version 7.1. An efficient design tool, known as central composite design (CCD), was chosen for this study.In this work, two factors were studied. The first was the BPF content (wt.%) in the epoxy blend (factor A). The second (factor B) was the stoichiometric ratio (g/g) between the amine hardener and epoxy. A DoE approach was carried out using a RSM assisted by Design Expertk, was 2, namely the BPF content in the epoxy blend (A) and the stoichiometric ratio of amine to epoxy groups (B). The CCD used had a core two-level factorial design, in which one level is referred to as a low level  and the other as the high level  of each variable. The number of experiments required for this CCD was calculated as 2k for the two-level factorial cube points, 2k for the axial or star points , and several replication samples at the center of the two-level experimental volume, to allow estimation of the curvature of standard error. It is recommended to have 3 or 5 replicates at the center of the experimental region [2 + 22 + 3 = 11. In this work, the number of variables (or factors), l region . In thisEach point for an actual value, as indicated in From From From the trigonometry calculation, the actual value for the cube point at point 7 with a coded value  was , and the values for the other 3 cube points  were determined using the same calculation. The actual values for the 3 replication points  with a coded point  in CCD were determined as the middle value in a range from minimum to maximum of the factor in actual values. For example, the actual value for the replication points for factor 1 (A) is 50%, when the range is 0% to 100% BPF of BPF/TGPAP content. These 11 values are listed in g and PW (pot life). For curing, blended epoxy was poured into a flexible mold made from polytetrafluoroethylene (PTFE)-coated fabric and degassed in a vacuum oven to eliminate trapped air at 100 °C for ~30 min. The degassed epoxy was then put into a circulating-air oven and heated following a cure cycle of 2 h at 130 °C, 2 h at 160 °C, and 2 h at 200 °C, followed by a post cure for 5 h at 200 °C. Tg was measured using a dynamic mechanical thermal analyzer (DMTA), where the Tg was determined from the onset of storage modulus curves. DMTA was performed using a Perkin Elmer 8000 from Perkin Elmer Ltd., Beaconsfield, United Kingdom, with a 3-point bending mode at a frequency of 1 Hz. Rectangular specimens (40 mm × 10 mm × 3 mm) ± 1 mm were cut using a Benetec sliding cutter. Specimens were heated from 30 °C to 300 °C at a rate of 5 °C/min. The curves of E′, E″, and tan δ were displayed as a function of temperature using Pyris software (Perkin Elmer). Three repetitions (n = 3) were performed for each run of experiments, to ensure reproducibility.The response from the input factors in this work was TThe PW of the epoxy blends was defined as the length of time for which the epoxy can retain a viscosity low enough for it to be processed. In this work, the PW was determined using rheometer and defined as the time for the epoxy blend to reach a viscosity of 100 Pa s, as stated in ASTM D 4473-95 . The rheg and PW, based on BPF content in the epoxy blend and stoichiometric ratio.The presented values shown in g was determined from the onset of the storage modulus curve. These Tg values are recorded in g was determined is presented in g is shown in g increases as the amount of TGPAP epoxy increases in the epoxy blend (factor A). For example, at a fixed stoichiometry ratio of 0.42, Tg increased by 336% when the TGPAP increased from 12.5% to 95% in the epoxy blend , the Txy blend b. Howevee groups . Thus, ae volume .g with a variation in stoichiometry ratio (r). They studied the effect of varying the amount of hardener added to DGEBA and showed that the highest Tg was obtained at 30 parts per hundred (pph), beyond which the Tg reduced. They mentioned that using the wrong stoichiometry ratio of curing agent (either more or less) caused the final structure of cured composites to have a lower crosslinking density, therefore lowering the Tg. Guerrero et al. [g for epoxy resin cured at a different stoichiometry ratio, r, which ranged from 0.3 to 1.0. They found the maximum Tg was obtained when r = 0.8–0.9, and decreased thereafter. The following equations are the final empirical regression models generated by the Design Expert® software for Tg in both coded and actual factors. Note that in the equation, BPF is the short term used to represent the weight of BPF in the total weight in the epoxy blend.This finding is in agreement with the study by Palmese and Mccullough , who repo et al.  also repg) can be identified by observing each factor coefficient. The coefficient as indicated in Equation (8) for the coded factor represents the expected change in response 1 (Tg) per unit change of each factor (A and B). For Equation (8) of the coded factor, A and B are the main effects, AB is the two-level interaction effect, and A2 and B2 are the second order effects. According to the regression model generated using Design Expert® software, the two-level interaction between BPF content in the epoxy blend (A) and stoichiometry (B) is the most significant factor associated with the Tg. This interaction produces the highest value of coefficient, of +14.39 of the coded value [g) for given actual values of each factor (A and B). This means that the value for each factor for Equation (9) should be specified in the actual values. The response (Tg) and factors (A and B), as indicated in the 3D plot and contour plot in The relative impact of each factor (A and B) to response 1  and the second-order effect are also significant. The coefficient of determination R2 value was high, close to 1, which is desirable. Adequate precision measures the signal-to-noise ratio, and a ratio greater than four is desirable [As mentioned previously, different amounts of TGPAP in the epoxy blend affect the Tn the Tg . Both faesirable ,34. For esirable .In this work, the PW was defined as the length of time until the viscosity of an epoxy blend increases and reaches 100 Pa s. Again, Small increases in PW are observed towards the end of the area in the top-left corner of the response plot in ® software for PW in both coded and actual factors:The following equations are the final empirical regression models generated by the Design Expertg) applies here for the explanation of Equations (10) and (11) for response 2 (PW).From the regression model, the most significant factor is the level of BPF in the epoxy blend at a coefficient of +18.87. The same explanation for Equations (8) and (9) of coded and actual factors for response 1  for the PW is shown in analysis .g and PW. It has been reported that for structural aerospace applications polymer composites should have a Tg value of at least 180 °C, due to high temperature applications during service [g was set to a range between 180 and 185 °C for the optimization. Finally, the PW was set to be “maximize”, as the epoxy resin system needs to be processed before it starts to cure and its viscosity increases significantly. The importance column , was determined in this work using RSM. The T


Janus wettability plays an important role in certain special occasions. In this study, field emission scanning electron microscopy (FESEM) was used to observe the surface microstructure of banana leaves, the static wettability of the banana leaf surface was tested, and the dynamic response of water droplets falling at different heights and hitting on the adaxial and abaxial sides was studied. The study found that the nanopillars on the adaxial and abaxial sides of the banana leaf were different in shape. The nanopillars on the adaxial side were cone-shaped with large gaps, showing hydrophilicity (Wenzel state), and the heads of the nanopillars on the abaxial side were smooth and spherical with small gaps, showing weak hydrophobicity (Cassie–Baxter state). Banana leaves show Janus wettability, and the banana leaf surface has high adhesion properties. During the dynamic impact test, the adaxial and abaxial sides of the banana leaves showed different dynamic responses, and the wettability of the adaxial side of the banana leaves was always stronger than the abaxial side. Based on the structural parameters of nanopillars on the surface of the banana leaf and the classical wetting theory model, an ideal geometric model around a single nanopillar on both sides of the banana leaf was established. The results show that the established model has high accuracy and can reflect the experimental results effectively. When the apparent contact angle was 76.17°, and the intrinsic contact angle was 81.17° on the adaxial side of the banana leaf, steady hydrophilicity was shown. The abaxial side was similar. The underlying mechanism of Janus wettability on the banana leaf surface was elucidated. This study provides an important reference for the preparation of Janus wettability bionic surfaces and the efficient and high-quality management of banana orchards. Opuntia) show age-dependent wetting properties [With the evolution of biology, a specific structure has been formed on the surface of animals and plants to adapt to the living environment, which has also become one of the hotspots of biologically efficient production management and bionics research. For example, the surface of rice leaves and butterfly wings show anisotropy ,2; the soperties ; the suroperties , and theoperties ,6. The “operties ,8,9.Janus wettability was an important wetting property discovered in recent years, and extensive research has been carried out ,11,12. IAs an important biological template, banana leaves have significant properties, such as self-cleaning  and enhaThe main work of this research is to reveal the wetting mechanism of the banana leaf surface, study the effect of water droplets on the dynamic wettability of the banana leaf surface, and clarify the Janus wettability transition from the Wenzel state to the Cassie-Baxter state and its underlying mechanism. This is of great significance for the bionic application on the surface of banana leaves and the efficient and high-quality production and management of banana orchards, especially for the guidance of pesticide spraying.Fresh banana leaves (plantain banana) that were in good health, free from pests, diseases, and mechanical damage were picked from South China Agricultural University, and the parts between the veins and edges of the banana leaves were selected as test samples. In order to ensure the cleanliness of the banana leaf surface and the accuracy of the experimental results, the test samples were taken from a banana plant aged between 7 and 9 months, and take the third healthy banana leaf from top to bottom. Deionized water was then used to ultrasonically clean the banana leaves for 60 s  to remove the physical attachments on the surface of the banana leaves.Using a contact angle measuring instrument  to measure the water contact angles (CAs) and rolling angles  of 6 μL droplets on the adaxial and abaxial sides of the banana leaves, they were measured at three different positions to obtain the average value. At the same time, the ultrasonically cleaned banana leaves were vacuum dried in a vacuum drying oven at 40 °C, then cut into 5 mm × 5 mm size, and gold sprayed through vacuum sputtering to make the sample conductive. Field emission scanning electron microscopy  was used to observe the microstructure of the adaxial and abaxial sides of the banana leaves. During the FESEM imaging process, the banana leaves were first pasted on the sample holder with conductive glue, and then sprayed gold with vacuum sputtering and observed under scanning electron microscopy. The detector type was SE, the electron beam intensity was 1.00 kV, and the working distance was 4.1 mm.V = 8 μL , and the surface tension of water was γ = 0.072 N·m−1. Water droplets fell freely from different heights (h) and impacted the surface of the banana leaf sample. Under the action of gravity, an impact velocity (v) was obtained, and v can be expressed as the following equation:g is the acceleration of gravity (9.8 m·s−2). Water droplets dropped from heights of 20 mm, 50 mm, 100 mm, 150 mm, and 200 mm to impact the banana leaf surface, and a high-speed camera  was used to record the impact process of water droplets on the banana leaf surface at a speed of 2000 fps, so as to test the dynamic wettability of the water droplets on the adaxial and abaxial sides of the banana leaf.The self-made dynamic impact test platform shown in Wettability is an important index to evaluate the surface properties of materials. The water contact angles (CAs) and rolling angles (RAs) of the banana leaf surface were measured at three different positions  on the adaxial and abaxial sides. The test results of the contact angles of 6 μL water droplets on the banana leaf surface showed that the contact angle on the adaxial side was 76.08° ± 2.01°, showing hydrophilicity, and the contact angle on the abaxial side was 94.32° ± 1.04°, showing weak hydrophobicity. In addition, the adaxial and abaxial sides of the banana leaf have high adhesion, the rolling angle is bigger than 90°, and the adaxial and abaxial sides of the banana leaves show Janus wettability.A large number of studies have shown that the surface microstructure is the main element that affects the wettability of the materials ,6,21. FiThe analysis of the test results shows that the main reason for the Janus wettability on the banana leaf surface was the difference in the structure and size of the nanopillars . The nanFrom −1, 0.99 m·s−1, 1.40 m·s−1, 1.71 m·s−1, and 1.98 m·s−1, respectively. A dynamic impact test was an important technical method to explore the robustness and dynamic stability of banana leaf surface wetting behavior. By adjusting the drop height of the water droplets, the impact velocity was controlled to be 0.63 m·sD0, and the diameter of the water droplets during the spreading process as D. The relation curve between the water droplets spreading factor Furthermore, we define the initial diameter of the water droplets as −1, the spreading factor of the droplets had a higher stability, and the spreading factor was obviously improved in the final steady-state. When the impact velocity was low, the kinetic energy of the droplet was small. During the energy-conversion process of the spreading stage, due to the conversion of kinetic energy and internal energy, the droplet shrinks and fluctuates greatly. However, when the impact velocity is high, the larger kinetic energy makes the droplets splash during the spreading and retracting stages, thus leading to the fluctuation in the spreading factor with time.In −1, the maximum spreading factor and the final steady-state spreading factor are relatively stable, and show a small peak. When the impact velocity is too low, there will be large fluctuations due to multiple fluctuations during the spreading phase of the droplets. When the impact velocity increases, the spreading factor will first decrease and then increase. If the velocity is too high, it may cause the droplets to break and splash. This provides an important reference for the design of pesticide spraying equipment and the adjustment of operating parameters in banana orchards.In order to further explore the dynamic impact behavior of water droplets on the surface of banana leaves, The test results of the impact behavior of water droplets on the surface of banana leaves show that in the final steady-state, the spreading factor of the adaxial side is always bigger than that of the abaxial side, indicating that the wettability of adaxial sides is always better than the abaxial side under the dynamic impact of water droplets. The banana leaf surface shows stable Janus wettability and better robustness.The adaxial side of banana leaves showed a hydrophilic Wenzel state, the nanopillars were completely infiltrated, and its hydrophilic state was analyzed by the Wenzel wetting mechanism. Assuming that the apparent contact angles (CAs) of the banana leaf surface are equation :(2)cosθ*On the rough banana leaf surface, According to the contact relationship between the nanopillars and water droplets in Thus, combining Equations (2)−(5), Equation (2) can be rewritten as:R, H, and S of the banana leaves’ adaxial side cosθ*Similarly, the Cassie–Baxter wetting state idealized geometric model can be used to describe the weakly hydrophobic wetting state of the abaxial side of the banana leaves.In this work, the wetting state of both the adaxial and abaxial sides of the banana leaves was studied. First, the surface of the fresh banana leaves was characterized by using advanced modern instrumental analysis techniques and methods. Second, the dynamic impact test was used to study the different dynamic responses of the banana leaf surface. Finally, an idealized geometric model of the banana leaf surface was established, revealing the underlying mechanism of the Janus wettability on the adaxial and abaxial sides of the banana leaves. The main results of this study are as follows:Scanning electron microscopy studies on the banana leaf surface found that the nanopillars on the adaxial side were cone-shaped with large gaps, and the heads of the nanopillars on the abaxial side were smooth and spherical with small gaps. The contact angle test found that the banana leaf surface has high adhesion properties (RA > 90°), and the banana leaf exhibits a Janus wetting state where the adaxial side was hydrophilic (CA = 76.08° ± 2.01°) and the abaxial side was weakly hydrophobic (CA = 94.32° ± 1.04°). This is caused by the different morphology and distribution of the nanopillars on the adaxial and abaxial sides of the banana leaf.−1, the maximum spreading factor and the final steady-state spreading factor showed a small peak, achieving the best effect.A dynamic impact test was carried out to study the wettability of the banana leaf surface. The study found that when water droplets hit the adaxial and abaxial sides of the banana leaves, they showed different dynamic responses, and the wettability of the adaxial side was always stronger than the abaxial side. The change trend of the spreading factor under different impact velocities was the same, and with the increase in the impact velocity, when the impact velocity reached 1.4 m·sAccording to the information of the parameters and structure of the nanopillars on the banana leaf surface, an idealized geometric model of both the adaxial and abaxial sides of the banana leaf was established, and the classical wetting theory model was introduced to reveal the Janus wettability transition from the Wenzel state to Cassie–Baxter states of the banana leaf surface and the underlying mechanism.The study of Janus wettability on banana leaf surface can provide an important reference for the design and manufacture of bionic nanostructured surfaces with Janus wettability and the exploration of efficient and high-quality production management of banana orchards. In the future, on the basis of current research, we will use banana leaves as a biological template to prepare a series of surface materials with Janus wettability and conduct practical production and application research in the fields of droplet condensation and fog harvest.


Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system. The Melanocortin Receptor Accessory Protein (MRAP) family, which includes two members, MRAP1 and MRAP2, binds G protein-coupled receptors (GPCRs) and modulates their trafficking and signaling transduction. MRAP2 is a small protein with a single hydrophobic transmembrane domain that can dimerize in parallel and antiparallel orientations. This dual topology is a unique feature in the eukaryote proteome [MRAP2 knock-out (KO) mice develop severe early onset obesity because MRAP2 is involved in the control of food intake . MRAP2 also regulates prokineticin receptors (PKRs) by reducing their signaling pathway and plasmatic membrane localization . It was αq/11, Gαs and Gαi after prokineticin 2 (PK2) binding [PKRs belong to GPCRs that are expressed both centrally and peripherally . PKR2 is binding , and med binding ,8. In neurons of the hypothalamic arcuate nucleus, PK2 induces STAT3 activation and melanocyte-stimulating hormone release by binding to PKR2, resulting in an anorectic effect and modulating thermoregulation and energy homeostasis. In adipocytes, PK2, which binds to PKR1, limits preadipocyte proliferation and differentiation, thereby controlling adipose tissue expansion .Despite recent advances in understanding the functions of MRAP2, little is known about the mechanism of the interaction of MRAP2 with the various GPCRs, except for data on melanocortin receptor 2 .In this study, we characterize the formation of the MRAP2–PKR2 complex using biochemical techniques.Escherichia coli (E. coli). We also analyze prokineticin-stimulated Gαi signaling in CHO cell lines expressing PKR2. Finally, to determine the role of the prokineticin transduction system on MRAP2 expression regulation, we analyze MRAP2 levels in mice hypothalamic explants and adipocyte tissue.Specifically, we determine the PKR2 region involved in MRAP2 binding using yeast as a heterologous expression system. To identify the MRAP2 regions required for PKR2 binding, we express the C-terminal soluble domain of MRAP2 in For the constitutive expression of MYC-DDk-tagged human MRAP2 in mammalian cell lines, RC203668 plasmid was purchased from Origene (Fisher Scientific).Saccharomyces cerevisiae (S. cerevisiae), the p413 MRAP2 plasmid was constructed. The cDNA of MRAP2 was amplified by PCR using a as template the RC203668 plasmid with the oligonucleotides MRAP2 BamHI up and MRAP2 EcoRI dw and cloned into the p413 digested with BamHI EcoRI under the control of the constitutive alcohol dehydrogenase promoter.For the expression of MRAP2 in E. coli expression of the carboxy-terminal domain of MRAP2, the pET 28 CT-MRAP2 construct was prepared. The DNA fragment of the C-terminal domain was amplified by PCR with gene-specific primers T70 BamHI and MRAP2 EcoRI dw and cloned into pBluescript to obtain PBS-CT_MRAP2. The resulting plasmid was digested with BamHI and EcoRI, and the fragment obtained was cloned into pET 28a. For the expression of the MRAP2 Δ131 deletion mutant, PBS CT-MRAP2 was digested with BamHI and HindIII, and the resulting fragment was cloned into pET 28a to obtain pet 28-131CT-MRAP2; both vectors express the recombinant protein fused to a poly-his tag. All oligonucleotides used for the plasmid constructions are listed in The procedure to construct pYESC3-PKR2, pYESC3-ΔN-PKR2 was previously described ,12. For 1/2 , mouse anti-pERK1/2 , mouse anti-STAT3 and rabbit anti-pSTAT3 (Tyr705) , monoclonal anti-polyhistidine-peroxidase  and rabbit anti-MRAP2 polyclonal antibody . After extensive washing with T-TBS, membranes were incubated with the appropriate IgG HRP-linked secondary antibody for 1 h at room temperature. The immunoreactive signals were visualized using an enhanced chemiluminescence system . After immunoblotting, digitized images of WB bands were acquired and the densitometric optical values were measured for each band using ImageJ software . The results were expressed as a percentage of control. The data were obtained from at least three independent experiments.An equal amount of proteins were separated by electrophoresis, then transferred to a nitrocellulose membrane (TCM) and blocked in 5% non-fat milk BSA/Tris-buffered saline containing 0.5% Tween-20 (TBS-T pH 7.4) for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with the appropriate primary antibodies in the blocking solution. The primary antibodies used were rabbit anti-ERKP. pastoris, PK2 was expressed in P. pastoris and purified, as described in Lattanzi et al., 2022 [w/v) polyacrylamide SDS–PAGE gel.In l., 2022 . BrieflyS. cerevisiae strain used for the co-expression of PKR2 and MRAP2 or ΔN-PKR2 and MRAP2 was Cy12946 (MATa FUS1p-HIS3 GPA1Gai2(5) can1 far1D1442 his3 leu2sst2D2 ste14::trp1::LYS2 ste3D1156 tbt1-1 trp1 ura3). The transformation of Cy12946 yeast cells was performed according to the lithium acetate protocol [The 7 cells/mL) were collected by centrifugation, and membrane homogenates were prepared by a glass bead method, as previously described [Yeast cells (1–2 × 10escribed .3 (Sigma) in 25 mM MOPS buffer pH 7.5 was freshly prepared. A total of 50 μL of membrane fraction (20 μg of protein) extracted from Cy12946 pYESC3 PKR2/p413 MRAP2 and Cy12946 pYESC3ΔN PKR2/ p413 MRAP2 was incubated together with BS3 for 30 min at room temperature. The reaction was stopped by the addition of 1 μL Tris–HCl 1.5 M. BS3 and control samples were analyzed by SDS–PAGE followed by immunoblotting with a monoclonal anti-His antibody (1:5000) and a polyclonal anti-MRAP2 antibody (1:1000) .A solution of 1 mM BSSolubilized membrane fractions co-expressing PKR2 and MRAP2 or ΔN-PKR2 and MRAP2 were co-precipitated by metal chelation chromatography using an Ni-NTA His-bind resin after batch purification under native conditions according to the manufacturer’s instructions . The eluates were fractionated by SDS–PAGE on a 12% gel and transferred to a polyvinylidene difluoride membrane. The membrane was coated with a monoclonal anti-His antibody (1:5000) or with a polyclonal anti-MRAP2 (1:1000) . E. coli, R2-GST fusion proteins were purified from cell extracts by affinity chromatography with glutathione-Sepharose beads , according to the manufacturer’s instructions. Briefly, 50 μL slurry of glutathione-Sepharose beads equilibrated in buffer A  were incubated with 2 mL R2-GST lysate for 1 h at 4 °C with constant stirring. Beads with bound R2-GST were collected by centrifugation, washed extensively in buffer A, and incubated overnight at 4 °C with total membrane extracted from yeast expressing MRAP2 or with purified MRAP2 CT domain or purified Δ131-MRAP2 expressed in E. coli. Beads were washed, as described above, and bound proteins were eluted with GSH according to the procedure of GE Healthcare and analyzed by Western blotting with anti-His antibody .The R2-GST proteins were obtained by a fusion of the 69 amino acids of the amino terminus of PKR2, with GST as described in ,15. AfteE. coli cultures expressing the carboxy-terminal domain and the Δ131-MRAP2 were grown at 37 °C until an optical density of 600 nm (OD600). Cultures were grown for an additional 4 h at 28 °C in the presence of isopropyl-β-D-1-thiogalactopyranoside (IPTG) 0.1 mM. The C-terminal domain of MRAP2 and the Δ131 -MRAP2 were purified using a His-tagged protein purification kit . The purified recombinant proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-His antibody .w/v Coomassie Blue G-250, 500 mM 6-aminohexanoic acid) was added to 20 μg of purified C-terminal MRAP2 domain and loaded on Native PAGE Novex 3% to 16% Bis–Tris gels . After electrophoresis at 0 °C  containing 100 U/mL penicillin/streptomycin, 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, and G418 200 ng/mL  at 37 °C and 5% CO2. Transient transfection with plasmid DNA of MRAP2 was performed in the presence of Lipofectamine 2000  according to the manufacturer’s instructions. Briefly, cells were incubated with lipo-DNA complex for 24 h at 37 °C and 5% CO2. The lipo–DNA complex was obtained by incubating 2 μg of plasmid DNA for MRAP2 with Lipofectamine 2000 diluted in Opti-MEM  for 20 min at room temperature. A total of 24 h after transfection cells were serum-starved and stimulated with PK2 (100 nM) for 10 min and 1 h at 37 °C, 5% CO2. At the end of the incubation period, cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail (1% v/v) (Sigma Aldrich), quantified, and used for WB analysis. CHO cells (Chinese hamster ovary) stably expressing human PKR2 were plated at a density of 4 × 10Epididymal adipose tissue was collected from 4- and 10-month-old WT (n = 4) and PKR1-KO (n = 4) mice, rapidly frozen on dry ice and stored at −80 °C until RNA or protein extraction. 2 and 95% O2 for 2 h, then serum-starved and treated with PK2 (100 nM) or PK2 plus WP1066 a STAT3 inhibitor (Merck KGaA) for 1 h. WP1066 (5 μM) was added 1 h before PK2 addition. At the end of the incubation period, the explants were quickly frozen on dry ice and stored at −80 °C until RNA or protein were extracted. Total cell protein was extracted using RIPA lysis buffer containing a protease inhibitor cocktail (1% v/v) , quantified, and used for WB analysis.Hypothalami were extracted from 4-month-old WT male mice (n = 12) immediately following decapitation, placed in oxygenated ice-cold Krebs–Ringer bicarbonate buffer  and successively cut into 300 μm thick slices using a vibratome. Hypothalamic slices were incubated in Dulbecco’s Modified Eagle Medium  supplemented with 1% of Fetal Bovine Serum  at 37 °C, 5% COTotal RNA was isolated from adipose tissue and hypothalamic explants using Trizol reagent  according to the manufacturer’s instructions. After determining its yield and purity by spectrophotometric absorbance at 260 and 280 nm, RNA was used for real-time PCR analysis.−ΔΔCt).One microgram of extracted RNA was reverse transcribed using SensiFAST cDNA Synthesis Kit  and the cDNA obtained was used to perform real-time PCR. All reactions were performed in triplicate using 25 ng of cDNA, iQ SYBR Green Supermix , and specific mouse primers . The reat-test or One-Way ANOVA followed by Tukey’s post-test when appropriate. Differences were considered significant at p < 0.05.Data were analyzed using GraphPad Prism 6 for Windows by Student 1/2 and STAT3 pathways [αi inhibitor pertussis toxin. We, therefore, examined ERK1/2 and STAT3 phosphorylation in CHO-PKR2 cells in the presence or absence of MRPA2, after PK2 incubation. MRAP2 is an important regulator of energy homeostasis that increases MC4R activation and inhibits PKR signaling [pathways ,15,17,181/2 and STAT3 phosphorylation. In contrast, in the presence of MRAP2, the effect of PK2 is significantly reduced. This suggests that the presence of MRAP2 reduces PK2-induced PKR2 activation and impairs Gi coupling  were expressed with MRAP2 in yeast Cy12946, and total membrane proteins were purified by Ni-NTA chromatography and subjected to Western blotting analysis. As shown in S. cerevisiae cells expressing MRAP2. As shown in S. cerevisiae cells co-expressing PKR2/MRAP2 or ΔN-PKR2/MRAP2 were fractionated into the membrane and cytosolic proteins and exposed to BS3 cross-linker. Proteins were separated using SDS−PAGE, blotted, and then probed with the anti-MRAP2 antibody ; the second exon contains a portion of the 5′ UTR sequence and a region encoding the first 44 amino acids; the third exon encodes a region comprising from the amino acid at position 45 to the amino acid at position 77; and the fourth exon encodes for the C-terminal cytosolic domain consisting of 129 amino acids and the 3′ UTR sequence A.To identify the MRAP2 region, which is important for PKR2 interaction, we generated an MRAP2 deletion mutant containing only the C-terminal region encoded by the fourth exon (CT-MRAP2) and a deletion mutant containing only the region extending from the residue at position 78 to the residue at position 131 (131CT-MRAP2) B.E. coli. The molecular mass of the purified proteins fused to His TAG was determined by SDS-PAGE and was approximately 20 kDa and 10 kDa, respectively, which is close to the values derived from the gene sequence. The identity of the purified proteins was also confirmed by Western blotting with an anti-His-tag antibody  pull-down experiments. Lysate from cells expressing the GST fusion protein PKR2-NT was incubated with glutathione-Sepharose resin in the presence of the purified CT-MRAP2 and 131CT-MRAP2 domains. As shown in Because MRAP2 is involved in energetic homeostasis, we examined the modulation of MRAP2 expression by the prokineticin system both in peripheral tissues, adipose tissue, and in the central nervous system, hypothalamus.In adipose tissue from wild-type (WT) and PKR1 knock-out (KO) mice, the expression of MRAP2 is up-regulated in adipocytes from PKR1 KO mice at 10 months of age at both mRNA and protein levels A,B.In ex vivo hypothalamic explants, PK2 treatment significantly reduces MRAP2 expression and pre-incubation with a STAT3 inhibitor restores MRAP2 expression to physiological levels C,D.αi coupling. The reduction in STAT3 and ERK1/2 activation in CHO cells expressing PKR2 and exposure to PK2 showed that MRAP2 also negatively modulates Gαi coupling. MRAP2 is a single transmembrane protein expressed mainly in the stomach and endocrine glands, in the hypothalamus, and in adipocytes . It is fS. cerevisiae. Using biochemical techniques, such as co-precipitation, GST pull-down and cross-linking, we demonstrated that MRAP2 binds specifically to PKR2, but does not interact with the ΔN mutant. These experiments showed that the N-terminal region of PKR2 is the essential part for interaction with MRAP2. This result may explain the differential ability of MRAP2 to regulate PKR1 and PKR2 in signal transduction and trafficking [E. coli: the entire C-terminal domain (76–205) and a limited region between amino acids at position 76 to the amino acid at position 131 (131CT domain). We demonstrated that the C-terminal domain of MRPA2 does not lose the ability to form a dimer structure. This is consistent with a dual topology of the protein and evidence that the C-terminal region is involved in parallel dimer formation [MRAP2 impairs PKR2 localization to the plasma membrane and inhibits PKR2 glycosylation in the correspondence of Asparagine at position 27 of the N-terminal extracellular region . To detefficking , becausefficking ,21. We eormation . Experimormation ,23.This effect could be mediated by the upregulation of PPARɣ in adipocytes of PKR1-KO mice , as PPARWe incubated mice hypothalamic explants with PK2 or PK2 in the presence of STAT3 inhibitor and we examined the expression of MRAP2 at the transcriptional and translational levels. Treatment with PK2 results in a significant reduction in the expression of MRAP2, and this reduction in expression is absent when explants were previously treated with the STAT3 inhibitor. This result suggests that STAT3 is involved in the regulation of MRAP2 expression.PKR2 has two putative glycosylation sites in the N-terminal region at position 7 and position 27. It was demonstrated that, by site-direct mutagenesis, glycosylation at position 27 is essential for the trafficking of PKR2 at the plasma membrane, and that this post-translational modification is absent in the presence of MRAP2 . Indeed,There is sufficient evidence that the C-terminus of MRAP2 is the most important domain for regulating GPCRs, but there is very little information about the structure of MRAP2, even if it seems that it does not contain an ordered secondary structure . The hyp