Vigna angularis is an edible crop and herbal medicine that is known to have antipyretic, anti-inflammatory, and anti-edema effects. Many studies have been conducted on the 95% ethanol extract of V. angularis, but there is little research on the 70% ethanol extract and hemiphloin, which is a new indicator component of the 70% ethanol extract of V. angularis. To investigate the in vitro anti-atopic effect and verify the mechanism action of 70% ethanol extract of V. angularis (VAE), TNF-α/IFN-γ-induced HaCaT keratinocytes were used. The VAE treatment alleviated TNF-α/IFN-γ-induced IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC gene expressions and productions. VAE also inhibited the phosphorylation of MAPKs, including p38, ERK, JNK, STAT1, and NF-κB in TNF-α/IFN-γ-induced HaCaT cells. 2,4-dinitochlorobenzene (DNCB)-induced skin inflammation mice model, and HaCaT keratinocytes were used. In the DNCB-induced mouse model, VAE treatment alleviated ear thicknesses and IgE levels. Furthermore, VAE decreased IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC gene expressions of DNCB-applied ear tissue. Additionally, we investigated the anti-atopic and anti-inflammatory effects of hemiphloin using TNF-α/IFN-γ-induced HaCaT keratinocytes and LPS-induced J774 macrophages. Treatment hemiphloin decreased gene expressions and productions of IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC in TNF-α/IFN-γ-induced HaCaT cells. The phosphorylations of p38, ERK, STAT1, and NF-κB were inhibited by hemiphloin in TNF-α/IFN-γ-induced HaCaT cells. Finally, hemiphloin showed anti-inflammatory activities in LPS-induced J774 cells. It decreased LPS-induced NO productions and iNOS and COX-2 expressions. Treatment of hemiphloin also inhibited LPS-induced TNF-α, IL-1β, and IL-6 gene expressions. These results suggest that VAE is an anti-inflammatory agent for inflammatory skin diseases and that hemiphloin could be a therapeutic candidate for inflammatory skin diseases. Previous studies have reported that it has excellent anti-oxidant  and filtered . Then, 1.6 kg of extract was obtained by concentration of filtrates under reduced pressure. The extract was suspended in distilled water, and the aqueous layer was partitioned with n-hexane, EtOAc, and BuOH. The n-hexane layer (1.1 kg) was subjected to silica gel column elution with CHCl3: MeOH  to obtain 16 fractions (PAH1 ∼ PAH16). Fraction 1 (PAH1) and fraction 12 (PAH12) were subjected to MPLC . Hemiphloin (3.3 mg) was obtained from fraction PAH12H (174.0 mg) by preparative HPLC , using isocratic elution with 17% CH3CN in H2O.The dried seeds of 2.1.121H22O10; ESI-MS m/z 435.4 [M+H]+; 1H NMR  δH 7.31 , 6.81 , 5.96 , 5.36 , 4.78 , 4.12 , 3.85 , 3.71 , 3.43 , 3.37 , 3.13 , 2.75 ; 13C NMR  δC 161.8 (C-5), 128.5 , 115.4 , 81.6 (C-5ʹʹ), 79.2 (C-3ʹʹ), 78.4 (C-2), 73.1 (C-1ʹʹ), 71.0 (C-4ʹʹ), 70.5 (C-2ʹʹ), 61.7 (C-6ʹʹ), 42.1 (C-3).Yellow powder; Chemical formula C2.2Six-week-old female BALB/c mice were obtained from Samtako . A total of 25 mice were divided into five groups (n = 5): the vehicle phosphate-buffered saline (PBS), DNCB vehicle (PBS), DNCB and VAE (100 and 300 mg/kg), and DNCB and dexamethasone  groups. For sensitization, 20 μL of DNCB was applied once each ear  in first week. Then, both ears of BALB/c mouse were challenged with DNCB  for 3 weeks. VAE (100 and 300 mg/kg) or DX (5 mg/kg) was orally administered by gavage for ﬁve consecutive days per week at the time of the DNCB challenge. Mice were provided with ad libitum access to food and water. The room was maintained with a 12 h light-dark cycle (lights on at 07:00 and lights off at 19:00) and controlled temperature (21 ± 2 °C). Mice were handle in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Korea Research Institute of Bioscience and Biotechnology (permission number #KRIBB-AEC-21259).2.3Mouse ear tissues were fixed in 4% (w/v) paraformaldehyde in PBS, (pH 7.4). The fixed samples were embedded in paraffin and sectioned. Then, the specimen were stained with hematoxylin and eosin (H&E).2.42.The human keratinocyte cell line, HaCaT  was maintained in Dulbecco's modiﬁed Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. The murine macrophage cell line J774 was maintained in DMEM, supplemented with 10% non-heated FBS and 1% penicillin–streptomycin. The cells were incubated at 37 °C in 90–95% humidity and 5% CO2.54 cells/well in 200 μL culture medium in 96-well plates and incubated for 24 h at 37 °C. The cells were treated with VAE or hemiphloin at several concentrations; 10, 30, 60, and 100 μg/mL VAE, and 1, 10, and 100 μM hemiphloin. After 24 h, the cells were treated with MTT solution for 3 h and supernatant was removed. Then, the crystallized formazan was dissolved in DMSO and the absorbance at 540 nm was measured using a microplate reader.Cell viability was determined by the 3--2, 5-diphenyl tetrazolium bromide (MTT) assay. HaCaT and J774 cells were seeded at a density of 3 × 102.6J774 cells were pretreated with hemiphloin or DX for 1 h and stimulated with 100 ng/mL lipopolysaccharide (LPS) for 24 h. After the incubation, nitrite levels in medium were determined using a NO Plus Detection Kit . Briefly, 50 μL of supernatant was mixed with an equal volume of NO Plus Detection kit reagent. The absorbance at 540 nm was measured using a microplate reader.2.7TRIzol regent was used for isolation of RNA. The complementary DNA (cDNA) was synthesized using a PrimeScript 1st Strand cDNA Synthesis Kit . Real-time qPCR was performed using a Bio-Rad T100 thermal cycler  with TaqMan probes and TaqMan Real-Time PCR Master mix  according to the manufacturer's protocol. The gene expressions were normalized by 18s rRNA and GAPDH, as an endogenous control. The information of TaqMan probe was provided in 2.8The level of cytokine  and chemokine  in cell conditioned medium, and serum IgE were determined using ELISA kits  according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader.2.96 cells/well in 6-well plates) were pretreated with VAE, hemiphloin, or cyclosporine for 1 h. For 30 min, stimulated with TNF-α (50 ng/mL)/IFN-γ (50 ng/mL). J774 cells (1 × 106 cells/well in 6-well plates) were pretreated with hemiphloin or DX for 1 h and then stimulated with LPS for 24 h. The cells were lysed with 100 μL of cell lysis buﬀer  and centrifuged at 13,000 rcf for 10 min at 4 °C. The supernatants were harvested and quantification were performed by DC protein assay kit . Equally quantified protein lysate was subjected to electrophoresis on an SDS-PAGE gel and then transferred onto a polyvinylidene ﬂuoride (PVDF) membrane. After blocking with 5% bovine serum albumin in Tris-buffered saline for 1 h, the membrane was incubated with primary antibody against the target protein, 3 times washed, and subsequently incubated with horseradish peroxidase-conjugated IgG secondary antibody. The bands were developed using a West-Queen RTS Western Blot Detection Kit . Antibodies purchased from Cell Signaling Technology and Santa Cruz were used  and statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.The data are presented as the mean ± SD of three for 33.1HaCaT cells, which are human keratinocytes, were treated with various concentrations to examine the effect of VAE on cell viability. After 24 h, cell viability was measured by MTT assay . VAE at 3.2To demonstrate the mechanism underlying the anti-inflammatory effect of VAE, the intracellular signaling pathway stimulated by TNF-α/IFN-γ was evaluated. MAPKs, including p38, EKR, and JNK, have been found to exert a wide spectrum of bioactivities at the cellular level, such as effect on the cell cycle and, metabolism and inflammation activity ,18. Phos3.3We evaluated the effects of VAE using and atopic-like animal model. The DNCB has been used to induce atopic skin lesions and is a powerful agent. ; H. N 2. As show3.4The effects of hemiphloin have not been reported. Thus, we conducted an experiment to confirm the activity of hemiphloin in HaCaT cells. To explore the effect of hemiphloin on the viability of HaCaT cells, we first treated cells with the indicated concentrations of hemiphloin and then measured cell viability after 24 h using the MTT assay. Hemiphloin at concentrations of 1, 10, and 100 μM did not have any significant toxic effects. . HaCaT c3.5To explore the mechanism of action of hemiphloin, the intracellular signaling pathway stimulated by TNF-α/IFN-γ was evaluated. It was shown that phosphorylation of signaling molecules was activated by TNF-α/IFN-γ in HaCaT cells. However, pretreatment with hemiphloin suppressed the TNF-α/IFN-γ-induced phosphorylation of signaling molecules . Further3.6To confirmed the efficacy of hemiphloin in marophage cells other than keratinocyte cells using J774 cells. As shown in HaCaT cells, hemiphloin was not cytotoxic to J774 cells. A. Additi4V. angularis on DNCB-induced skin inflammation using in vitro and in vivo models.Our results showed that VAE inhibited TNF-α/IFN-γ-stimulated production of cytokines in keratinocytes. In addition, the level of MAPKs, STAT-1, and NF- κB was alleviated by VAE. In the inflammatory environment, damaged keratinocytes release the many inflammatory cytokines such as IL-1β, IL-6, and IL-8, which leads to pathogenesis of skin inflammation [V. angularis contains several compounds, and these compounds have been reported to exert various pharmacological effects [V. angularis. In addition, we investigated the anti-inflammatory effects of hemiphloin in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Hemiphloin exerted inhibitory effects on cytokine production and the phosphorylation of MAPKs, STAT-1, and NF-κB in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Our results indicated that the anti-inflammatory effects of VAE are due to the inhibition of cytokine expression through suppressing the cell signaling pathway. Inflammatory mediators and cytokines exacerbate the inflammatory response to skin lesions in skin diseases such as atopic dermatitis [2 and PGE2, which play a role in the allergic and inflammatory responses [Atopy is one of the most common forms of chronic eczema and affects thousands of people worldwide. Atopic dermatitis is an allergic reactions in the skin barrier. The lesion of acute atopic dermatitis is confined to hives, rashes, and itching. However, in the chronic state occurs to severe skin damage, resulting in a breakdown of the patient's skin immune system and psychological distress. Therefore, the development of therapeutic agents with safe and high efficacy was required. We demonstrated the pharmacological effects of 70% ethanol extract of ammation . These cammation . Therefo effects , 25, 26.rmatitis . In addirmatitis . Clinicarmatitis ,30. In trmatitis . In atoprmatitis . Morevoermatitis . COX-2, esponses . Therefo5in vitro and in vivo. Hemiphloin which was isolated from V. angularis extract, also significantly decreased chemokine and cytokine production by TNF-α/IFN-γ in vitro. Hemiphloin reduced the levels of these mediators of atopic dermatitis-like inflammation, such as NO, COX-2, iNOS, and cytokines in LPS-stimulated J774 cells. These results indicate that VAE and hemiphloin can act as preventive and therapeutic agents for inflammation-related skin diseases.In this study VAE significantly inhibited chemokine and cytokine production induced by TNF-α/IFN-γ and DNCB Seon Gyeong Ba, Hyung Jin Lim: Conceived and designed the experiments; Wrote the paper.Eun Jae Park, Yeong Seon Won, Sang Ik Park: Performed the experiments; Analyzed and interpreted the data.Seung Woong Lee, Soyoung Lee, Mun-Chual Rho: Contributed reagents, materials, analysis tools or data.Seung-Jae Lee: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Korea Institute for Advancement of Technology (P0010383), Ministry of Oceans and Fisheries and the Korea Research Institute of Bioscience and Biotechnology (KGM5242221).This work was supported by the Data included in article/supplementary material/referenced in article.The authors declare no competing interests.


Ipomoea batatas, a medicinal food plant used in ethnomedicine for centuries. In the current study, in vitro antiproliferative potential against PCa cells (DU145 and PC3) via 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay; Western blot protein expression analysis; absorption, distribution, metabolism, excretion (ADME); pharmacokinetic prediction studies; and bisphenol A (BPA)-induced prostate inhibition in Sprague Dawley rats were conducted to gauge the anti-cancer ability of NA. Significant (p < 0.05 and p < 0.01) time- and dose-dependent reductions in proliferation of PCa cells, reduced migration, invasion, and increased apoptotic cell population were recorded after NA treatment (3–50 µM). After 72 h of treatment, NA displayed significant IC50 of 21.18 ± 3.43 µM against DU145 and 24.21 ± 3.38 µM against PC3 cells in comparison to the controls cabazitaxel (9.56 ± 1.45 µM and 12.78 ± 2.67 µM) and doxorubicin (10.98 ± 2.71 µM and 15.97 ± 2.77 µM). Further deep mechanistic studies reveal that NA treatment considerably increased the cleavage of caspases and downstream PARP, upregulated BAX and P53, and downregulated BCL-2 and NF-κB, inducing apoptosis in PCa cells. Pharmacokinetic and ADME characterization indicate that NA has a favorable physicochemical nature, with high gastrointestinal absorption, low blood–brain barrier permeability, no hepatotoxicity, and cytochrome inhibition. BPA-induced perturbations of prostate glands in Sprague Dawley rats show a potential increase (0.478 ± 0.28 g) in prostate weight compared to the control (0.385 ± 0.13 g). Multi-dose treatment with NA (10 mg/kg) significantly reduced the prostate size (0.409 ± 0.21 g) in comparison to the control. NA-treated groups exhibited substantial restoration of hematological and histological parameters, reinstatement of serum hormones, and suppression of inflammatory markers. This multifaceted analysis suggests that NA, as a novel small molecule with a strong pharmacokinetic and pharmacological profile, has the potential to induce apoptosis and death in PCa cells.Prostate cancer (PCa) is the most common cancer in men, accounting for approximately 10% of all new cases in the United States. Plant-derived bioactive compounds, such as pentacyclic triterpenoids (PTs), have the ability to inhibit PCa cell proliferation. We isolated and characterized nummularic acid (NA), a potent PT, as a major chemical constituent of  Prostate cancer (PCa) is one of the most common cancers worldwide and has a high mortality rate. Enlarged prostate, with reduced volume and intensity of the urinary stream as a result, is considered a leading cause of illness and death in PCa. It is the fourth most significant type of cancer on Earth, prevailing in 1.41 million people (WHO). Surveys suggest that by 2030, PCa will cross 1.7 million new cases and 499,000 new deaths ,6. It isκB (NF-κB). PTs such as oleanolic acid (OA) and glycyrrhizic acid (GA) are extensively used to treat liver disorders, as well as betulinic acid for prostatitis, corosolic and gymnemic acids for diabetic complications, and Asiatic acid in wound healing. Thus, the whole class of PTs is a worthy treasure of novel multi-targeted bioactive components with commendable therapeutic potential , one methyl doublet [δ 1.03 (J = 6.3 Hz)], one carbinol methine [δ 3.45 ], and one olefinic proton [δ 5.51 ] typical of hydroxyursane skeleton were prominent in 1H NMR spectra. The HMBC correlation of H3 (δ 3.45) with C2 (δ 27.25), C4 (δ 38.55), C23 (δ 28.73), and C24 (δ 16.65) suggested OH at C3. The coupling constants of H3 proposed β and equatorial positioning of OH at C3. The HMBC interface of H18 (δ 2.63) with C13 (δ 138.41), C17 (δ 31.23), C19 (δ 46.13), and C29 (δ 181.18) and of H19 (δ 1.85) with C18 (δ 55.53), C20 (δ 38.56), C21 (δ 30.22), C29 (δ 181.06), and C30 (δ 21.58) collectively showed the location of a carboxyl group at C19 locus. From the NMR data, the compound was identified as a known compound 3β-Hydroxyurs-12-en-29-oic acid [The purified compound was obtained as a white amorphous powder with molecular formula Cspectrum , COOH atcarboxyl 93 cm−1, κB (PDB-ID 1NFK), and −8.4 kcal/mol E-value with P53 (PDB-ID 1AIE). In the current study, NA binding affinities with ligands for target proteins and amino acid residues involved in the binding pocket interactions were comparable to selective standard inhibitors, i.e., doxorubicin, lapatinib, and vincristine. This study strongly suggests that NA has significant potential to be developed as a selective inhibitor of extrinsic and intrinsic apoptotic proteins. All 2D and 3D images of binding interactions of NA with target proteins are represented as The affinity among the protein targets and the ligands was investigated using molecular docking. AutoDock Vina program was used for the docking analysis through the PyRx user interface. E-value  was used to assess the protein affinity and best-docked pose complex. It provided a prediction of binding free energy and binding constant for docked ligands. In the current study, NA has the most stable complexes, showing −8.5 kcal/mol binding energy  with BAX (PDB-ID: 2K7W), −8.2 kcal/mol E-value with BCL-2 (PDB-ID 1K3K), −1.9 kcal/mol E-value with NF-50 of 41.23 ± 4.84 µM and 21.18 ± 3.43 µM. In contrast, IC50 of NA was recorded 46.18 ± 4.80 µM and 24.21 ± 3.38 µM at 48 h and 72 h exposure time, respectively, against PC3. Standard drugs (Cabazitaxel and Doxorubicin) exhibited maximum cytotoxicity against DU145  and PC3  after maximum exposure time (72 h). All results are displayed in Significant reductions in cell viability were observed upon exposure to NA for 24, 48, and 72 h. NA showed significant time-dependent inhibition against DU145 after 48 h and 72 h exposure time, with ICIn successive experiments, the inhibitory effect of NA on the multiplication of prostate cancer cells was assessed by clonogenic assay. DU145 cells were treated for 48 h and permitted to grow until untreated cells formed adequate colonies. Visual observation of cells demonstrated that cells could not develop sufficient colonies in the presence of NA. In contrast, there was an unchecked proliferation of untreated prostate cancer cells and those treated with 1% DMSO as a negative control . As deteκB and BCL-2) proteins were analyzed through Western blotting  and anti-apoptotic , and pharmacokinetic prediction studies were conducted for the compound NA. The physico-chemical characteristics of NA are discussed in NA has a good gastrointestinal absorption (HIA) and no BBB permeability, as seen in p < 0.05) increased, measuring 0.478 ± 0.28 g in comparison to the control (0.385 ± 0.13 g) with ROW (0.274 ± 0.16). Maximum protection was examined in the BPA + NA (10 mg/kg) group followed by the BPA + NA (5 mg/kg) group, showing significant (p < 0.05) decreases in prostate size . The test group receiving NA (10 mg/kg) showed no obvious increase in prostate size (0.393 ± 0.16 g), validating its safety in multidose treatment.Excised gonads on day 21 of experimentation were measured. The results  depict t6/µL), WBC (4.19 ± 0.11 and 4.76 ± 0.12 × 103/µL), platelet count (468.1 ± 9.81 and 437 ± 03.21 × 103/µL), Hb level (11.33 ± 0.47 and 10.31 ± 0.54 g/dL), and ESR (4.18 ± 0.53 and 5.09 ± 0.19 mm/h) of test groups BPA + NA (10 mg/kg) and BPA + NA (5 mg/kg), respectively, were significantly restored in comparison to control. BPA intoxication considerably altered the hematological profile of treated rats.The blood profiling of the BPA-intoxicated rats treated with NA (10 mg/kg and 5 mg/kg) doses is listed in Histological investigations ascertain the gonadoprotective potential of NA and validate its anti-prostate carcinoma potential. Slides attest that NA at high (10 mg/kg) and low (5 mg/kg) doses is proactive against BPA-induced toxicity . The conp < 0.05) alteration in rats receiving NA (10 mg/kg) in comparison to the control and vehicle, hence validating their nontoxic behavior towards gonads and sex hormones. On the other hand, test groups BPA + NA (10 mg/kg) and BPA + NA (5 mg/kg) showed significant protection against BPA-induced gonadotoxicity as testosterone, FSH, LH, and estradiol were recorded as 4.02 ± 0.09 ng/mL, 10.12 ± 0.27 mIU/mL, 2.90 ± 0.13 mIU/mL, and 21.07 ± 1.17 pg/mL, respectively, and 4.37 ± 0.11 ng/mL, 11.09 ± 0.42 mIU/mL, 3.22 ± 0.10 mIU/mL, and 20.39 ± 1.12 pg/mL, respectively. Raised estradiol levels (26.19 ± 2.16 pg/mL) in rats receiving BPA indicate gonadotoxicity, which ultimately leads to prostatitis (p < 0.05) restoration of these biochemicals.Levels of circulating hormones  were recorded, with no marked (statitis . NA signThe significant findings of the current study are (i) the characterization of Nummularic acid, specifically that (ii) NA reduces cell proliferation, migration, and invasion of prostate cancer cells; (iii) induces apoptosis at very low concentrations; (iv) has excellent pharmacokinetic and ADME properties; and (v) reduce prostatitis in vivo.30H48O3 and a molecular weight of 456.3 g/mol. The presence of hydroxyl (3421 cm−1), carboxyl (1693 cm−1), and olefinic double bond (1652 cm−1) in FTIR and the presence of 30 C signals in 13C NMR spectrum confirm the triterpenoid structure of its ursane skeleton. Ursane triterpenoids have been reported to suppress the proliferation of various tumor cells; induce apoptosis; and inhibit tumor promotion, metastasis, and angiogenesis in animal cancer models. The presence of the OH group at C-3, COOH at C-29, and dimethyl substitution at C-17 and C-20 of NA plays a vital role in enhancing their cytotoxic effect [Compounds of natural origin with fewer side effects and admirable therapeutic efficacy have gained a repute in anti-cancer drug development. In recent years, various potent plant-based triterpenoids have been discovered, which have shown great promise as chemopreventive and therapeutic agents. They were principally inhibiting key signaling molecules, inflammatory mediators, tumor cell proliferation, invasion, metastasis, and angiogenesis in various in vitro and in vivo models of cancer. NA is obtained as needle-shaped white crystals with molecular formula CκB is a protein complex that monitors the transcription of specific genes, cytokine production, and cell survival. NF-κB regulates anti-apoptotic genes, particularly TRAF1/TRAF2, and blocks the caspase family of enzymes involved in various apoptotic pathways. Thus, downregulation of NF-κB will trigger caspases and ultimately upregulate apoptosis [κB) indicate that the compound induced apoptosis in PCa cells. Previously, a team of researchers reported that NA isolated from F. xanthoxyloides induces apoptosis in prostate cancer (DU145 and C4-2) cells by activating AMP-kinases, altering the metabolic rate, and triggering an immediate energy crisis that causes the ultimate death of the cells [κB levels. Ursolic acid (UA) has been repeatedly reported to induce apoptosis in various cancer cells by activating caspase cascade and upregulating pro-apoptotic (BAX and P53) while downregulating anti-apoptotic (BCL-2 and NF-κB) proteins. UA has the ability to trigger the activation of protein kinase C (PKC) which in turn is involved in the apoptosis of many cancer cells, including PCa cells [Apoptosis is a complex pathway to thoroughly understand, but is generally divided into extrinsic (FAS/FasL ligand) and intrinsic  pathways. Caspase activation by intrinsic or extrinsic pathways is mediated by the activation of caspase 8 and caspase 9, respectively. These can activate and cleave downstream protein caspase 3 that can elicit morphological hallmarks of apoptosis, including DNA fragmentation by proteolytic cleavage of PARP. NF-poptosis ,19. It whe cells . Earlierhe cells , reporteCa cells .Pharmacokinetic and ADME profiles are key steps in the biotransformation of any biomolecule into a medication . Accordiα-reductase (5αR) plays a vital role in converting testosterone to 5α-dihydrotestosterone (DHT) inside the stroma and basal cells. DHT has a 10× greater affinity with androgen receptors than testosterone and plays a vital role in prostate enlargement. So, BPA could compete with DHT to bind to the androgen receptor and initiate anti-androgenic activity in cell systems by forming an AR/BPA complex that prevents endogenous androgens from regulating androgen-dependent gene transcription. Continuous exposure to xenobiotics such as BPA causes oxidative stress, leading to genitourinary abnormalities, sperm deformity, epigenetic variations, enlarged prostate mass, and reduced epididymal weight [BPA is a weak agonist to the androgen receptor (AR); thus, it can disrupt the estrogen-triggered pathways by forming a transcriptional complex that can bind the estrogen-responsive element (ERE). Intracellular enzyme 5l weight .p < 0.05) increases prostate mass (0.478 ± 0.28 g) of male Sprague Dawley rats and reduced epididymal weight to 0.334 ± 0.17 g. Triterpenoids with oleanane, ursane, and lupine skeleton exhibit significant anti-oxidant potential and have a greater tendency to mitigate ROS- and iNOS-induced pathological conditions in the body. The presence of the OH group at position C-3 and COOH at C-29 turn NA into more anti-oxidant molecules as OH and COOH have a commendable H+ donating feature [S. cumini have shown promising anti-oxidant and gonadoprotective potential. Similarly, Olasantan et al. [A. floribunda triterpenoids.Currently, multidose intoxication of BPA significantly  in comparison to the control and vehicle. ROS is the ultimate product of sustained bodily exposure with endocrine-disrupting chemicals (EDC) such as BPA that cause oxidative stress. As a result, hypomethylation, a mutation in genetic makeup, and testicular disruption occur, which cause immature and de-morphed spermatogenesis [Hematology is the most important predictive tool for determining the existence and intensity of any type of inflammation. Persistent oxidative stress damages vital organs such as the testes, causing inflammation that can be identified with hematological tests. It is evident in the literature that raised ESR, higher WBC count, lower platelet numbers, and elevated levels of circulatory NO indicate bodily inflammations and uremic toxicity . All theogenesis . BPA-intI. batatas, and its stock solution was prepared in dimethyl sulfoxide (DMSO) and stored at –20 °C. Doxorubicin and Cabazitaxel  were used as positive control and their stock solutions were also prepared in DMSO. Primary antibodies Cleaved-PARP (Asp214) (E2T4K) Mouse mAb #32563, Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb # 9664, BCl-2 (124) Mouse mAb #15071, BAX (2D2) Mouse mAb #89477, P53 (1C12) Mouse mAb #2524, and NF-κB p65 (D14E12) XP® Rabbit mAb #8242 were supplied by Cell Signaling Technology . Anti-mouse and anti-rabbit secondary antibodies were purchased from GE healthcare . Prostate cancer cell lines DU145 (HTB-81) and PC3 (CRL-1435) were purchased from American Type Culture Collection . Dimethyl sulfoxide (DMSO), phosphate buffer, and Folin–Ciocalteu reagent were purchased from Riedel-de Haen . Bisphenol A (BPA), trichloroacetic acid (TCA), and tryptone soy broth (TSB) were procured from Sigma-Aldrich (USA), and Tween-20 from Merck-Schuchardt . We also used Medium 199, heat-inactivated Biowest FBS (South America), and RPMI-1640 culture media . Dulbecco’s Modified Eagle Medium (DMEM), DMEM/F1 supplemented with L-glutamine and 2.438 g/l H2CO3 , 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder, phosphate buffer saline (PBS), SRB (sulforhodamine B), and acetic acid  were also purchased locally. Pre-coated silica gel 60 F254 TLC plates, normal phase silica gel 60 (63–200 µm particle size) and silica gel 60 (particle size = 40–60 µm), and chromatography columns were purchased from Merck . Nummularic acid was isolated from ethyl acetate extract of the aerial part of I. batatas (IPA-EAf) was dissolved in ethyl acetate. The slurry was adsorbed on silica gel 60  and loaded on a gravity column packed with 580 g silica gel 60 for chromatography. The amorphous powder as a purified compound was eluted with DCM and EA (1:0–0:1) using flash column, and purity was observed using the LC-PDA-ELSD method from a single peak obtained during analysis. The isolated compound was characterized using 1D and 2D NMR, FTIR, and mass spectroscopy.Ethyl acetate fraction from the aerial part of κB (Musmusculus PDB-ID 1NFK), and P53  were accessed from Protein Data Bank (PDB)  with PDB IDs of 1M9K and 1O86, respectively. The Autodock Tools program was used to prepare target proteins to be docked. The proteins were energy-minimized, and Gasteiger charges were added and saved in PDBqt format. Discovery Studio 4.1 Client (2012) was used to generate the hydrophobicity and Ramachandran graphs. The protein construction and statistical percentage values of helices, β-sheets, coils, and turns were assessed by VADAR 1.8 [The three-dimensional (3D) structures of BAX , BCL2  NF-ADAR 1.8 .κB, and P53 using the PyRx virtual screening tool with the Auto Dock VINA Wizard approach [κB (center X = −10.754 center Y = 12.598 center Z = 112.161) and size values were adjusted . P53 (center X = 6.586 center Y = 22.334 center Z = −0.939) was adjusted for better conformational position in the active region of the target protein. Ligands were docked individually against nitric oxide synthase and angiotensin-converting enzyme with a default exhaustiveness value of 25. The predicted docked complexes were evaluated based on the lowest binding energy values . The 3D graphical depictions of all the docked complexes were accomplished by Discovery Studio (2.1.0) .The compounds were designed in Discovery Studio Client and saved in PDB format as ligands after energy minimization. Autodock Tools were used to prepare ligands in their most stable conformation. After the addition of the Kolman and Gasteiger charges, the ligands were saved in PDBqt format. Molecular docking analysis was used for all the synthesized ligands against BAX, BCL-2, NF-approach . The griβ-sheets, 30% coils (63 residues), 14% turns (30 residues), and a total of 212 amino acid residues R = 0.210 and resolution A° = 1.22. Unit cell dimensions for the lengths were observed to be a = 69.786, b = 91.573, and c = 156.096 with 90° angle for α, β, and γ. The Ramachandran plot confirmed that 96% amino acids were in the allowed regions for the phi (φ) and psi (ψ) angles. BCL2 consisted of 69% helices (101 residues), 0% β-sheets, 30% coils (45 residues), 35% turns (52 residues), and a total of 158 amino acid residues R = 0.150 and resolution A° = 1.19. Unit cell dimensions for the lengths were observed to be a = 69.786, b = 91.573, and c = 156.096 with 90° angles for α, β, and γ. The Ramachandran plot confirmed that 96% amino acids were in the allowed regions for the phi (φ) and psi (ψ) angles. NF-κB consisted of 90% helices (59 residues), 45% β-sheets (287 residues), 44% coils (278 residues), 7% turns (44 residues), and a total of 672 amino acid residues R = 0.340 and resolution A° = 2.30. Unit cell dimensions for the lengths were observed to be a = 84.2, b = 132.1, and c = 80.1 with 90° angles for α = 90, β = 93.1, and γ = 90. The Ramachandran plot confirmed that 97% amino acids were in the allowed regions for the phi (φ) and psi (ψ) angles. P53 consisted of 64% helices (20 residues), 0% β-sheets, 35% coils (11 residues), 0% turns, and a total of 31 amino acid residues R = 0.252 and resolution A° = 1.50. Unit cell dimensions for the lengths were observed to be a = 69.786, b = 91.573, and c = 156.096 with 90° angle for α, β, and γ. The Ramachandran plot confirmed that 93% amino acids were in the allowed regions for the phi (φ) and psi (ψ) angles. The Ramachandran plots for target proteins are presented in the BAX consisted of 70% helices (148 residues), 0% 50 was determined after exposing cells with extracts for 24, 48, and 72 h. To assess the cytotoxic ability of NA against prostate cancer cell lines DU145 (HTB-81) and PC3 (CRL-1435), MTT assay was carried out according to the pre-established protocol . Brieflyn = 3) designated as the control were seeded with freshly prepared media without any extract or drug. Test group wells (n = 3) were supplied with freshly prepared media containing 20 µM of each extract. The whole event was photographed at intervals of 0, 12, and 24 h on an inverted microscope . The distance between migration edge and wound edge was measured with Image Pro Plus software, and the % scratch area of test groups compared to controls was plotted.After assessing cytotoxic potential, NA was tested for cell migration of DU145 using an established in vitro scratch test for 24 h ,32. In s2 for 2 days. Cells were then treated with 10 µM and 20 µM concentrations of NA for 48 h. After treatment for 48 h, the cells were allowed to form colonies for 5–7 days and media in the test and control groups were regularly changed on alternate days. Following the methodology of [The prostate cancer cell line (DU145) was plated on 6-well tissue culture plates with 1 million cells/well and placed at 37 °C in 5% COology of , cells w6/flask). After 48 h, cells were treated with NA (10 μM and 20 μM) for 24 h. After treatment, media were aspirated, cells were washed with cold PBS with pH maintained at 7.4, trypsinized and pelleted in 15 mL falcon tubes, and cold lysis buffer was added to the pellet. To perform Western blotting, 40–60 μg of protein was mixed with an equal volume of sample buffer and denatured for 10 min on a heat block at 95 °C. Samples after cooling at room temperature were centrifuged for a split second. Marker (5 μL) and denatured samples loading were carried out on 4–20% mini-protean TGX® stain-free gels (50 μL well) fixed in a mini-Protean® Tetra system vertical electrophoresis tank. The tank was filled with running buffer and proteins were resolved with Power Pac Firmware version 1.07 at 3 A and 100 V for 1.5 h. After separation, the protein transfer was conducted to a 0.2 μm nitrocellulose membrane on a Trans-Blot turboTM transfer pack at 2.5 A for 10 min using the Trans-Blot Turbo transfer system. After transferring the proteins, blots were washed with 1× wash buffer for 5 min, blocked for 30 min by blocking buffer, and probed with the appropriate primary monoclonal antibody (3 μL/3 mL blocking buffer) overnight at 4 °C. Later, 5 min washing of blots was carried out with 1× wash buffer, probed with specific secondary antibody (rabbit/mouse IgG) at room temperature for 2 h and developed with ECLTM Prime Western blotting detection reagent for 5 min. Protein bands were detected by chemiluminescence autoradiography using the ChemDocTM MP imaging system.Protein extraction and Western blot analysis were performed following the protocol described previously . Cells  was used to obtain ADME properties of NA [http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 20 February 2022) was used to predict the pharmacokinetic scores.ADME  are the essential measurement tools for any compound before it is elected as a drug candidate. The online web tool swiss ADME , Islamabad, Pakistan, and housed at Primate Facility of Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan. The approved guidelines of the ethical committee of Quaid-i-Azam University, Islamabad, Pakistan, for animal care and experiments (letter number # QAU-PHM-017/2016) were strictly followed. The experimental design was executed according to the guidelines of NIH, Islamabad. During the study, it was ensured that the test animals experienced minimum distress, discomfort, and pain. Euthanasia and blood sampling for hematological, biochemical, and serological studies were performed under anesthesia.Rattus norvegicus) weighing approximately 170–220 g/each. All test animals were properly fed and supplied with plentiful fresh water. To gauge the protective ability of NA, a detailed investigation was conducted (n = 7) against BPA-induced gonadotoxicity and prostatitis. A total of 21 days of experimentation were carried out as previously described by following published protocol [Standardized conditions  were provided to the experimental animals (protocol .Group I. (Control) group remained untreated.Group II. (Vehicle) group received 10% DMSO in water, administered orally at 10 mL/kg body weight (BW) on alternate days.Group III. (BPA) group received 50 mg/kg BW dissolved in 10% DMSO and injected intraperitoneally on alternate days.Group IV. (NA) group received 10 mg/kg in 10% DMSO given orally on alternate days.Group V. (BPA + NA) group received 10 mg/kg . Group VI. (BPA + NA) group received 5 mg/kg .On the final day of the study, rats were weighed and euthanized by cervical dislocation under chloroform anesthesia. The blood samples were collected under anesthesia via the abdominal aorta for hematological, biochemical, and serological investigations.Rats from each experimental group were anesthetized adequately before euthanizing on the final day of the experiment. Gonads were excised, cleaned out of extra connective tissues, weighed, and measured for size. ROW was calculated as follows:The blood samples were collected under anesthesia via the abdominal aorta in specific tubes (BD vacutainer) for hematological, biochemical, and serological investigations. Serum was separated by centrifuging blood samples at 6000 rpm for 15 min at 4 °C that were either analyzed or stored at −20 °C. A Neubauer hemocytometer  was used to count platelets, red blood cells (RBC), and white blood cells (WBCs). Sahli’s hemoglobin meter was used to estimate hemoglobin (Hb) content. A modified Westergren method was followed to measure erythrocyte sedimentation rate (ESR) . Afterwa2O2 hydrolysis at 240 nm after every minute, and one unit of catalase activity was determined as an absorbance change of 0.01 units per minute. The results are expressed as unit per milligram protein (U/mg protein). GSH was measured at 405 nm by oxidizing the serum with DTNB as μM/mg protein and NO was measured by reducing Griess reagent at 540 nm. A sodium nitrite curve was used to quantify NO amount in serum as μM/mg protein.To investigate the fate of sex hormones in BPA intoxication and the protective ability of NA, testosterone, FSH (follicle stimulating hormone), LH (luteinizing hormone), and estradiol were quantified according to a well-established method as previously described . Quantifp < 0.05 and p < 0.01.Data obtained in this study are presented as mean ± SD. One-way analysis of variance was performed to determine the variability among groups by Statistix 8.1. Graph Pad Prim 8.1 was used to construct different graphs. Tukey’s multiple comparisons were used to calculate significant differences among groups at I. batatas in prostatitis and suggests that NA, as a small, potent molecule, has the ability to induce apoptosis and death in PCa cells. We created a simple, quick, and easy approach for multifaceted screening of compounds for antiproliferative and apoptotic potential. ADME and pharmacokinetic profile also reveal that NA can be a potential biomolecule for anti-cancer pharmaceutical preparations with higher bioavailability and less toxicity. Targeted and specified oncological formulations can also be used to evaluate the anti-cancer potential of the compound. Therefore, more detailed investigations are warranted to declare NA as a potential therapeutic agent in formulations for the prevention and treatment of prostate cancer and other types of cancers.The present study validates the ethnomedicinal use of


Their structures were determined by single crystal X-ray diffraction and characterized by elemental analysis, infrared spectroscopy and thermogravimetric analysis. Dy-1 and Tb-3 are isostructural with a conjugate bimolecular four-nuclear cluster structure constructed with intramolecular hydrogen bonds and they form a 3D supramolecular structure with intermolecular hydrogen bonding. Sm-2 is a one-dimensional chain structure and is further connected by intricate hydrogen bonds into a three-dimensional supramolecular structure. These three compounds exhibit significant characteristic luminescence from the ligand to the central Ln(iii) ion, which is found by solid-state photoluminescence measurement. Sm-2 exhibits a long luminescence lifetime and high fluorescence quantum yield. A slow relaxation phenomenon is observed for the dysprosium compound by measuring the alternating-current susceptibility at low temperature and the underlying mechanism was further confirmed by theoretical calculations.Three lanthanide compounds have been synthesized, namely, {[Dy The structures, fluorescence and magnetic properties of series three lanthanide complexes have been investigated. Dysprosium complex exhibit the single-molecule behavior. This disadvantage can be overcome by using highly absorbent ligands to efficiently sensitize lanthanide ions. After the introduction of suitable organic ligands, lanthanide compounds exhibit unique photophysical properties, such as strong luminescence, high quantum yield, long luminescence life and large Stokes shifts.12,13 Experiments show that H2bpda ligand is an efficient sensitizer for the lanthanide ions because the energy gaps between the ligand and lanthanum ions is suitable for the effective ligand-to-metal energy transfer. Moreover, H2bpda ligand can be coordinate to lanthanide ions in various modes, such as unidentate coordinating, bidentate chelating and bridging coordination since the carboxyl group of the ligand can be partially or completely dehydrogenated by adjusting the pH value.14 Thereby, the rich diversity of structures have the potential to have some fascinating properties. Meanwhile, the magnetic properties of lanthanide compounds are also an attractive field15,16 and they have provided an opportunity to shed light on tuning of the magnetic properties of Ln(iii) compound.17 Single-molecule magnets (SMM) exhibit magnetic bistability and quantum magnetic properties due to the existence of magnetic anisotropic energy barrier, which makes it a candidate material for ultra-high density information storage, quantum computing and molecular spintronic.18 The magnetic measurements in this article reveal that Dy-1 displays weak magnetic relaxation under a zero dc field. Combined with the ab initio calculations, the magnetic anisotropy and magnetic dynamic of Dy-1 were studied.Through the past decades, lanthanide compounds including europium spectra (4000–400 cm−1) were collected in the solid state on an Avatar™ 360 E. S. P. IR spectrometer using KBr pellet. Using SDT 2960 thermogravimetric analyzer, the temperature rise rate is 10 °C min−1 (Al2O3 ceramic disc is the support) when nitrogen flow is 40 mL min−1 in the range of 30–800 °C, and thermogravimetric analysis (TGA) is carried out. Solid-state luminescence spectra, luminescence lifetimes and luminescence quantum yield (QY) of the three compounds were measured with an Edinburgh instrument FLS1000 fluorescence spectrometer at room temperature. Luminescence QY was also collected by the same Edinburgh FLS1000 which equipped with an integrating sphere. Magnetic susceptibility were measured with Quantum Design PPMS-XL9 VSM. DC variable-temperature magnetic susceptibilities were measured under a 0.1 T applied magnetic field in 2–300 K. The diamagnetic contribution calculated by Pascal constants was used to correct all the data.H2.22bpda , 0.5 mL Ln(NO3)3  (0.1 mol L−1) and 12 mL deionized water were mixed together and stirred for several minutes, adjusting the pH value to 4 with hydrochloric acid. The mixture was placed in a 25 mL Teflon-lined autoclave, heated at 150 °C autogenic pressure for 3 days, and then cooled to room temperature at a rate of 1 °C h−1. After filtration, washing and drying, crystals of 1–3 were obtained suitable for X-ray diffraction analysis.H144H98Dy8N24O61 (%): C, 38.95; H, 2.22; N, 7.57. Found: C, 38.57; H, 2.26; N, 7.58. IR (cm−1): 3074–3380 br, 2351 m, 1630–1556 vs., 1465 s, 1415 s, 1380 vs., 1272 s, 1192 s, 1160 s 1087 m, 1018 s, 914 m, 856 s, 775 s, 678 s, 644 s, 570 s, 428 s.Dy-1: colorless block crystals. Yield: 0.032 g (58%) based on dysprosium element. Anal. calcd for C24H14N4O9Sm (%): C, 44.16; H, 2.16; N, 8.58. Found: C, 44.17; H, 2.16; N, 8.59. IR (cm−1): 3075–3380 br, 2350 m, 1630–1554 vs., 1465 s, 1415 s, 1380 vs., 1271 s, 1190 s, 1160 s 1085 m, 1018 s, 915 m, 856 s, 775 s, 678 s, 645 s, 570 s, 428 s.Sm-2: yellow primrose blocks crystals. Yield: 0.0251 g (77%) based on samarium element. Anal. calcd for C144H98N24O61Tb8 (%): C, 39.20; H, 2.24; N, 7.62. Found: C, 39.19; H, 2.25; N, 7.60. IR (cm−1): 3070–3460 br, 2353 m, 1630–1558 vs., 1465 s, 1419 s, 1381 vs., 1273 s, 1192 s, 1161 s, 1087 m, 1018 s, 914 m, 856 s, 775 s, 678 s, 644 s, 570 s, 428 s.Tb-1: colorless block crystals. Yield: 0.021 g (75%) based on terbium element. Anal. calcd for C2.3λ = 0.71073 Å) at 296(8) K. Structures have been solved with olex2.solve and refined with ShelXL (2014) and olex2.refine.19 Crystal analytical data are shown in †The X-ray intensity data of compounds were collected on an Oxford Diffraction Super Nova area-detector diffractometer using mirror optics monochromatic MoKα radiation  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O)) and bending vibration (δ(O–H)) of carboxylic acid, respectively. These two bands disappeared in complexes 1–3 and two new ones of 1545–1667 cm−1 and 1378–1457 cm−1 appeared. The bands at 1545–1667 cm−1 could be attribute to the asymmetric stretching vibrations of carboxylate (νas(COO−)), and the bands at 1378–1457 cm−1 could be ascribed to the symmetric stretching vibrations of carboxylate (νs(COO−)) in the complexes.20 The separation (Δν) between νas(COO−) and νs(COO−) can be used to explain the coordination types of carboxyl groups in ligand. Therefore, the Δν values of 167–210 cm−1 in the spectra of compounds 1–3 suggest that the carboxylate groups may coordinate to the lanthanide ions via monodentate and bidentate coordination modes.21,22In the IR spectrum of H2bpda appeared at 302 nm, which could be ascribed to the n–π* or π–π* absorption of conjugate pyridine ring of the ligand. This peak disappears in the complexes and new absorption peaks appeared at 305–317 nm. Relative to the ligand, the peak red shift in the complexes indicated that the ligand coordinated to the lanthanide ions and lower the energy.23 In addition, compared with the ligand, the absorption spectra of lanthanide compounds changing, indicated that the coordination of the lanthanide ions significantly influence the energy levels of the ligands.24The UV-vis absorption spectra of the ligand and the three complexes were measured in DMF solvent. As shown in 3.2iii) ion, three bpda2− ligand, three coordinated water molecules. Dy1 is eight-coordinated of which four oxygen atoms and four nitrogen atoms come from two bpda2− ligands, respectively. Dy2 is also eight coordinated, where two oxygen atoms and two nitrogen atoms come from the same bpda2− ligand and three oxygen atoms from three water molecules and the rest of the oxygen atom from another bpda2− ligand. Dy–O bond distances are in the range of 2.277(2)–2.390(3) Å and Dy–N lengths in the range of 2.457(2)–2.501(3) Å, which indicate that oxygen atom has stronger coordination capacity than nitrogen atom. The adjacent dysprosium ions Dy1 and Dy2 are connected by O9 of the carboxyl group. The distance between Dy1 and Dy2 is 6.278 Å. The [Dy2(bpda)3(H2O)3] units are connected by hydrogen bonds between lattice water molecules and coordinated water molecules /n space group. As shown in 2− ligands and another oxygen atom from one water molecule, resulting in a distorted tri-capped trigonal prismatic coordination sphere around Sm1 ion. The Sm–O bond lengths are in the range of 2.357(3)–2.659(3) Å and Sm–N ones in 2.545–2.659 Å. The bond angles around Sm ion vary from 62.09(9) to 157.96(9) . The second main mass loss (58.43%) occurs in 395–491 °C corresponding to the decomposition of the residual organic components of the compound . The residue is thought to be Sm2O3 .3.44F9/2 → 6H15/2 (magnetic-dipole), 4F9/2 → 6H13/2, 4F9/2 → 6H11/2  and 4F9/2 → 6H9/2, corresponding emission peaks are 487, 546, 577 and 662 nm, respectively. The emission band at 577 nm (4F9/2 → 6H11/2) is the strongest among the four bands, which is strongly influenced by the local environment prevailing around Dy(iii) ions.25 The emission intensity ratio of 4F9/2 → 6H11/2vs.4F9/2 → 6H15/2 is 2.6 and so high value indicates that this complex lacks a centre of symmetry,26,27 since 4F9/2 → 6H11/2 is probed to determine coordination symmetry around Dy(iii) system.28 To Sm-2, the sharp peaks at 590, 616, 652 and 700 nm should be ascribed to the Sm(iii) transitions of 4G2/5 → 6HJ . The emission band at 616 nm (4G5/2 → 6H7/2) is the strongest among the four bands  of the Tb(iii) ions, respectively.29,30 Among the four bands, the emission band at 547 nm is the strongest, emitting green light visible to the naked eye under laser lamp.The solid-state emission spectra of the compounds 1–3 were measured at ambient temperature and shown in ur bands . The lumur bands  shows foτ) obtained by fitting curves of the decay curves on FLS1000 Photoluminescence Spectrometer are listed in τ for 2 is 847 μs at 616 nm and it has the longest fluorescence lifetime among the three complexes. It is also a rather better value with comparison to some previous Sm-complex.31,3213,14,31 though a small amount of water molecules are involved in the coordination sphere.The data of solid quantum efficiency measured in the condition of maximum emission for 1–3 are reported in 3.5χMTvs. T in χMT value is 51.55 cm3 K mol−1 at 300 K, which is lower than the expected value of 56.68 cm3 K mol−1 for four uncoupled Dy(iii) ions . Upon cooling, χMT gradually decreases until 12 K and then drops rapidly to reach a minimum of 42.67 cm3 K mol−1 at 2 K. The susceptibility χM (blue line in 3 mol−1 at 2 K. This behavior is attributed to antiferromagnetic exchange interaction between the Dy(iii) ions and Stark energy level degeneracy caused by spin–orbit coupling.33,34The direct current (dc) magnetic susceptibility of Dy-1 was studied in an applied magnetic field of 2000 Oe and the temperature range 300–2 K and plotted as The variation of alternating current (ac) susceptibility with frequency and temperature for Dy-1 under 2.2 Oe ac oscillating field was studied, and the magnetization kinetics was investigated  and 8. Tχ′′ or χ′ vs. frequency curves. As shown in χ′ and χ′′ keep increasing on cooling, which indicates that there is a slow relaxation of magnetization phenomenon in Dy-1 expected for a single-molecule magnet. Furthermore, the phenomenon of magnetic relaxation becomes more obvious with the increase of magnetic field intensity and the decrease of temperature. However, slow relaxation of magnetization for Dy-1 is observed experimentally only in a narrow temperature range, and no maximum of χ′′ is observed in the temperature window, in which the energy barrier and corresponding relaxation time could not be calculated. Alternatively, a method employed by G. Y. Yang et al.35 can be used to evaluate roughly the energy barrier Ea and relaxation time τ0 based on the following relationship  vs. 1/T at different frequencies, we obtained an estimate of the activation energy Ea/kB = 1.14 K and τ0 = 1.2 × 10−6 s. A more precise result must wait for very low temperature measurements (T < 1 K) by using a micro-SQUID.By nonlinear fitting the experimental ln(iii) coordinating to nitrogen or oxygen atoms, the symmetry and intensity of Dy(iii) coordination field are different, which may strongly impact on magnetic anisotropy, leading to distinct dynamic behavior.36,37 It is visible that magnetic relaxation mainly results from Dy(iii) anisotropy that is very sensitive to changes of the coordination geometry.As described in 3.2 structural descriptions section, Dy1 is coordinated to four nitrogen atoms and four oxygen atoms while Dy2 is connected to two nitrogen atoms and six oxygen atoms. Due to the bond lengths are different after Dy(M) for Dy-1 were collected in the field range 0–70 kOe and at 2, 3 and 5 K, as shown in the 38 At 2 K, the M versus H data of Dy-1 exhibit slim butterfly-shaped hysteresis loops without a remanence and a coercive field  compounds base on H2bpda ligand. Complexes 1, 2 and 3 exhibit strong f–f transition and long luminescent lifetime and high quantum efficiency, which indicated that the ligand H2bpda was a good organic chelator to absorb and transfer energy to Dy(iii), Sm(iii), Tb(iii) and could be considered as promising candidate in the design of photoluminescence devices. Dy-1 exhibit slow magnetization relaxation and single molecular magnet behaviour except showing obvious yellow emission, which it may be a candidate of photo-magnetic functional.This article reports the syntheses, crystal structures, fluorescence and magnetic properties of three Ln(There are no conflicts to declare.RA-010-C9RA10975G-s001RA-010-C9RA10975G-s002


Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.Pullulanase , a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava  It is synthesised in plastids, including chloroplasts in leaves and amyloplasts in tubers and grain. Starch consists of two α-glucans: amylose and amylopectin. The amylose mainly presents as a linear glucan structure comprised of α-1,4 glycosidic bond with a limited α-1,6-linked branching (less than 1%). This glucan is synthesised by the concerted action of ADP-glucose pyrophosphorylase (AGPase) and granule-bound starch synthase I (GBSSI). In contrast, amylopectin is much more complex since it harbours highly branched structures generated by the action of AGPase, soluble starch synthases (SSs), starch branching enzymes (SBEs), and starch debranching enzymes (DBEs) . In addisex phenotype arises , members of glycoside hydrolase family 13 (GH13), are found in plants and microorganisms. They could be categorised into two major types: isoamylase  and pullulanase  according to their peptide sequences and substrate specificities. Although they both can act on α-1,6 glucosidic linkages , only PUe arises . Furthere arises . Deficiee arises . To dateHordeum vulgare) (HvLD), a monocotyledon, has been biochemically and structurally characterised  located between N-terminal domain and catalytic domain (Solarium tuberosum L.) (StPUL) (Spinacia oleracea L.) (SoPUL)   and puri (SoPUL)  were bio (SoPUL) .Manihot esculenta Crantz), one of the most important economic crops in Asia, Africa and South America, in particular, possesses a very high starch content in its tubers (in vivo (Manihot esculenta Crantz (MePUL), with a view to determining its biological role in starch metabolism which remained unclear.Cassava . The RNA was converted into cDNA using a SMARTer® RACE 5′/3′ Kit (Clontech®) followed the manufacturer’s manual. Full length MePUL was amplified by PrimeSTAR® HS DNA Polymerase (Clontech®) using the 5′ RACE cDNA as a template. 5′ RACE forward primer provided in the RACE kit and a gene specific reverse primer (5′ GTAAGCTTAAATTTTCCTAGGCTCAACAAACACAG 3′) were used for PCR. The PCR product was cloned into a pJET1.2/blunt (Thermo Fisher Scientific™) and the putative gene was confirmed by DNA sequencing (1st BASE DNA sequencing). A new forward primer (5′ CTCTA GCTAGCGGTTCCACTCCCACTTCTGAGTTGC 3′) excluding a transit peptide sequence predicted by ChloroP1.1 server and a new reverse primer (5′ AAGGAAAAAATGCGGCCGCAATTTTCCTAGGCTCAACAAACAC 3′) were specifically designed. The gene excluding a stop codon was subcloned into a pET21b vector via NheI and NotI restriction sites in-framed with C-terminal 6x His sequence of the plasmid. The recombinant plasmid (pET21b-Mepul) was transformed into E. coli TOP10 (Invitrogen™). The plasmid was extracted and subjected to DNA sequencing.Total RNA was extracted from nine-month-old cassava 2.2Mepul was transformed into E. coli SoluBL21 (DE3) and grown on an LB agar plate containing 100 μg/mL ampicillin. Then, the transformant was cultured in LB broth containing 100 μg/mL ampicillin until OD600 reached ~ 0.6. Cells were induced for protein production by adding 0.4 mM IPTG and continuously cultured at 16°C with shaking at 250 rpm for overnight. Cells were collected by centrifugation at 8,000 x g for 10 min at 4°C. After that, the cell pellet was suspended in buffer A  and lysed by ultrasonication. After that, cell debris was removed by centrifugation at 10,000 x g for 10 min at 4°C. The supernatant was loaded onto a Histrap™ FF column equilibrated with the buffer A. The column was washed by the same buffer and the rMePUL was eluted with a linear gradient of buffer B . Finally, the purified fractions containing MePUL activity were pooled and subjected to dialysis against 25 mM phosphate buffer pH 7.2 at 4°C for overnight. SDS-PAGE and western blot analysis were used for evaluating protein purity.The pET21b-2.3MePUL was analysed with an MAbPac™ SEC-1 column . The system was run in 25 mM phosphate buffer pH 6.8 containing 300 mM NaCl with a flowrate of 0.2 mL/min at 30°C. The signal was monitored by measuring A280. Gel Filtration Standards (Bio-Rad) were used for molecular weight calibration. To investigate the multimeric state of rMePUL/rMeISA3  pullulan (Megazyme®) in 25 mM acetate buffer pH 6.0 in the total volume of 0.2 mL at 50°C to determine debranching activity. The reaction was terminated by adding an equal volume of 2,4-dinitrosalicylic (DNS) acid reagent  pullulan in 25 mM buffer at various pH values [acetate buffer (pH 4.0 – 6.0) and phosphate buffer (pH 6.0 – 7.5)] at 30°C.The purified r2.5.2MePUL was assayed in reactions containing 0.75% (w/v) pullulan in 25 mM acetate buffer pH 6.0 incubated at various temperatures (30 - 60°C).The purified r2.5.3MePUL was measured in the reaction containing 0.75% (w/v) pullulan in 25 mM acetate buffer pH 6.0 and 1 mM EDTA at 50°C. The activity was compared with the reaction without EDTA.To evaluate the effect of EDTA, the activity of purified rMePUL was dialysed against 1 mM EDTA in 25 mM phosphate buffer pH 7.2 at 4°C overnight. Next, the pretreated enzyme was incubated with 0.75% (w/v) pullulan substrate in 25 mM acetate buffer pH 6.0 at 50°C with the presence of 10 mM metal ion .The effect of various metal ions was observed. The purified r2.5.4MePUL was incubated with 0.75% (w/v) of different types of substrates  in 25 mM acetate buffer pH 6.0 at 50°C.The purified r2.5.5MePUL was incubated with pullulan or β-limit dextrin in 25 mM acetate buffer pH 6.0 at 50°C in 0.1-mL reactions under enzyme initial velocity of 15 min. The reactions were initiated by addition of 0.75 μg of the purified rMePUL and then analysed by DNS method. To determine the effect of some maltooligosaccharides on enzyme activity, 1 - 3 mM maltose or maltotriose were mixed in the reactions. The kinetic parameters were fitted and determined by the OriginPro 2017 software.To assess enzyme kinetics, the purified r2.5.6MePUL was incubated with 0.75% (w/v) pullulan and 0.01% (w/v) β-CD in 25 mM acetate buffer pH 6.0 in the total volume of 0.2 mL at 50°C. The activity was assayed by DNS method. Relative activity was compared with the control reaction without adding β-CD.The r2.62.6.1MePUL, purified rMePUL was incubated with 2.5 mg/mL various oligosaccharides  in 25 mM acetate buffer pH 6.0 at 50°C for 2.5 hr.To determine the substrate specificity of KpPUL  and the purified rMePUL were also compared. 0.2 U/mL of both PULs were incubated with 0.5% (w/v) pullulan in 25 mM acetate buffer pH 6.0 at 25°C and at 50°C for KpPUL and rMePUL, respectively. The products were analysed at 0, 0.5, 1, 3.5, 6, and 24 hr after incubation.In addition, to confirm the specificity of pullulan cleavage, products produced by 254, Merck). The TLC was run in a TLC tank equilibrated with acetonitrile:ethylacetate:1-propanol:water (85:20:50:60). Finally, the result was visualised by heating at 110°C for 10 min after staining with orcinol solution orcinol solution (sulphuric acid:ethanol:water (5:27:13) containing 0.2%(w/v) orcinol) (The products from the enzymatic reactions were spotted onto TLC plates (TLC Silica gel 60 Forcinol) .2.6.2KpPUL and rMePUL, respectively. The products were analysed at 0.5 hr after incubation.To analyse the product pattern according to the debranching activity of PUL on pullulan substrate, The 0.2 U/mL of both PULs were incubated with 0.5% (w/v) pullulan in 25 mM acetate buffer pH 6.0 at 25°C and at 50°C for The enzymatic reactions were analysed by HPAEC-PAD using a CarboPac™ PA-100 column with a flowrate of 1 mL/min at 30°C. The products were eluted with a linear gradient of 0 – 0.5 M sodium acetate in 150 mM NaOH solution for 35 min, followed by 0.5 M sodium acetate in 150 mM NaOH for 5 min.2.6.3Each sample obtained from PUL reactions (section 2.6.2) was desalted by using a mixed bed resin . The samples were diluted 10-fold prior to mixing with 10 mg/mL of 2,5-dihydroxybenzoic acid with the ratio of 1:1 and spotted onto a MALDI target plate. Masses were analysed with a JEOL SpiralTOF MALDI Imaging-TOF/TOF Mass Spectrometer using spiral mode.2.7MeISA3 and rMePUL activities were defined under the same condition; 0.75% (w/v) β-limit dextrin (Megazyme®) in 25 mM acetate buffer pH 6.0 at 37°C. For the individual enzyme reactions, 0.2 U/mL of either rMeISA3 or rMePUL was incubated with 0.5% (w/v) β-limit dextrin in 25 mM acetate buffer pH 6.0 at 37°C. For the mixed-enzyme reaction, 0.1 U/mL of each enzyme was added to the reaction and then incubated under the same conditions. The reactions were collected at the time points of 0, 0.5, 1, 3.5, 6, and 24 hr and analysed by HPAEC-PAD and MALDI-TOF MS.To investigate the possible relationship between PUL and ISA3 in starch degradation process, r2.8MePUL using crystal structure of barley limit dextrinase (E510A mutant) in complex with a 6II-O-maltotriosylmaltotriose   were added to neutralise the systems. PMEMD module of AMBER 20 was then used to minimise and simulate the system under the periodic boundary condition as previously described (oc). RMSD value of all atoms of the protein or ligands were calculated with respect to the minimised structure. The structure was found to be stable in the range of 40 to 50 ns. Therefore, this trajectory was used for analysis. Hydrogen bonds will present if the following conditions are met: (i) a donor-acceptor distance of less than 3.5, and (ii) a donor H-acceptor bond angle of less than 120°. H-bond occupations > 50% were taken into account (SWISS-MODEL server was employed to constructed homology model of mature D: 4J3W)  as a temt pH 6.0 . LEaP mot pH 6.0  with theescribed . In the escribed  of AMBER account . The bin account  using MM account  of AMBER2.9Mepul was cloned into a pCR™8/GW/TOPO® TA vector (Invitrogen™) based on circular polymerase extension cloning technique (® LR Clonase® II (Invitrogen™). The pGWB5-Mepul was transformed into an Agrobacterium tumefaciens GV3101 by electroporation.The full-length echnique . The genechnique  using GaA. tumefaciens harbouring pGWB5-Mepul and A. tumefaciens containing the silencing suppressor p19 gene were co-infiltrated into 4-week Nicotiana benthamiana leaves . GFP fluorescence signal was monitored at 488/510 nm of excitation/emission, while chloroplast autofluorescence was observed at 633/664 nm.a leaves . A pGWB233.1Mepul sequence with an open reading frame of 2,904 bp  transformant. After purification by a metal ion affinity column, SDS-PAGE and western blot analyses showed a single band of the purified rMePUL. The apparent molecular mass of ~ 100 kDa corresponded to that calculated for monomeric protein the by Expasy Compute pI/Mw tool (https://web.expasy.org/compute.pi/) which was 100776.98 Da (including 6x His). Nevertheless, rMePUL exhibited two protein peaks by gel filtration chromatography with the calculated sizes of 197 and 84 kDa, as shown in The full-length 3.2MePUL exhibited optimal temperature and pH at 50 °C and pH 6.0, respectively , were as expected rather similar, as shown in the TLC image  simulation analysis. The RMSD plots showed that MePUL was likely stable at 10 ns of simulation, while branched maltohexaose was stable after 20 ns of simulation  longer than 7, while products of rMeISA3 were mainly short maltooligosaccharides (DP <5) as shown by HPAEC-PAD  than a summary of the peak area obtained from each single rMePUL and rMeISA3 reaction , Oryza sativa pullulanase , Solanum tuberosum pullulanase , Arabidopsis thaliana pullulanase  Zea mays pullanase , and Spinacia oleracea pullulanase , and Klebsiella pneumoniae pullulanase, a bacterial pullulanase . The sequence alignments are presented in MePUL showed very low similarity compared with other types of Manihot esculenta debranching enzymes  shown in MePUL belongs to GH 13 subfamily 13 (pullulanase subfamily) based on sequence similarity  transformant. Noticeably, protein MW obtained from gel filtration chromatography under non-denaturing condition was ~ 2-fold higher than the MW shown by SDS-PAGE analysis . Notably, maltotriose, the main product of pullulan hydrolysis, was a potential positive effector of the rMePUL action, since it could clearly promote binding affinity (k or mK) of the enzyme to pullulan substrate. However, maltose and maltotriose could also increase catk around 1.2 - 1.7 folds for β-limit dextrin hydrolysis -6-deoxy-6-thiocyclomaltoheptaose , panose or isopanose. The experimental data demonstrated that the active site of rMePUL required longer substrates in order to make productive contacts with the longer binding site. This information acorrelated with computational analysis, which showed that the MePUL-branched maltohexaose complex exhibited high binding energy at +2, +1, -1, -2, and -3 sites  was apparently increased (MeISA3 and MePUL worked synergistically, possibly by providing favourable substrates for each other by removal of some unfavourable branched points due to their different substrate preferences. Importantly, considering the prospect of an heterocomplex between rMeISA3 and rMePUL, no multimeric rMeISA3/rMePUL complex was observed by gel filtration chromatography (To analyse the relationship between ubstrate Figure 7in vivo revealed that deficiency of PUL in rice (MeISA3 and MePUL seemed to exhibit overlapping activities in debranching processes, cassava starch catabolism may not be efficient when only either PUL or ISA3 are present, which contrasts with some previous reports from other plant species (in vivo studies showed the relationship between these two enzymes, but they did not clearly unravel the reason for the conservation of both enzymes. Nevertheless, our results show that both PUL and ISA3 are necessary for efficient starch degradation in vitro: their synergistic action may account for why these two types of debranching enzymes are typically present in plant cells.Some previous studies  in rice  and maiz in rice  slightly species . Importa5Manihot esculenta Crantz ‘KU50’ was successfully cloned, expressed, and biochemically characterised. This debranching enzyme was clearly categorised as pullulanase type I due to its preference for cleaving α-1,6 linkages of pullulan and β-limit dextrin. The debranching action of PUL is conserved in most plants, both mono- and di-cotyledons, and is located in plastids. The present work demonstrates the synergistic action between MePUL and MeISA3, which is important for efficient starch degradation in cassava and which may have implications for biotechnology innovation in relation to harnessing starch for food and materials applications.Pullulanase from The original contributions presented in the study are included in the article/KW, TC, and PP: conceptualization. KW, TC, MK, and PP: methodology. KW, TC, and PP: formal analysis. KW, TC, MK, RF, and PP: investigation. TC and PP: resources. TC and PP: data curation. KW, TC, and MK: writing - original draft. KW, TC, RF, and PP: writing - review & editing. KW, TC, MK, and PP: visualization. KW and PP: supervision. PP: funding acquisition. PP: Project Administration. All authors contributed to the article and approved the submitted version.


Garcinia hanburyi tree, exhibits various pharmacological properties, including anti-inflammation, antioxidant, anti-proliferation, and anti-cancer activity. However, its effect on inhibiting cell proliferation in PKD is still unknown. This study aimed to determine the pharmacological effects and detailed mechanisms of GA in slowing an in vitro cyst growth model of PKD. The results showed that GA (0.25–2.5 μM) significantly retarded MDCK cyst growth and cyst formation in a dose-dependent manner, without cytotoxicity. Using the BrdU cell proliferation assay, it was found that GA (0.5–2.5 μM) suppressed MDCK and Pkd1 mutant cell proliferation. In addition, GA (0.5–2.5 μM) strongly inhibited phosphorylation of ERK1/2 and S6K expression and upregulated the activation of phosphorylation of AMPK, both in MDCK cells and Pkd1 mutant cells. Taken together, these findings suggested that GA could retard MDCK cyst enlargement, at least in part by inhibiting the cell proliferation pathway. GA could be a natural plant-based drug candidate for ADPKD intervention.Abnormal cell proliferation and accumulation of fluid-filled cysts along the nephrons in polycystic kidney disease (PKD) could lead to a decline in renal function and eventual end-stage renal disease (ESRD). Gambogic acid (GA), a xanthone compound extracted from the brownish resin of the  Autosomal dominant polycystic kidney disease (ADPKD), a common renal genetic disorder, is usually found in adults and has an incidence of 1:1000 . TherapePKD1 gene or PKD2 gene, which encode the proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively [ADPKD is caused by mutations of either the ectively . Dysfuncectively . Previouectively , mTOR siectively , and Wntectively . In addiectively . Thus, r38H44O8) is the principal bioactive and caged xanthone compound isolated from the brownish/orange gamboge resin of the Garcinia hanburyi tree [Pkd1 mutant cell proliferation.Gambogic acid . It was dissolved in 100% DMSO to prepare a stock solution. Trypsin, fetal bovine serum, penicillin, streptomycin, and DMEM/F-12 Ham were purchased from Invitrogen . Protease inhibitor cocktail was purchased from Hoffman-La Roche Ltd. . The BrdU cell proliferation assay kit and enhanced chemiluminescence ECL solution were obtained from Calbiochem . Collagen type I (PureCol) was purchased from Advanced BioMatrix . Primary antibodies , including anti-p-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-p-AMPK (Thr172), anti-AMPKα, anti-p-S6K (Thr421/Ser424), anti-S6K, anti-β-actin, and anti-rabbit IgG HRP-linked antibody, were purchased from Cell Signaling .+Pkd1−/  and −Pkd1−/  cells were kindly given by Prof. Stefan Somlo, Yale University School of Medicine, Connecticut, USA. The MDCK cells were cultured at 37 °C and the Pkd1 mutant cells were cultured at 33 °C in a 95% humidified and 5% CO2 atmosphere in a 1:1 DMEM/F-12 Ham medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL of insulin, 5 µg/mL of transferrin, 5 µg/mL of selenium X, and 5 µg/mL interferon γ for the Pkd1 mutant cells. The cells were trypsinized with 0.25% trypsin and centrifuged at 600× g before seeding.Madin-Darby canine kidney (MDCK) renal epithelial cells were kindly provided by Prof. David N. Shephard, University of Bristol, Bristol, UK, and mouse renal cystic epithelial cells 3, 100 U/mL penicillin, and 10 µg/mL streptomycin, and were incubated at 37 °C for 90 min. After gelation, MDCK medium containing 10 µM forskolin was added to each well to stimulate cAMP-induced cystogenesis from day 0 to day 6, and the media was changed every two days. To evaluate the inhibitory effect of GA on MDCK cyst formation, GA at a dose of 0, 0.25, 0.5, 1, and 2.5 µM was added to the culture medium containing 10 µM forskolin from day 0. The MDCK medium containing forskolin and GA was changed every two days. On day 6, the number of cysts (with diameters ≥ 50 µm) and non-cyst (with diameters of < 50 µm) colonies were counted and captured at 10× magnification using an inverted microscope . For MDCK cyst growth, cells were plated and sustained in collagen gel from day 0 to day 6. GA  was added to the medium containing 10 µM forskolin from day 6 to day 12. The MDCK medium containing forskolin and GA was changed every 2 days from 6 days onwards. Photographs of individual cysts  were taken on days 6, 9, and 12 by an inverted microscope . Cyst diameter (µm) was measured using Image J software. At least 30 cysts were found per condition.Type I MDCK cells were added to an individual well of a 24-well plate and suspended in 0.4 mL of ice-cold collagen containing 10% 10× minimum essential medium (MEM), 10 mM HEPES, 27 mM NaHCOThe MTT assay was used to assess GA cytotoxicity. MDCK cells  were grown in a 96-well plate for 24 h and were exposed to GA at doses of 0, 0.25, 0.5, 1, 2.5, and 5 µM in serum free media for 24 h. A 10% MTT solution (5 mg/mL) was added and maintained without light for 4 h. The medium was then removed, and 100 µL DMSO was added. The absorbance at 530 nm was measured and the percentage of cell viability was expressed as 100% of control.Pkd1 mutant cells (8000 cells/well) were seeded in 96-well plates in DMEM/F-12 Ham medium supplemented with 10% FBS and grown for 24 h. Cells were treated with serum-free medium containing GA at doses of 0, 0.25, 0.5, 1, and 2.5 µM, and blasticidin (20 µg/mL) treatment was used as a positive control for 24 h. BrdU reagent solution was added 18 h later and incubated for 6 h. The absorbance was measured at 490 nm by an automated microplate reader, and BrdU cell proliferation was calculated as 100% of control.A BrdU cell proliferation kit was used to assay the cell proliferation. MDCK and +Pkd1−/, and −Pkd1−/ treated cells with various concentrations of GA (0–2.5 µM for 24 h) were washed with PBS and lysed with ice-cold RIPA buffer  containing a protease inhibitor cocktail. Samples were centrifuged at 10,000× g and the supernatant proteins (60 µg) were separated in 10% SDS-PAGE gels. Samples were then transferred to a nitrocellulose membrane. After blocking non-specific binding with 10% non-fat dry milk at room temperature for 1 h, membranes were incubated with primary antibodies  overnight at 4 °C. The membranes were washed by TBS-Tween 20 solution three times, followed by incubation with secondary antibody for 1 h. After that, the membranes were washed with TBS-Tween 20 solution three times, and the band intensity of the proteins of interest was developed by chemiluminescence (ECL solution).Western blot analysis was performed as described previously . MDCK, Pp < 0.05.All data are expressed as the mean ± SEM. The statistical significance of data between the treatment and control groups was determined by one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Differences were determined to be statistically significant at To rule out cytotoxicity, the effect of GA  or GA at doses of 0.25, 0.5, 1, 2.5 μM from day 6 to day 12 onwards (for the assessment of MDCK cyst growth). The result showed that GA (0.5–2.5 µM) significantly retarded MDCK cyst enlargement in a dose-dependent manner c,e. In a+Pkd1−/ and −Pkd1−/ cells), a BrdU cell proliferation assay was performed. MDCK cells were treated with DMSO (control) or GA at doses of 0.25, 0.5, 1, and 2.5 μM, and evaluated by this assay. The results showed that GA at doses of 0.5–2.5 µM significantly inhibited MDCK and Pkd1 mutant cell proliferation in a dose-dependent manner compared to the control, as well as 20 µg/mL blasticidin as a positive control  ,25,26,27Pkd1 mutant cells by using BrdU incorporation. The results showed that GA (0.5–2.5 μM) significantly suppressed cell proliferation (Pkd1 cell monolayers in a dose-dependent manner (The cAMP-dependent B-Raf/MEK/ERK signaling pathway is one of the key regulators of cell proliferation in PKD pathogenesis . Previouferation . The ERKt manner . This fiPkd1 mutant cell monolayers (Hyperactivated ERK also affects the upregulation of the mTOR signaling pathway. mTORC1 regulates many cell processes, including cell growth, cell survival, and cell metabolism . mTORC1 nolayers . PreviouIn conclusion, our study elucidated that GA retards MDCK cyst enlargement by inhibiting phosphorylation of the ERK1/2 and mTOR/S6K signaling pathways. Furthermore, GA significantly increases activation of phosphorylation of AMPK. Therefore, the combination treatment of an approved drug with GA could be useful for inhibiting renal cystogenesis. However, further study is still needed to investigate the inhibitory effect of GA in slowing renal cystogenesis in an animal model of PKD. Taken together, our findings suggest that GA could be a novel nutraceutical for PKD intervention in combination with other compounds.