Biological effect of alkaloid enriched fractions and reticuline from the Stephania dielsianaY. C. Wu on promyelocyte HL-60 cell line

Biological effect of alkaloid enriched fractions and reticuline from the Stephania dielsianaY. C. Wu on promyelocyte HL-60 cell line | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biological effect of alkaloid enriched fractions and reticuline from the Stephania dielsianaY. C. Wu on promyelocyte HL-60 cell line Petya Dimitrova, Tsvetelina Doncheva, Nadezhda Kostova, Iveta Uzunova, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5585865/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 May, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 6 You are reading this latest preprint version Abstract Stephania dielsiana Y. C. Wu is a traditional medicinal plant that, like the other species of the genus Stephania , is used in some Asian countries for the treatment of various conditions and diseases. Phytochemical studies have revealed that the species’ therapeutic activity is mainly due to isoquinoline alkaloids. This study focused on the biological effects of S. dielsiana root tuber extract, alkaloid enriched fractions and the isolated alkaloid reticuline on promyelocytic cell line HL-60. Systematic chromatographic techniques were applied to obtain three alkaloid-enriched fractions and eleven pure alkaloids from the methanolic extract of S . dielsiana root tubers. The quantities of alkaloids in the plant extract and its fractions were determined using HPLC method. The biological effects of the extract, alkaloid fractions and the reticuline were evaluated using colorimetric and flow cytometric assays. The crude extract and alkaloid enriched fractions inhibited cell growth at concentrations higher than 50 µg/ml. One of the alkaloid enriched fractions (St-2) affected the synchronicity of proliferation till day 3. Based on the quantification analysis, reticuline was found as the predominant alkaloid in this fraction with the relative content of 64.9%. Reticuline (9.5 µg/ml) modestly affected mitochondrial functions, decreased the level of phosphorylated p38 kinase in non-stimulated cells, but increased stimulus-induced p38 phosphorylation and elevated the CD11b + cells, indicative for myeloid differentiation. In conclusion, we provide evidence that reticuline derived from S. dielsiana root tubers affected growth, proliferation, apoptosis and differentiation of HL-60 cell line. Stephania dielsiana isoquinoline alkaloids reticuline p38 kinase CD11b HL-60 cell line Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Genus Stephania (Menispermaceae) consists of more than 60 species distributed in the tropical and subtropical areas of Asia and Africa and some regions of Oceania (Semwal et al., 2010 ). The Stephania plants are herbaceous lianas with large root tubers and have been widely used in the traditional Chinese medicine for the cure of various health disorders and diseases. The crude plant extracts show a wide range of pharmacological properties, including antimicrobial, antiparasitic, antitumor, antihyperglycemic, antioxidant, anti-inflammatory, antinociceptive, antipyretic, analgesic, neuroprotective, and cardioprotective activities (Semwal et al., 2010 ; Wang et al., 2022 ). So far, the majority of the studies have been focused on S. tetrandra S. Moore, S. cepharantha Hayata, S. glabra (Roxb.) Meyers, S. japonica (Thunb.) Meyers and S. venosa (Blume) Spreng (Semwal et al., 2010 ). The species are rich in isoquinoline alkaloids and in particular the derivatives of aporphine, proaporphine, morphinan, hasubanan, protoberberine, benzylisoquinoline and bisbenzylisoquinoline (Wang et al., 2022 ; Thien et al., 2018 ; Knockleby et al., 2020 ; Thi Thu Hien et al., 2023 ). The content of isoquinoline alkaloids was found to be highest in the root tubers of the plants, reaching in some cases up to 3–4% of the plant material (Rong et al., 2016 ). Stephania dielsiana Y. C. Wu is a medicinal plant used in traditional Vietnamese medicine for thousands of years to treat insomnia, relieve pain, dispel toxins, improve circulation, inflammation and injuries. The main part of the plant used is the tuberous roots, which are dried, ground into a powder and used to make a decoction. Similarly to other species of the genus Stephania , the biologically active components in the extracts of the species have been found to be the aporphine alkaloids. In previous studies, were found that the alkaloid extract and the alkaloids, crebanine and stephanine, isolated from the Stephania dielsiana root tubers exhibit high inhibitory activity against various pathogens (Deng et al., 2011 ). The same alkaloids isolated from the leaves and stems of the species, as well as the alkaloids oxostephanine and thailandine, have been shown to have potential as anticancer and anti-inflammatory agents (Thien et al., 2018 ; Knockleby et al., 2020 ; Thi Thu Hien et al., 2023 ). A large number of the alkaloids identified from Stephania species have shown a potential to inhibit survival, proliferation and inflammatory responses in various cell lines and models of inflammation-mediated diseases (Semwal et al., 2010 ; Wang et al., 2022 ). The Stephania derived cepharanthine was able to suppress the production of pro-inflammatory cytokines interleukin (IL)-1β or tumor necrosis factor (TNF)-α thought the suppression of mitogen-activated protein kinase p38 (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), part of the activation pathway related to the production of more than 1000 different mediators involved in inflammation, cell migration and regulation of autophagy (Yao et al., 2022 ). The same signaling pathway has been inhibited by the isoquinoline alkaloid, reticuline in the model of obesity-related asthma (Lyu et al., 2024 ). Despite that the antiproliferative and anti-inflammatory activities of some Stephania species has been well characterized and explained their efficacy in phytotherapy, the biological activities of S. dielsana root tubers have been less studied. Herein, we investigated the effect of the methanolic extract, alkaloid enriched fractions and alkaloids from S. dielsana root tubers collected in Vietnam on the growth, survival, proliferation, apoptosis and differentiation of the promyelocytic HL-60 cell line. Material and methods Plant material The root tubers of S . dielsiana Y. C. Wu were collected from Quan Ba commune, Ha Giang province, Vietnam in January 2023. The plant material was identified by Dr. Nguyen Cuong from Institute of Ecology and Biological Resources, Vietnam Academy of Sciences (VAST). A voucher specimen with reference number QTBG-0622 was deposited at the Laboratory of Agro-Pharmaceutical Research, Center for High Technology Research and Development, VAST. Alkaloid extraction and isolation The air-dried and powdered roots (500 g) of S . dielsiana Y. C. Wu were extracted with MeOH (3x 1000 ml) and evaporated to dryness to obtain the crude extract (St-extr.) (15 g). The crude extract was further separated by vacuum liquid chromatography (VLC) on silica gel and eluted with CHCl 3 –MeOH to give fractions St-1 (5.2 g) (CHCl 3 ), St-2 (4.3 g) (CHCl 3 /MeOH, 1/1, v/v) and St-3 (3.1 g) (MeOH). СС on silica gel with eluent petroleum/CHCl 3 /Me 2 CO/MeOH, 4/8/1/2, v/v/v/v of fraction St-1 give 3 subfractions St-1A (582 mg), St-1B (423 mg) and St-1C (125 mg). Subfraction St-1A was subjected to PTLC on silica gel plates (Kieselgel 60 F 254 , Merck) using petroleum/CHCl 3 /Me 2 CO/MeOH, 14/4/1/1, v/v/v/v as mobile phase in NH 3 atmosphere to give the alkaloids oxostephanine ( 1 ) (12.5 mg) and O-Me-bulbocapnine ( 2 ) (13.6 mg). Subfraction St-1B was subjected to PTLC using petroleum/CHCl 3 /Me 2 CO/MeOH, 4/4/1/1, v/v/v/v as mobile phase in NH 3 atmosphere to give the alkaloids: crebanine ( 3 ) (28.2 mg), tetrahydropalmatine ( 4 ) (1.6 mg) and stephanine ( 5 ) (14.2 mg). Subfraction St-1C was subjected to PTLC using petroleum/CHCl 3 /Me 2 CO/MeOH, 4/8/1/2 (v/v/v), as mobile phase in NH 3 atmosphere to give dehydrocrebanine ( 6 ) (8.5 mg). From fraction St-2 using PTLC on silica gel plates (Kieselgel 60 F 254 , Merck) with mobile phase petroleum/CHCl 3 /Me 2 CO/MeOH 4/8/1/2 (v/v/v) the alkaloids reticuline ( 7 ) (14.5 mg), ushinsunine ( 8 ) (6.1 mg), ayuthianine ( 9 ) (7.2 mg), liriodenine ( 10 ) (3.5 mg) and stesakine ( 11 ) (1.2 mg) were isolated. Using the same PTLC (as for St-2) the alkaloid reticuline ( 7 ) (3.9 mg) was purified from the fraction St-3. The presence of alkaloids was visualized by Dragendorff’s spray reagent. The structures of isolated alkaloids 1 – 11 were elucidated by comparison of their 1D and 2D NMR spectra with the available data and resources (Lee et al., 1992; Zhou et al., 2018 ; Thien et al., 2018 ). Purity of isolated alkaloids (from 90 to 98%) was determined by high-performance liquid chromatography. Quantification of alkaloids by HPLC Liquid chromatographic analyses were performed on a Shimadzu (Nexera-LC40 XS, Kyoto, Japan) HPLC system equipped with a PDA detector (М40) managed by LabSolutions software. The chromatographic elution was carried on a core-shell Restek Raptor RP-C18 column (L150 x i.d. 4.6 mm x 2.7 µm) using 0.25 ml/minute flow rate, 5 µl injection volume and 40ºC, oven temperature. The target isoquinoline alkaloids were successfully separated with a step-wise gradient using (A) 100 mM ammonium acetate with 0.2% triethylamine (adjusted to pH 5.2 with a glacial acetic acid) and (B) 100% acetonitrile as follow: 0–3 minutes, 3% B; 3–7 minutes, 3→7% B; 7–20 minutes, 7→18% B; 20–35 minutes, 18→25% B; 35–40 minutes, 25→30% B; 40–50 minutes, 30→33% B; 50–55 minutes, 33→55% B; 55–65 minutes, 55% B; 65–70 minutes, 3% B (B = 3% for 10 minutes.). The absorbance of UV spectrum was read in the range of 200 ÷ 400 nm and the peak area at 280 nm was taken for the calculations. All determinations were performed in triplicate. The concentrations were expressed in mg/g dry weight. The method was validated by following parameters – system suitability, linearity, range, limit of detection (LOD), limit of quantification (LOQ) and precision in compliance with the International Conference on Harmonization (ICH) guidelines Q2 R1 ( Suppl. Material S1 ). Preparation of stock solutions of the extract, fractions and compounds Dry methanolic extract (St-extr.), fractions (St-1, St-2 and St-3) and reticuline ( 7 ) were dissolved to stock concentration of 10 mg/ml in endotoxin-free dimethyl sulfoxide (DMSO, #D4540, Sigma-Aldrich, Munich, Germany). The initial stocks were further diluted to 1 mg/ml in endotoxin-free phosphate buffered saline (PBS; 8 g NaCl; 0.2 g KCl; 2.81 g Na 2 HPO 4 .12H 2 O; 0.24 g KH 2 PO 4 , all from Sigma-Aldrich, Munich, Germany) to reach DMSO concentration below 0.01%. Etoposide was purchased from Sigma-Aldrich (#341205, Munich, Germany), dissolved in 0.2% DMSO/PBS to 1 mM. The p38 MAPK Inhibitor SB203580 was purchased from Invivogene (#inh-sb20-5, InvitroFit™, Thermo Fisher Scientific, Waltham, MA, USA) and dissolved in DMSO/PBS to 1 mg/ml. All stock solutions were aliquoted and stored at -20°C. Cell line and culturing Promyelocyte HL-60 cell line (#CCL-240, ATCC; USA; gift from Prof. Giovanni Bernardini from University of La Sapienza, Rome, Italy), 3th cell passage, was stored into the nitrogen cryopreservation tank (Taylor-Wharton, Livermore, CA, USA) in cryotubes (#430659, Corning, Sigma-Aldrich, Germany) with 1 ml cryopreservation media containing 20% DMSO, 10% fetal bovine serum (FBS; #P40-37100, PAN, India) and culture medium Roswell Park Memorial Institute 1640 (RPMI) with L-Glutamine and NaHCO 3 (#RPMI-A, Capricorn Scientific, Germany). HL-60 cells were thawed, centrifuged at 200 x g, and resuspended in pre-warmed 10% FBS/RPMI medium supplemented with amino acids (#R7131, Sigma-Aldrich, Germany) and antibiotics (#R8758, Sigma-Aldrich, Germany). The cells were counted, resuspended at concentration 1x10 6 /ml, cultured in T25 flasks (#83.3910.002, Sarstedt, Germany) in cell incubator (Memmert, Schwabach, Germany) at 37°C, 5% CO 2 . The cells were let to growth exponentially for 48 hours and then used in the experiments. MTT assay for cell growth Colorimetric MTT kit (#CT01, Merck KGaA, Darmstadt, Germany) was used to evaluate the cleavage of the yellow tetrazolium salt − (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) to purple formazan crystals by metabolically active cells. The method was performed according the manufacturer’s protocol. Briefly, HL-60 cells (1x10 5 cells/ml, 100 µl/well) were seeded at 96-well plates (#3596, Corning, Sigma-Aldrich, Germany) and cultured in the presence the extract, fractions and reticuline ( 7 ). They were diluted at concentrations from 2 to 2000 µg/ml, 100 µl/well. After 48 hours of culture, 0.01 ml MTT/PBS solution was added to each well, incubated for 4 h at 37°C for cleavage of MTT and production of the formazan black crystals in live cells. Then 100 µl/well isopropanol with 0.04 N HCl was added to each well and mixed well to dissolve the crystals. The absorbance was measured within 20 minutes using ELISA plate reader (BioTek EL 800, Winooski, Vermont, USA) with a wavelength of 595 nm and a reference wavelength of 630 nm. The data were acquired using GEN 5.0 software. The cell growth was calculated as a percentage (%) of the control cultures incubated with the vehicle – 0.01% DMSO/PBS. Crystal Violet staining for cell viability Crystal Violet staining is used for assessing cell density and viability, as the dye binds to the basic amino acid residues in cellular proteins, enabling the cell quantification. The Crystal violet Assay Kit of Abcam (#ab232855, Abcam, UK) was used. The cells (1x10 5 cells/ml, 100 µl/well) were seeded in 96-well plate and then incubated with increasing concentrations of St-extr., St-1, St-2 and St-3 (diluted in PBS) for 24 hours, at 37°С, 5% СО 2 . The control of vehicle − 0.01% DMSO/PBS, and a background control with growth media was performed. After 24 hours the plate was centrifuged at 200 x g for 10 minutes, the cells were washed with 200 µl/well PBS and then fixed with 100 µl/well of fixation buffer containing 4% paraformaldehyde (PFA)/PBS (#420801, Biolegend, UK) for 10 minutes, at room temperature. The cells were washed 2 times with PBS and the Crystal Violet/MEOH solution was added in each well (50 µl/well) and incubated by orbital shaking for 20 minutes. The plate was washed 3 times with PBS, then 1 time with dH 2 O to remove any excess dye and let dry at room temperature. Then 100 µl/well of the 1% Sodium dodecyl sulphate (SDS) solution was added for 1 hour to dissolve the dye. The adsorption of the samples was measured at 595 nm using microplate reader (INNO, LTEK, China) with INNO-X software. Flow cytometry Flow cytometry to evaluate cell proliferation Cell proliferation was evaluated by flow cytometry using CellTrace™ Violet proliferation kit (#C34571, Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). The dye binds permanently to cellular proteins rendering the cells fluorescent but after cell division, each daughter cell contains half of the parent cell’s fluorescence, enabling quantification of proliferation and number of cell generations (G). The cells (1х10 6 /ml) were resuspended in 1 ml PBS in FACS tubes (#352052, Falcon; Belgium) and were labelled with 5 µМ CellTrace™ for 20 minutes, at 37°С, in the dark. The unbound dye was eliminated by addition of 5% FBS and incubation for 5 minutes, at room temperature. The cells were centrifuged at 150 x g for 10 minutes, at 10°С and then were resuspended in 5 ml 10% FBS/RPMI and were let to rest for 10 minutes more. Part of the cells, 10 µl, were used for flow cytometry to prove cell labelling with the dye while the rest were seeded at 48-well plate in the presence of the extract (St-extr.), fractions (St-1, St-2 and St-3) or reticuline ( 7 ). The cells were collected every day for 3 days, washed 3 times with 2 ml PBS, resuspended in 300 µl/tube PBS and subjected to flow cytometry using BSR II flow cytometer (BD Biosiences, San Jose, CA, USA) with DIVA 6.0 software. The analyses were performed after collecting 30 000 events/tube. Mitochondrial staining by flow cytometry The cells (1х10 6 /ml) treated with 0.2% DMSO/PBS (Vehicle), the extract (St-extr.), fractions (St-1, St-2 and St-3)(4 or 10 µg/ml) and reticuline ( 7 ) at appropriate concentrations for 24 hours, were collected in FACS tubes (#352052, Falcon, Belgium), washed with 1 ml warm PBS and resuspended at concentration 1х10 5 /ml in warm 2% Bovine serum albumin (BSA, #A2153, Sigma-Aldrich, Germany)/PBS. The MitoSpy™ NIR DiIC1 (#424807, BioLegend, London, UK) stock solution of 10 µM was prepared in DMSO and was further diluted in warmed 2% BSA/PBS and added to a final concentration of 20 nM. The cells were incubated for 30 minutes, at 37°C, 5%CO 2 . The cells were then washed twice with warm PBS, finally resuspended in 300 µl/tube PBS and analysed by flow cytometry. Surface expression of TRAIL and CD11b by flow cytometry Cultured HL-60 cells (1x10 5 /ml) were collected, washed 3 times with 2 ml PBS, resuspended in 2% BSA/PBS, 100 µl/tube and stained with 0.1 mg/ml antibody against human CD11b (clone QA20A58, #379905, Biolegend, London, UK) labelled with allophycocyanin (APC) or antibody against human TRAIL (clone RIK-2, # 308205, Biolegend, London, UK) labelled with phycoeritrin (PE) or isotype controls of mouse IgG1 labelled with PE (clone MOPC-21, # 400111, Biolegend, London, UK) or mouse IgG1 labelled with APC (clone MOPC-21, #400119, Biolegend, London, UK) (10 µl/tube). After incubation for 20 minutes, the cells were washed with 2%BSA/PBS, resuspended in 300 µl/tube PBS and then subjected to flow cytometry. Phosphorylation of p38 MAPK evaluated by flow cytometry HL-60 cells (1x10 6 /ml), at exponential phase of growth, were incubated with extract (St-extr.), fractions (St-1, St-2 and St-3) or reticuline ( 7 ) as described above and activated or not with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, #Р185, Sigma-Aldrich, Germany). After 30 minutes, the cells were washed with 2 ml cold PBS freshly supplemented with phosphatase and proteins inhibitors (#PPC1010, Sigma-Aldrich, Minich, Germany), then fixed with fixation buffer containing 4%PFA/PBS (#420801, Biolegend; London, UK) for 10 minutes, room temperature and washed 3 times with 2% BSA/PBS. The cells were permeabilized with 0.5% Triton X-100/2% BSA/PBS for 10 minutes and or purified antibodies against p38 (clone MK14, #ab170099, anti-p38 alpha/MAPK14 antibody [E229]), the phosphorylated proteins, pp38 (clone MK14 against phospho Y182 + T180, #ab278674), all from Abcam (Cambridg, UK) and corresponding isotype controls. After incubation for 1 hour, in the dark, 4°C, the cells were washed with 2% BSA/PBS buffer containing 0.05% Tween-20 and then stained with anti-rabbit IgG (clone Poly4064, #406403, Biolegend, London, UK) or anti-mouse IgG antibodies labelled with FITC (clone Poly4060, #406001, Biolegend, London, UK) for 1 hour, 4°C, in the dark. After washing 4 times with PBS, 300 µl/tube of PBS was added and cells were subjected to flow cytometry. Differentiation of HL-60 cells HL-60 cells (1x10 6 /ml), at exponential phase of growth, were cultured for 3 days in the presence of medium or 1.5% DMSO/medium to initiate differentiation towards granulocyte lineage. Each day within 3-day culturing, the cells were collected, washed with 1 ml/tube PBS and used in flow cytometry analyses. Statistical analysis Standard deviations (SD) were calculated for the parameters where applicable. ANOVA test with Bonferroni post-hoc correction was used to compare the data. Box-and-whisker plot chart was used to show the data distribution in the groups. The p-values calculated for p < 0.05 (5%) or p < 0.083 (Bonferroni corrections) were considered as statistically significant. The quantitative HPLC results are expressed as the mean value ± relative standard deviation (% RSD, n = 3). Statistical analyses, including one-way ANOVA, Tukey's HSD post-hoc tests, were conducted using StatPlus 7.7.00 (StatPlus Inc., Taipei, Taiwan). Results showing differences were considered statistically significant at p < 0.05 (95% confidence level). Results Phytochemical analysis of the S. dielsiana root extract The carried out phytochemical analysis of the S. dielsiana root tuber extract led to the isolation of 11 isoquinoline-type alkaloids: oxostephanine ( 1 ), O-Me-bulbocapnine ( 2 ), crebanine ( 3 ), tetrahydropalmatine ( 4 ), stephanine ( 5 ), dehydrocrebanine ( 6 ), reticuline ( 7 ), ushinsunine ( 8 ), ayuthianine ( 9 ), liriodenine ( 10 ) and stesakine ( 11 ). The structure characterization of the alkaloids was determined based on NMR spectral data and comparison with the literature [3,10, 11]. All isolated alkaloids have been previously identified in the genus Stephania (Wang et al., 2022 ). Among them, alkaloids 7 and 8 were identified in this study as novel compounds in the root of the S. dielsiana (Fig. 1 ). In addition, the content and the yield of the alkaloids in the crude extract of S. dielsiana roots and the alkaloid enriched fractions St-1, St-2 and St-3 was determined. The quantitative data for alkaloids in extract and fractions are expressed as mg per g dry weight (mg/g DW) (Table 1 ) . Based on the quantitative analysis, the alkaloids found in the largest amount in the root extract were crebanine ( 3 ) (41.74 ± 0.40 mg/g DW) and ayuthianine ( 9 ) (35.36 ± 0.11 mg/g DW), followed by ushinsunine ( 8 ) (24.74 ± 0.22) and reticuline ( 7 ) (19.75 ± 0.44). Alkaloid 3 was determined as a main compound in fraction St-1 with a quantity of 503.50 ± 0.13 mg/g DW. Reticuline ( 7 ) was found as the predominant isoquinoline alkaloid in a fraction St-2 (512.052 ± 0.52 mg/g DW), followed by the fraction St-3 (184.49 ± 0.70 mg/g DW). Table 1 Quality assessment of the S. dielsiana Y. C. Wu crude root extract (St-extr.) and its fractions St-1, St-2 and St-3. Isoquinoline alkaloids St-extr. (mg/g DW ± SD) St-1 (mg/g DW ± SD) St-2 (mg/g DW ± SD) St-3 (mg/g DW ± SD) Oxostephanine ( 1 ) 8.845 ± 0.23 f 77.62 ± 0.42 d 12.71 ± 1.68 d BQL O-me-bulbocapnine ( 2 ) 3.089 ± 0.87 i 107.55 ± 0.35 c 0.137 ± 1.55 f 0.1350 ± 1.64 d Crebanine ( 3 ) 41.74 ± 0.40 a 503.50 ± 0.13 a BDL BDL Tetrahydropalmatine ( 4 ) 1.193 ± 0.36 k 1.554 ± 0.58 i BQL BQL Stephanine ( 5 ) 13.59 ± 0.40 e 135.019 ± 0.07 b BDL BDL Dehydrocrebanine ( 6 ) 7.136 ± 0.21 g 21.62 ± 0.79 e 0.1242 ± 1.89 f BQL Reticuline ( 7 ) 19.75 ± 0.44 d BQL 512.052 ± 0.52 a 184.49 ± 0.70 a Ushinsunine ( 8 ) 24.74 ± 0.22 c 7.269 ± 1.49 f 99.80 ± 0.49 c 0.6120 ± 0.48 c Ayuthianine ( 9 ) 35.36 ± 0.11 b 4.156 ± 0.09 g 151.50 ± 0.67 b BQL Liriodenine ( 10 ) 2.283 ± 0.25 j 2.660 ± 1.33 h 12.61 ± 0.36 d 3.934 ± 0.28 b Stesakine ( 11 ) 5.861 ± 1.76 h BQL 0.4431 ± 0.36 e BQL Results were presented as mean ± RSD; BQL – below quantification limit; BDL – below detection limit; DW - dry weight; RSD – relative standard deviation Different letters in the same column indicated significant differences at p < 0.05 (α = 0.05) according to the ANOVA, Tukey’s HSD test Antiproliferative effects of S. dielsiana root extract, alkaloid-enriched fractions and reticuline ( 7 ) In order to evaluate the biological activity of S. dielsiana crude extract (St-extr), we designed an experiment with promyelocyte HL-60 cell line. The cells in the exponential growth phase were cultured for 48 hours in the presence/absence of St-extr and fractions (St-1, St-2 and St-3) at concentrations varied from 2 to 2000 µg/ml. Cell growth was inhibited by the St-extr and the fractions at concentrations higher than 50 µg/ml (Fig. 2 a), most likely due to the presence of various isoquinoline alkaloids (Fig. 1 ; Table 1 ; Suppl. S2.1. ). While St-extr and St-1 inhibited cell growth at lower concentrations, St-2 and St-3 increased cell growth at concentrations below 50 µg/ml (Fig. 2 a). Crebanine ( 3 ), tetrahydropalmatine ( 4 ) and stephanine ( 5 ) were under the detection minimum in the St-2 and St-3 (Table 1 ) and highly abundant in the St-extr and St-1. A high relative content of 25.5% and 58.4% for crebanine ( 3 ) and 8.5% and 15.7% of stephanine ( 5 ) in St-extr and St-1, respectively, resulted in cytotoxic activity. The relative content of reticuline ( 7 ) was enriched from 12.1% in St-extr to 64.9% in St-2 and 97.5% in St-3 and most likely contributed to a cytotoxic effect of the highest concentrations and a stimulatory action of the lower concentrations between 2 and 20 µg/ml of St-2 and St-3 (Fig. 2 a). Indeed, we evaluated a dose-dependent activity of the isolated compounds on cell growth and we confirmed that reticuline ( 7 ) at low concentrations (less than 50 µg/ml and between 2 and 10 µg/ml) failed to inhibit markedly cell growth ( Suppl. S2.1. ). To provide additional evidence that the St-extr and St-1 were not toxic at lower concentrations, the crude extracts and fractions were further dissolved in endotoxin-free PBS to concentrations varied from 0.0002 to 200 µg/ml. The cells were cultured for 24 hours in the presence/absence of the root extract and the fractions and then the cell viability was evaluated by Crystal Violet Assay Kit (Fig. 2 b). The dye staining is directly proportional to the cell biomass and thus can be used to detect cell cytotoxicity. The S. dielsiana root extract and St-1 fraction showed similar dose-dependent pattern with a cytotoxic effect at concentrations higher than 20 µg/ml (Fig. 2 b) and an increased vitality at concentration between 0.02-2 µg/ml. Both, St-2 and St-3 increased cell viability reaching peaks at concentrations 0.2 and 20 µg/ml. They showed a lack of significant cytotoxic activity at 20 µg/ml by opposite to St-extr and St-1 (Fig. 2 b). The results showed that during the first 24 hours of incubation, St-2 and St-3 did not have a cytotoxic effect at doses lower than 20 µg/ml. Although St-extr and St-1 concentrations between 2 and 20 µg/ml did not significantly reduce cell vitality (Fig. 2 b), those concentrations may have an impact on cell growth for a longer duration of culture 48 hours as determined by the MTT experiment (Fig. 2 a). The anti-proliferative effect of the alkaloids isolated from the Stephania species have been well documented (Semwal et al., 2010 ; 4. Knockleby et al., 2020 ; 5. Thi Thu Hien et al., 2023 ). However less is known about the specific actions of some alkaloids on cell proliferation, especially when used at low concentrations. Based on the data from the alkaloid pattern in the extracts (Table 1 ), cell growth (Fig. 2 a) and vitality (Fig. 2 b) we focused on studying the biological effect of both, St-2 and St-3 which contain mainly reticuline ( 7 ). We compared that activity with the crude extract and isolated reticuline ( 7 ). We calculated the corresponding relative amount of reticuline ( 7 ) in St-extr, St-2 and St-3 used at two concentrations (Table 2 ). Table 2 Concentrations of St-2 and St-3 corrected to the corresponding relative amount of reticuline (7) Fractions Concentration of fraction (µg/ml) Concentration of reticuline in the fraction (µg/ml) St-extr 10 1.3 St-2 10 6.5 4 2.6 St-3 10 9.8 4 3.9 In the experimental setting, cells were treated with the extract and fractions at a concentration of 10 µg/ml (Table 2 ). That concentration did not inhibit cell growth and failed to have a significant cytotoxic activity (Fig. 2 a, b). The cells were labelled with a fluorescent dye, then cultured in the presence of St-extr, St-2, St-3 and reticuline ( 7 ). The proliferation was daily tracked for 3 days using a flow cytometer (Fig. 3 ). Four generations were detected in a 3-day-culture of the Vehicle-treated group and were assigned as generation G1 to G4 (Fig. 3 a). On day 1, most of the cells were distributed in G1 gate and less in G2 and G3 gates (Fig. 3 b). On day 2 and 3, the cells shifted to G3 or G3/G4 gates, respectively (Fig. 3 b, vehicle group). The St-2 and St-3 fractions and mostly St-extr restricted the cells in G1 and G2 on day 1, but this effect was reversed on day 2 and 3, when the cells moved to G3 and G4, similarly to the vehicle group (Fig. 3 b). The intensity of the staining with the dye was daily monitored and showed a reduction of the staining intensity by each generation, as expected for the daughter’s populations (Fig. 4 a). Additional analyses using the Cyflogic software (Fig. 4 b) was performed to study the cell distribution in each generation for 3 days. The effect of the St-2 and St-3 fractions was compared to that of reticuline ( 7 ) at corresponding concentrations (Table 2 ). The etoposide (1 µM) was used as a negative control, because it inhibits G2/M phases of HL-60 cell division (Żuryń A et al., 2016 ). While, the cells reached 55–65% in G1 gate in the Vehicle, St-extr, St-3 and reticuline-treated group on day 1, they were at the highest frequency of 81% in the St-2-treated group (Fig. 4 b). Reticuline ( 7 ) derived of S. dielsiana at concentration of 6.8 µg/ml, that corresponds to its amount in St-2, was unable to increase G1 indicating that the interaction of 7 with other alkaloids in St-2 can shape St-2 biological activity. However, the cells in G1 were reduced almost twice on day 2 and only 3–9% were at G1 gate on day 3 in all groups proposing that they still underwent divisions and proliferated (Fig. 4 b). On day 1, the distribution of the cells in G2 remained the highest in the St-2 group reaching 91% (Fig. 4 b). As in this group only 4% of the cells shifted from G1 to G2 in comparison to around 30% in other groups (except the negative control of etoposide) on day 2, we concluded that St-2 fraction but not reticuline ( 7 ), itself, inhibited cell proliferation by increasing the G1 and G2 generations (Fig. 4 b). On day 1, very few cells were in G3 in all groups but they proliferated and 55–60% were found in G3 on day 2, and 28–33% on day 3. The G4 generation was mainly detected on day 3. The only exception was observed in the St-2-treated group, where 45% were distributed in G3 and 50% in G4. However, St-2 was unable to suppress severely cell proliferation unlike etoposide (Fig. 4 b). Notably, the percentages of cells in G4 reached 95% in the St-3 group and the group treated with 9.5 µg/ml reticuline ( 7 ) (Fig. 4 b) indicating that the growth and proliferation of HL-60 cells can be maintained by reticuline ( 7 ). Similar data we observed in the MTT assay, where St-3 increased cell growth at concentrations between 2 and 10 µg/ml (Fig. 2 a). In conclusion, the data showed that St-2 enhanced the G1 and G2 cell pools. St-2 may affect the initial phases of cell division, thus changing the synchronicity of cell proliferation. The fraction can slow the shift from G1/G2 towards G3/G4 generations in some cells, lowering the percentages of stained cells in G4 gate. By opposite, St-3 and reticuline ( 7 ) failed to have such inhibitory effect. Effect of the S. dielsiana root extract, fractions and reticuline (7) on cell apoptosis The biological effects of the root extract, fractions and isolated reticuline ( 7 ) might affect not only cell growth and proliferation but they might possess pro- or anti-apoptotic properties. The mitochondria activity declines in apoptotic cells and dyes binding the mitochondria are used to discriminate the live from apoptotic cells (Glover et al., 2024 ). Such dye is the MitoSpy™ NIR DiIC1, which localized in mitochondria depending on the membrane potential and cell vitality. The mitochondrial activity of HL-60 cells was evaluated after 24 hours of culturing in the absence/presence of the S. dielsiana crude root extract, fractions St-2 and St-3 at concentration of 4 and 10 µg/ml and reticuline at concentrations indicated in Table 2 . St-extr at concentration 10 µg/ml decreased the mitochondrial staining on day 1 (24 hours) (Fig. 5 a, Suppl. Fig. S2 .1. and S2.2. ). St-2 showed significantly reduced mean fluorescence intensity (MFI) at concentration of 4 µg/ml. However, reticuline ( 7 ) at concentration corresponding to its relative amount in St-2 (2.6 µg/ml) was unable to diminish markedly the mitochondrial activity indicating that other alkaloids can also contribute to the pro-apoptotic effect of St-2. Correspondingly, St-2 increased G1 and G2 generations at day 1 (Fig. 4 ) probably triggering initial apoptosis (Fig. 5 a). St-3 (10 µg/ml) and reticuline ( 7 ) (9.5 µg/ml) demonstrated similar ability to decrease the staining intensity proposing that the cells might loss the mitochondrial membrane potential. Reticuline ( 7 ) at low concentration (3.9 µg/ml) sustained mitochondrial activity, but it can initiate the mitochondrial dysfunction at concentrations higher than 6.8 µg/ml (Fig. 5 a). However, the compound was unable to diminish significantly the frequency of live cells (positive for MitoSpy™ NIR DilC1) (Fig. 5 b). Apoptosis can be also activated via the death receptor-mediated pathways. TNF-related apoptosis-inducing ligand (TRAIL; Apo2 ligand (Apo2L)) is expressed on cell surface as a monomer or is secreted in the environment forming homotrimers to crosslink the receptor ligands on responder cells by which can induce and accelerate cell apoptosis (Shiiki et al., 2000 ; Pimentel et al., 2023 ). According to our data, TRAIL + cells in HL-60 culture were around 14% (Fig. 5 b) supporting the notion that HL-60 cells can express TRAIL (Shiiki et al., 2000 ). These cells were probably apoptotic as they were negative for MitoSpy™ NIR DilC1 staining live cells (Fig. 5 b). The frequency of TRAIL + cells were slightly increased by St-extr and St-2 or were maintained by St-3 and reticuline ( 7 ) (3.9 µg/ml) at low concentration in comparison to control group (Fig. 5 c). However, reticuline ( 7 ) at high concentration and the fractions diminished TRAIL + cells (Fig. 5 c) probably affecting apoptosis. Effect of the fractions St-2 and St-3, and reticuline ( 7 ) on the expression of phosphorylated form of p38 MAPK We observed that reticuline ( 7 ) can affect TRAIL + cells but it can maintain the cell survival. TRAIL and its receptors can be involved in several intracellular pathways involving proteins such as MAPKs (Johnstone et al., 2008 ). Therefore, we delineated the cytoplasmic level of non-phosphorylated and phosphorylated p38 MAPK in HL-60 cells cultured in the presence/absence of high concentration of the fractions St-2 and St-3 (10 µg/ml) and reticuline ( 7 ) at corresponding concentrations of 6.8 and 9.5 µg/ml. The cells were incubated for 24 hours, then stimulated with PMA for 30 minutes and the intracellular level of the kinase was evaluated by flow cytometry (Fig. 6 ). The expression level of p38 was similar in non-stimulated and PMA-stimulated cells in control group and changed its pattern in all treated groups in respect to intensity of fluorescence (MFI; Fig. 6 a). PMA stimulation induced the phosphorylation of р38 kinase and elevated the level of pp38 in the control group (MFI; Fig. 6 a) leading to significantly decreased pp38/p38 ratio. St-2 and St-3 reduced the level of pp38 vs p38 in non-stimulated cells and failed to change that ratio upon PMA-stimulation (Fig. 6 b). Reticuline ( 7 ) at low concentration showed similar effect to St-2 and St-3. The compound 7 at higher concentration decreased the phosphorylated vs non-phosphorylated form in non-stimulated cells but elevated significantly that ratio in PMA-stimulated group (Fig. 6 b). Indeed, we observed an increased fold of PMA-induced pp38 levels by reticuline ( 7 ) indicating that the compound at high concentration can affect the intrinsic activation pathways leading to enhanced p38 phosphorylation (Fig. 6 C). Effect of the fractions St-2 and St-3 and reticuline (7) on the expression of CD11b Our data showed that reticuline ( 7 ) (9.5 µg/ml) derived from S. dielsiana failed to affect significantly the proliferation of HL-60 cells within 3 days but can initiate mitochondrial dysfunction and can decrease p38 phosphorylation in non-stimulated cells at the first 24 h of culture. As proliferation and apoptosis involving p38 signalling are key processes that regulate the lineage commitment of promyelocytes towards granulocytes, we investigated the effect of the fractions and reticuline on the expression of the characteristic differentiation marker - CD11b (Fig. 7 a). The cells were cultured in medium or differentiation stimulus, 1.5% DMSO, and in the presence of St-2 (10 µg/ml), St-3 (10 µg/ml), reticuline (6.8 and 9.5 µg/ml) and p38 MAPK inhibitor (SB203580; 9.5 µg/ml). Each day the cells were collected and the expression of CD11b was evaluated by flow cytometry (Fig. 7 b). DMSO initiated granulocyte differentiation and elevated CD11b positive cells in all group on day 1. St-2 modestly inhibited CD11b cells on day 2 of culture in the absence/presence of DMSO (Fig. 7 b). This effect was not observed when reticuline at corresponding concentration has been used showing that other alkaloids in the St-2 fraction can affect CD11b expression. DMSO increased the CD11b + cell frequency on day 3. Reticuline ( 7 ) at high concentration (9.5 µg/ml) elevated further the percentage of positive cells, similarly to SB203580. Discussion The literature data demonstrates that the aerial parts and root tubers of the genus Stephania are a rich source of isoquinoline alkaloids used to treat infections and relieve inflammatory symptoms (Semwal et al., 2010 ; Wang et al., 2022 ). Since the biological activity of alkaloids isolated from S. dielsana has been studied to a greater extent in the leaves and stems of the plant, we aimed to investigate the effect on the growth, survival, proliferation, apoptosis and differentiation of the promyelocytic HL-60 cell line of the methanolic extract, the alkaloid-enriched fractions and the alkaloids obtained from root tubers. Eleven isoquinoline alkaloids were identified from the methanolic root tuber extract of S. dielsiana . Nine of these alkaloids are of the aporphine type ( 1 – 3 , 5 – 6 , 8 – 11 ), one is of the protoberberine ( 4 ), and one is of the benzylisoquinoline type ( 7 ). All of the isolated compounds have also been identified in other species of the genus, such as S. venosa , S. tetrandra, S. cepharantha ets., which means that there is some genetic stability in the genus in terms of alkaloid biosynthesis (Wang et al., 2022 ). Previously, alkaloids 1 – 6 and 9–11 have been also detected in the methanolic leaf extract from S . dielsiana (Knockleby et al., 2020 ; Thi Thu Hien et al., 2023 ). The presence of the same metabolites in the leaves and roots indicates that isoquinoline alkaloids are key metabolites in S. dielsiana and probably, accumulate there during plant growth and adaptation (Yu et al., 2023 ). Quantitative analysis indicated the highest abundance of crebanine ( 3 ) and ayuthianine ( 9 ) in the root tuber extract, followed by ushinsunine ( 8 ) and reticuline ( 7 ). The St-extr and St-1 had high relative content of 25.5% and 58.4% for crebanine ( 3 ), respectively and 8.5% and 15.7% of stephanine ( 5 ), respectively. We found that St-extr and St-1 inhibited cell vitality for 24 hours (Fig. 2 a) at concentrations higher than 20 µg/ml and inhibited significantly cell growth for 48 hours (Fig. 2 b) at concentrations higher than 50 µg/ml. The high abundance of crebanine ( 3 ) and the presence of oxostephanine ( 1 ) and stephanine ( 5 ) in St-extr and St-1 was associated with cytotoxic activity ( see Suppl. Fig. S2 .1. ). Similar biological effect of the extract from the stems and leaves of S. dielsiana has been observed on cancer cell lines (HepG2, MCF7 and OVCAR8) (Tran et al., 2022 ). Correspondingly, Knockleby et al. ( 2020 ) have reported a dose-dependent increase in the anti-proliferative activities of the S. dielsana derived oxostephanine ( 1 ), crebanine ( 3 ) and stephanine ( 5 ) in several cancer (HeLa, MDA-MB231, MDA-MB-468, MCF-7) and non-cancer cell lines (184B5 and MCF10A) with inhibitory concentration (IC) 50 ranging from 17.82 to 48.17 µM for crebanine ( 3 ); 1.66 to 4.35 µM for oxostephanine ( 1 ) and 3.33 to 7.19 µM for stephanine ( 5 ). The authors have found that oxostephanine ( 1 ) is two-times more potent than stephanine ( 5 ) against cancer cells (Knockleby et al. 2020 ). In our study crebanine ( 3 ), tetrahydropalmatine ( 4 ) and stephanine ( 5 ) were under the detection minimum in the St-2 and St-3 (Table 1 ) but fraction St-2 contained oxostephanine ( 1 ), ayuthianine ( 9 ) and stesakine ( 11 ). The biological action of ayuthianine ( 9 ) and stesakine ( 11 ) has not been thoroughly studied while oxostephanine ( 1 ) is known to suppress Aurora kinase activity and to cause aneuploidy in cancer cells (Knockleby et al. 2020 ; Tran et al., 2022 ). We observed that St-2 (10 µg/ml) enhanced the G1 and G2 cell pools and we proposed that St-2 may affect the initial phases of cell division, thus changing the synchronicity of cell proliferation. The fraction also retarded the shift from G1/G2 towards G3/G4 generations in some cells, lowering the percentages of stained cells in G4 gate (Fig. 4 ). We have to note that St-2 was unable to suppress severely cell proliferation unlike our control treated with the topoisomerase inhibitor, etoposide (Fig. 4 b) and despite its inhibitory effects failed to be toxic for the cells. Oxostephanine ( 1 ) may contribute directly to the inhibitory effect of St-2 on cell proliferation as it has been shown to induced G2/M cycle arrest (Knockleby et al. 2020 ), however the synergistic antiproliferative effect of all alkaloids or of reticuline ( 7 ) in the presence of oxostephanine ( 1 ) /ayuthianine ( 9 ) /stesakine ( 11 ) in St-2 cannot be excluded. The anti-proliferative activity of the alkaloids in St-2 (4 and 10 µg/ml) might be also related with a decreased mitochondrial activity indicative for initiation of apoptosis. Indeed, oxostephanine ( 1 ) at concentration of 2.5-5 µM can induce early apoptosis as evaluated by Annexin V staining (Tran et al., 2022 ). Reticuline ( 7 ) was detected for the first time in the S . dielsiana . The alkaloid was predominant in a fraction St-2 followed by the fraction St-3. Reticuline ( 7 ) and the alkaloids derived from (S)-reticuline can be found in leaves and stems, but the roots are the predominant site of their biosynthesis and the preferred time-dependent location for alkaloid biosynthetic enzymes (Zeng et al., 2013 ; Gorpenchenko et al., 2019 ). This may explain why reticuline ( 7 ) was highly abundant in the root extract of S. dielsana and correspondingly in the St-2 and St-3 fractions. The compound was enriched from 12.1% in St-extr to 64.9% in St-2 and 97.5% in St-3 and most likely contributed to a cytotoxic effect of the highest concentrations (50 µg/ml) and a stimulatory action on cell growth and vitality at concentrations between 2 and 20 µg/ml (Fig. 2 ). While St-2 contained other alkaloids (as we discussed above) which can have cytotoxic activity and interfere with proliferation and apoptosis, reticuline ( 7 ) was at high relative content in St-3. We also compared the biological activity between the crude extract, fractions and isolated reticuline ( 7 ) by using the concentrations of reticuline ( 7 ) corresponding to its relative amount in them (Table 2 ). St-3 and reticuline ( 7 ) at concentration 9.5 µg/ml failed to show inhibitory effect on cell proliferation as the percentages of cells in G4 on day 3 reached 95% (Fig. 4 b). Our data indicated that the growth and proliferation of HL-60 cells can be maintained by reticuline and this notion has been supported by the data from the MTT assay, where St-3 increased cell growth at concentrations between 2 and 20 µg/ml (Fig. 2 a). Interestingly, St-3 (10 µg/ml) and reticuline ( 7 ) (6.8 and 9.5 µg/ml) demonstrated a decreased mitochondrial staining intensity proposing that the cells might loss the mitochondrial membrane potential and might undergo apoptosis. The mitochondrial-dependent apoptotic pathway includes a disturbed balance between the pro-apoptotic (Bax, Bad, Spike) and the pro-survival (Bcl-2, Bcl-XL, Mcl-1) proteins that was followed by caspase activation. Reticuline ( 7 ) derivative have cytotoxic and pro-apoptotic effects above 30 µM in epithelial like cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) (Kim et al., 2010 ). However, reticuline ( 7 ) at similar concentration of 9.5 µg/ml (30 µM) in our study showed milder effect on mitochondrial staining without a significant increase in apoptosis and reduction in the frequency of live cells (positive for MitoSpy™ NIR DilC1) (Fig. 5 b). Indeed, reticuline ( 7 ) at low concentration (3.9 µg/ml) sustained mitochondrial activity (Fig. 5 a) proposing that its action on mitochondria is dose-dependent and might be reversible at low concentrations. Apoptosis can be also activated via TRAIL and the death receptor-mediated pathways. TRAIL can crosslink the receptor ligands on responder cells by which can induce and accelerate cell apoptosis (Shiiki et al., 2000 ; Pimentel et al., 2023 ). We observed that reticuline ( 7 ) can affect TRAIL + cells, but we did not show its direct role on TRAIL-induced apoptosis. It has been observed that other alkaloid, tetrandrine, found in Stephania spp, can upregulate the ligands of TRAIL and can elevate the sensitivity of chemotherapy resistant tumor cell lines to TRAIL-induced apoptosis (Shishodia et al., 2018 ). It is possible that reticuline ( 7 ) may affect TRAIL expression and pathways indirectly via an effect on mitochondrial functions, as the link between both pathways exists (Johnstone et al., 2008 ). TRAIL can also have apoptosis-independent functions on myeloid precursors related to the ability of myeloid cells to differentiate to osteoclast or granulocyte lineages (Shiiki et al., 2000 ; Dimitrova et al., 2012 ; Liao et al., 2019 ). It will be interesting to delineate the action of reticuline ( 7 ) on TRAIL in respect to osteoclasts differentiation in the future. TRAIL and its receptors can be involved in several intracellular pathways engaging the activation, phosphorylation and ubiquitination of proteins such as MAPKs and in particular p38 (Johnstone et al., 2008 ). Therefore, we studied the cytoplasmic level of non-phosphorylated and phosphorylated p38 MAPK in HL-60 cells. The cells were pre-cultured in the presence of St-2, St-3 (10 µg/ml) and reticuline ( 7 ) (6.8 and 9.5 µg/ml) for 24 hours, then they were stimulated with PMA for 30 minutes and the intracellular level of the kinase was evaluated by flow cytometry (Fig. 6 ). St-2 and St-3 reduced the level of pp38 vs p38 in non-stimulated cells and failed to change that ratio upon PMA-stimulation (Fig. 6 a). Reticuline ( 7 ) at low concentration showed similar effect to St-2 and St-3. At higher concentration, it decreased the phosphorylated vs non-phosphorylated form in non-stimulated cells and elevated significantly that ratio in PMA-stimulated group (Fig. 6 b). Indeed, we observed an increased fold of PMA-induced pp38 levels by reticuline ( 7 ) indicating that the compound at high concentration can affect the intrinsic activation pathways leading to enhanced p38 phosphorylation (Fig. 6 c). Similarly, other study using the immunoblot has demonstrated the elevated phosphorylated levels of p38 MAPK and of a downstream transcription factor NF-κB p65 in vivo , in two experimental models of asthma and obesity‐related asthma mice (Lyu et al., 2024 ). This effect on p38 phosphorylation was also related to the nature of the stimulus applied and was affected by a loop regulating not only the phosphorylation but also the activation of p38 MAPK and entire NF‐κB signaling pathway (Lyu et al., 2024 ). We also observed a different effect of reticuline ( 7 ) in non-stimulated and PMA-stimulated cells, proposing that effect of reticuline ( 7 ) on p38 MAPK might depend on overall functional cellular state. As proliferation and apoptosis involving p38 MAPK are key processes that regulate the lineage commitment of promyelocytes towards granulocytes, we investigated the effect of the fractions and reticuline ( 7 ) on the expression of the characteristic differentiation marker - CD11b (Fig. 7 a). We observed that reticuline ( 7 ) at high concentration (9.5 µg/ml) elevated DMSO-induced increase in the percentage of positive cells. This effect was similar to that of p38 MAPK inhibitor, SB203580. Indeed, this inhibitor has been shown to upregulated the expression of the CD11b gene in HL-60 and NB4 promyelocyte cell lines on day 3 (Suzuki et al., 2024 ) and was used to drive myelocyte differentiation in acute leukemia (Takahashi, 2022 ). Similarly, in our study, reticuline ( 7 ) can regulate CD11b expression on differentiating cells. However, its effect might be dependent of its purity as the presence of other alkaloids in the St-3 fraction interfered with the frequency of CD11b positive cells. Conclusion In conclusion, the methanolic root tuber extract of S. dielsiana Y. C. Wu from Vietnam contained isoquinoline alkaloids inhibiting growth, survival and proliferation of HL-60 cell line. For the first time, reticuline ( 7 ) was identified in the root extract of S. dielsana . Reticuline-enriched fractions exhibited less cytotoxic effects at concentrations between 0.2 and 20 µg/ml than the individual alkaloid 7 . However, the presence of other alkaloids in the fractions masked the biological activity of reticuline ( 7 ). For example, St-2 that consist around 65% reticuline ( 7 ), enhanced the G1 and G2 cell pools and changed the synchronicity of cell proliferation while the compound 7 at the same concentration did not show such effect. St-3, containing around 95% reticuline ( 7 ), showed similar effect on mitochondrial function and TRAIL + cells to the individual compound 7 , but it was less effective on altering p38 phosphorylation in non-stimulated and stimulated cells. Finaly, reticuline ( 7 ), unlike St-3, elevated the frequency of differentiating CD11b positive cells. This effect was similar to that of the p38 inhibitor but not to St-3. Our study demonstrated that reticuline ( 7 ) derived from S. dielsiana Y. C. Wu was able to affect growth, proliferation, apoptosis and differentiation of HL-60 cell line. Declarations Funding This work was supported in part by the Vietnam Academy of Science and Technology (QTBG01.01/23-24) and the Bulgarian Academy of Sciences (IC-VT/03/2023-2025) under the International Scientific Cooperation. Ethics approval Not applicable for this study. Authorship contribution statement Petya Dimitrova : Writing – review & editing, Writing – original draft, Data curation. Tsvetelina Doncheva : Writing – review & editing, Resources, Project administration, Data curation. Nadezhda Kostova : Writing – review & editing, Methodology, Data curation, Formal analysis. Iveta Uzunova: Methodology, Data curation. Nikol Latinova: Methodology, Data curation. Vanya Gerasimova : Writing – review & editing, Data curation, Formal analysis. Nguyen Tien Dat : Project administration, Writing – review & editing. Do Hoang Giang : Formal analysis, Data curation. Nguyen Thi Luyen : Writing – review & editing, Data curation. 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Acta Histochem 118(5):537–543. 10.1016/j.acthis.2016.05.010 Supplementary Files GraphicalabstractStephania10112024.tif SUPPLEMENTARYMATERIALStephania19122024.docx Cite Share Download PDF Status: Published Journal Publication published 28 May, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Editorial decision: Major revisions 03 Feb, 2025 Reviewers agreed at journal 08 Jan, 2025 Reviewers invited by journal 27 Dec, 2024 Editor invited by journal 23 Dec, 2024 Editor assigned by journal 20 Dec, 2024 First submitted to journal 19 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5585865","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":394997431,"identity":"5abccd2d-5e2c-438f-b692-880e5d1ec875","order_by":0,"name":"Petya Dimitrova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYPACZh4GZghLjoGBB4zwqYZpgegxJloLjMGQ2EBIizl7/zGJHwzWMrrt/Mc+fGyzS99wvPcAw5sK3Fosew6zSfYwpPOYHWZmnjmzLTl3w5lzCYxzzuDWYnAjmU2Ch+EwWAszzxnm3G03cgyYedvwaLn/mE3yD0JLfbrZ/TdALf/w2cLMJo2wpeJwgtkNHqCWBnx+STa2loH4xZhxRsVxw/1n8hIOzjmGW4s5+8GHN98wWNubnT/4mOGDQbW8ZPvZgw/e1OBxGAMDiwQjussP4NYA1sL8AZ+CUTAKRsEoGAUMANvDSXfBQzYWAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0920-3712","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Petya","middleName":"","lastName":"Dimitrova","suffix":""},{"id":394997432,"identity":"3597bffe-366a-4dc7-ac1d-ed9c9ee1cc84","order_by":1,"name":"Tsvetelina Doncheva","email":"","orcid":"","institution":"Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tsvetelina","middleName":"","lastName":"Doncheva","suffix":""},{"id":394997433,"identity":"496d3c95-c750-4d01-a9bb-7691120606fb","order_by":2,"name":"Nadezhda Kostova","email":"","orcid":"","institution":"Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nadezhda","middleName":"","lastName":"Kostova","suffix":""},{"id":394997434,"identity":"e3d08730-2dcf-4360-b986-a55715de683f","order_by":3,"name":"Iveta Uzunova","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Iveta","middleName":"","lastName":"Uzunova","suffix":""},{"id":394997435,"identity":"88a348d8-6475-4f2d-a415-c11f32ad1b67","order_by":4,"name":"Nikol Latinova","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nikol","middleName":"","lastName":"Latinova","suffix":""},{"id":394997436,"identity":"8b5cf0b6-3b11-4046-8efa-f36751076a64","order_by":5,"name":"Vanya Gerasimova","email":"","orcid":"","institution":"Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vanya","middleName":"","lastName":"Gerasimova","suffix":""},{"id":394997437,"identity":"54337981-3845-4a63-940c-2053635f6b8c","order_by":6,"name":"Nguyen Tien Dat","email":"","orcid":"","institution":"Vietnamese Academy of Science: Vietnam Academy of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Tien","lastName":"Dat","suffix":""},{"id":394997438,"identity":"563081bf-5faa-489f-a351-936ef0c69bb0","order_by":7,"name":"Do Hoang Giang","email":"","orcid":"","institution":"Vietnamese Academy of Science: Vietnam Academy of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Do","middleName":"Hoang","lastName":"Giang","suffix":""},{"id":394997439,"identity":"75d4f738-b399-4a33-8c08-80cb698bf6ed","order_by":8,"name":"Nguyen Thi Luyen","email":"","orcid":"","institution":"Vietnamese Academy of Science: Vietnam Academy of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Thi","lastName":"Luyen","suffix":""}],"badges":[],"createdAt":"2024-12-05 09:55:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5585865/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5585865/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-025-00657-5","type":"published","date":"2025-05-28T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72597053,"identity":"71e6b0d1-9685-46ba-9e1c-d691b7f8d7d0","added_by":"auto","created_at":"2024-12-30 08:11:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":775404,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of isolated alkaloids from \u003cem\u003eS. dielsiana\u003c/em\u003e root extract.\u003c/p\u003e","description":"","filename":"Figure1Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/0bbe2cebc80bae66d4154a2c.png"},{"id":72597355,"identity":"2cf8d88b-c826-43aa-b6e9-89126643697a","added_by":"auto","created_at":"2024-12-30 08:19:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":464455,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effect of \u003cem\u003eS. dielsiana\u003c/em\u003e crude root extract (St-extr), fractions (St-1, St-2 and St-3) on growth and vitality of HL-60 cell line. \u003cstrong\u003ea.\u003c/strong\u003eEffect of the extract and fractions on cell growth. Cells were cultured in the presence of the St-extr and St-1, St-2 and St-3 fractions at concentration varied from 2 to 2000 µg/ml. Cell growth was evaluated by colorimetric MTT assay after 48 hours and presented as a percentage (%) of the control (0.02% DMSO/PBS). Data represent mean ± standard deviation (SD) of 3 repeats from 2 individual experiments. \u003cstrong\u003eb.\u003c/strong\u003e Cell viability of HL-60 cell line. Cells were cultured in the presence of the St-extr and fractions at concentrations varied from 0.0002 to 200 µg/ml. Cell viability was evaluated by colorimetric Cristal Violet assay after 24 h and the absorption at 595 nm was measured at microplate reader. Data represent mean ± SD of 3 repeats.\u003c/p\u003e","description":"","filename":"Figure2Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/fc38867ce8530e7fb562af39.png"},{"id":72597056,"identity":"951d339e-4b92-4e4f-aa2f-0f616342d8b1","added_by":"auto","created_at":"2024-12-30 08:11:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1974315,"visible":true,"origin":"","legend":"\u003cp\u003eGating strategy for Cell-Trace Violet dye labelled cells. HL-60 cells (1x10\u003csup\u003e6\u003c/sup\u003e/ml) were labelled with 5 µM Cell-Trace Violet dye. \u003cstrong\u003ea.\u003c/strong\u003e Dot-plots showing cell gating (Forward scatter (FSC) vs Side scatter (SSC)) and histograms showing expression (counts) of Cell-Trace labelled cells and gating strategy for positive cells and generations\u003cstrong\u003e. b.\u003c/strong\u003e Representative histograms showing the distribution of cells in G1, G2, G3 and G4 gates on day 1, 2, 3 of cultures with Vehicle (0.02% DMSO/PBS), St-2 and St-3 fractions and St-extr.\u003c/p\u003e","description":"","filename":"Figure3Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/c82c9b04496ae8011848bf00.png"},{"id":72597063,"identity":"552982be-196e-43d6-b858-d18544a078b9","added_by":"auto","created_at":"2024-12-30 08:11:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1299340,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eS. dielsiana\u003c/em\u003e root extract, alkaloid enriched fractions and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) on cell proliferation \u003cstrong\u003ea\u003c/strong\u003e. Intensity of the Cell-Trace Violet staining in the generations was measured by flow cytometry and represented as a mean of fluorescence (MFI). \u003cstrong\u003eb.\u003c/strong\u003e Cell proliferation of HL-60 cell line in the presence of 10 µg/ml of the \u003cem\u003eS. dielsiana\u003c/em\u003ecrude root extract (St-extr) and its fractions (St-2 and St-3), reticuline (\u003cstrong\u003e7\u003c/strong\u003e) at concentrations 1.3, 6.8, 9.5 µg/ml (Reticuline 1.3; Reticuline 6.8; Reticuline 9.5). The negative control of etoposide (1 µM) was used in the experiment. Cell proliferation is evaluated by CellTrace™ Violet Cell Proliferation Kit using flow cytometer with DIVA 6.0 software (BSR II, BD Biosciences, San Jose, CA, USA). The data are analysed by Cyflogic software (Turku, Finland). The frequency of the labelled generation (in %) are presented as a proportion of each generation.\u003c/p\u003e","description":"","filename":"Figure4Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/cb00c8f07086592d471eecd2.png"},{"id":72598541,"identity":"d95d22e2-e35f-47db-a64a-52269ce6421e","added_by":"auto","created_at":"2024-12-30 08:27:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2051295,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of St-2 and St-3, and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) on mitochondrial activity of HL-60 cells. \u003cstrong\u003ea. \u003c/strong\u003eCells were\u003cstrong\u003e \u003c/strong\u003ecultured for 24 hours in the presence of medium (Control), vehicle (0.01% DMSO/PBS),\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eS. dielsiana\u003c/em\u003e crude root extract (St-extr), fractions St-2 and St-3 at concentrations 4 and 10 µg/ml and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) at concentrations 2.6, 3.9, 6.8 and 9.5 µg/ml. Then cells were labelled with MitoSpy™ NIR DilC1 and the mean of fluorescence (MFI) was evaluated by flow cytometry. Data represent mean ± SD of 4 samples/group indicated in a column and individual values indicated by circles. *p\u0026lt;0.083 vs Control group; ANOVA test with Bonferroni correction. \u003cstrong\u003eb.\u003c/strong\u003e Representative dot-plots showing the percentages of positive cells for TRAIL and MitoSpy™ NIR DilC1.\u003cstrong\u003e c.\u003c/strong\u003e The percentages of TRAIL positive cells assessed by flow cytometry. Data represent mean ± SD of 4 samples/group indicated in a column and individual values indicated by circles, *p\u0026lt;0.083 vs Control group; ANOVA test with Bonferroni correction.\u003c/p\u003e","description":"","filename":"Figure5Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/725fad4560e0888b8e8c5d4f.png"},{"id":72597357,"identity":"f53682a3-36db-4d2d-88d9-a0fa3aaa6680","added_by":"auto","created_at":"2024-12-30 08:19:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1876011,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of St-2, St-3 and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) on p38 MAPK phosphorylation. \u003cstrong\u003ea.\u003c/strong\u003eRepresentative overlayed histograms showing the mean of intensity in non-stimulated or PMA-stimulated (100 ng/ml) cells pre-incubated for 24 hours with media, Vehicle, St-2, St-3 and reticuline at two concentrations (6.8 or 9.5 µg/ml). \u003cstrong\u003eb.\u003c/strong\u003e Wisker plots showing the distribution of the values of the ratio between phosphorylated vs non-phosphorylated p38 forms. *p\u0026lt;0.083 when compared non-stimulated vs PMA-stimulated groups, #p\u0026lt;0.083 vs control group; n=4/group, ANOVA test with Bonferroni corrections. \u003cstrong\u003ec.\u003c/strong\u003e Effect of St-2, 3 and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) on PMA-induced p38 phosphorylation. Data represent mean ± SD of 4 samples/group; *p\u0026lt;0.05 vs Vehicle group; ANOVA test.\u003c/p\u003e","description":"","filename":"Figure6Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/d022ca8968a98c5ddd66e24f.png"},{"id":72598540,"identity":"ef510333-86c4-47bd-8818-cac204623606","added_by":"auto","created_at":"2024-12-30 08:27:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1141562,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of St-2, St-3 and reticuline (\u003cstrong\u003e7\u003c/strong\u003e) on the frequency of CD11b+ differentiating myeloid cells. \u003cstrong\u003ea.\u003c/strong\u003e Gating strategy of showing live cell gate, singlets and positive and negative staining for CD11b. \u003cstrong\u003eb.\u003c/strong\u003e HL-60 cells were treated with medium or 1.5% DMSO in the presence of St-3 and St-3 fractions (10 µg/ml), reticuline (6.8 or 9.5 µg/ml) and SB203580 (9.5 µg/ml). The percentage of the CD11b positive cells was obtained upon flow cytometry analyses on day 1, 2 and 3; n=3/group, ANOVA test, *p\u0026lt;0.05 vs DMSO control group, #p\u0026lt;0.05 vs Media control group.\u003c/p\u003e","description":"","filename":"Figure7Stephania10112024.png","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/beae074a8fb1fb0f6f59da5b.png"},{"id":83783011,"identity":"094d3b04-2e5e-4e9f-9131-cee42d53f520","added_by":"auto","created_at":"2025-06-02 16:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12642100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/b54eb748-434c-4190-be69-5be32156779d.pdf"},{"id":72597367,"identity":"c0fe274c-7a41-4fd8-b60c-e6864046b31a","added_by":"auto","created_at":"2024-12-30 08:19:38","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":719164,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractStephania10112024.tif","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/f49b96928c035494e6e92845.tif"},{"id":72597371,"identity":"ef98697a-a6e8-4c23-a954-952939944e9e","added_by":"auto","created_at":"2024-12-30 08:19:39","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":1136660,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIALStephania19122024.docx","url":"https://assets-eu.researchsquare.com/files/rs-5585865/v1/2087456e71533776d5b49043.docx"}],"financialInterests":"","formattedTitle":"Biological effect of alkaloid enriched fractions and reticuline from the Stephania dielsianaY. C. Wu on promyelocyte HL-60 cell line","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenus \u003cem\u003eStephania\u003c/em\u003e (Menispermaceae) consists of more than 60 species distributed in the tropical and subtropical areas of Asia and Africa and some regions of Oceania (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The \u003cem\u003eStephania\u003c/em\u003e plants are herbaceous lianas with large root tubers and have been widely used in the traditional Chinese medicine for the cure of various health disorders and diseases. The crude plant extracts show a wide range of pharmacological properties, including antimicrobial, antiparasitic, antitumor, antihyperglycemic, antioxidant, anti-inflammatory, antinociceptive, antipyretic, analgesic, neuroprotective, and cardioprotective activities (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). So far, the majority of the studies have been focused on \u003cem\u003eS. tetrandra\u003c/em\u003e S. Moore, \u003cem\u003eS. cepharantha\u003c/em\u003e Hayata, \u003cem\u003eS. glabra\u003c/em\u003e (Roxb.) Meyers, \u003cem\u003eS. japonica\u003c/em\u003e (Thunb.) Meyers and \u003cem\u003eS. venosa\u003c/em\u003e (Blume) Spreng (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The species are rich in isoquinoline alkaloids and in particular the derivatives of aporphine, proaporphine, morphinan, hasubanan, protoberberine, benzylisoquinoline and bisbenzylisoquinoline (Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Thien et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Knockleby et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Thi Thu Hien et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The content of isoquinoline alkaloids was found to be highest in the root tubers of the plants, reaching in some cases up to 3\u0026ndash;4% of the plant material (Rong et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStephania dielsiana\u003c/em\u003e Y. C. Wu is a medicinal plant used in traditional Vietnamese medicine for thousands of years to treat insomnia, relieve pain, dispel toxins, improve circulation, inflammation and injuries. The main part of the plant used is the tuberous roots, which are dried, ground into a powder and used to make a decoction. Similarly to other species of the genus \u003cem\u003eStephania\u003c/em\u003e, the biologically active components in the extracts of the species have been found to be the aporphine alkaloids. In previous studies, were found that the alkaloid extract and the alkaloids, crebanine and stephanine, isolated from the \u003cem\u003eStephania dielsiana\u003c/em\u003e root tubers exhibit high inhibitory activity against various pathogens (Deng et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The same alkaloids isolated from the leaves and stems of the species, as well as the alkaloids oxostephanine and thailandine, have been shown to have potential as anticancer and anti-inflammatory agents (Thien et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Knockleby et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Thi Thu Hien et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA large number of the alkaloids identified from \u003cem\u003eStephania\u003c/em\u003e species have shown a potential to inhibit survival, proliferation and inflammatory responses in various cell lines and models of inflammation-mediated diseases (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The \u003cem\u003eStephania\u003c/em\u003e derived cepharanthine was able to suppress the production of pro-inflammatory cytokines interleukin (IL)-1β or tumor necrosis factor (TNF)-α thought the suppression of mitogen-activated protein kinase p38 (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), part of the activation pathway related to the production of more than 1000 different mediators involved in inflammation, cell migration and regulation of autophagy (Yao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The same signaling pathway has been inhibited by the isoquinoline alkaloid, reticuline in the model of obesity-related asthma (Lyu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite that the antiproliferative and anti-inflammatory activities of some \u003cem\u003eStephania\u003c/em\u003e species has been well characterized and explained their efficacy in phytotherapy, the biological activities of \u003cem\u003eS. dielsana\u003c/em\u003e root tubers have been less studied. Herein, we investigated the effect of the methanolic extract, alkaloid enriched fractions and alkaloids from \u003cem\u003eS. dielsana\u003c/em\u003e root tubers collected in Vietnam on the growth, survival, proliferation, apoptosis and differentiation of the promyelocytic HL-60 cell line.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eThe root tubers of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003edielsiana\u003c/em\u003e Y. C. Wu were collected from Quan Ba commune, Ha Giang province, Vietnam in January 2023. The plant material was identified by Dr. Nguyen Cuong from Institute of Ecology and Biological Resources, Vietnam Academy of Sciences (VAST). A voucher specimen with reference number QTBG-0622 was deposited at the Laboratory of Agro-Pharmaceutical Research, Center for High Technology Research and Development, VAST.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlkaloid extraction and isolation\u003c/h3\u003e\n\u003cp\u003eThe air-dried and powdered roots (500 g) of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003edielsiana\u003c/em\u003e Y. C. Wu were extracted with MeOH (3x 1000 ml) and evaporated to dryness to obtain the crude extract (St-extr.) (15 g). The crude extract was further separated by vacuum liquid chromatography (VLC) on silica gel and eluted with CHCl\u003csub\u003e3\u003c/sub\u003e\u0026ndash;MeOH to give fractions St-1 (5.2 g) (CHCl\u003csub\u003e3\u003c/sub\u003e), St-2 (4.3 g) (CHCl\u003csub\u003e3\u003c/sub\u003e/MeOH, 1/1, v/v) and St-3 (3.1 g) (MeOH). СС on silica gel with eluent petroleum/CHCl\u003csub\u003e3\u003c/sub\u003e/Me\u003csub\u003e2\u003c/sub\u003eCO/MeOH, 4/8/1/2, v/v/v/v of fraction St-1 give 3 subfractions St-1A (582 mg), St-1B (423 mg) and St-1C (125 mg). Subfraction St-1A was subjected to PTLC on silica gel plates (Kieselgel 60 F\u003csub\u003e254\u003c/sub\u003e, Merck) using petroleum/CHCl\u003csub\u003e3\u003c/sub\u003e/Me\u003csub\u003e2\u003c/sub\u003eCO/MeOH, 14/4/1/1, v/v/v/v as mobile phase in NH\u003csub\u003e3\u003c/sub\u003e atmosphere to give the alkaloids oxostephanine (\u003cb\u003e1\u003c/b\u003e) (12.5 mg) and O-Me-bulbocapnine (\u003cb\u003e2\u003c/b\u003e) (13.6 mg). Subfraction St-1B was subjected to PTLC using petroleum/CHCl\u003csub\u003e3\u003c/sub\u003e/Me\u003csub\u003e2\u003c/sub\u003eCO/MeOH, 4/4/1/1, v/v/v/v as mobile phase in NH\u003csub\u003e3\u003c/sub\u003e atmosphere to give the alkaloids: crebanine (\u003cb\u003e3\u003c/b\u003e) (28.2 mg), tetrahydropalmatine (\u003cb\u003e4\u003c/b\u003e) (1.6 mg) and stephanine (\u003cb\u003e5\u003c/b\u003e) (14.2 mg). Subfraction St-1C was subjected to PTLC using petroleum/CHCl\u003csub\u003e3\u003c/sub\u003e/Me\u003csub\u003e2\u003c/sub\u003eCO/MeOH, 4/8/1/2 (v/v/v), as mobile phase in NH\u003csub\u003e3\u003c/sub\u003e atmosphere to give dehydrocrebanine (\u003cb\u003e6\u003c/b\u003e) (8.5 mg). From fraction St-2 using PTLC on silica gel plates (Kieselgel 60 F\u003csub\u003e254\u003c/sub\u003e, Merck) with mobile phase petroleum/CHCl\u003csub\u003e3\u003c/sub\u003e/Me\u003csub\u003e2\u003c/sub\u003eCO/MeOH 4/8/1/2 (v/v/v) the alkaloids reticuline (\u003cb\u003e7\u003c/b\u003e) (14.5 mg), ushinsunine (\u003cb\u003e8\u003c/b\u003e) (6.1 mg), ayuthianine (\u003cb\u003e9\u003c/b\u003e) (7.2 mg), liriodenine (\u003cb\u003e10\u003c/b\u003e) (3.5 mg) and stesakine (\u003cb\u003e11\u003c/b\u003e) (1.2 mg) were isolated. Using the same PTLC (as for St-2) the alkaloid reticuline (\u003cb\u003e7\u003c/b\u003e) (3.9 mg) was purified from the fraction St-3. The presence of alkaloids was visualized by Dragendorff\u0026rsquo;s spray reagent. The structures of isolated alkaloids \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e were elucidated by comparison of their 1D and 2D NMR spectra with the available data and resources (Lee et al., 1992; Zhou et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Thien et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Purity of isolated alkaloids (from 90 to 98%) was determined by high-performance liquid chromatography.\u003c/p\u003e\n\u003ch3\u003eQuantification of alkaloids by HPLC\u003c/h3\u003e\n\u003cp\u003eLiquid chromatographic analyses were performed on a Shimadzu (Nexera-LC40 XS, Kyoto, Japan) HPLC system equipped with a PDA detector (М40) managed by LabSolutions software. The chromatographic elution was carried on a core-shell Restek Raptor RP-C18 column (L150 x i.d. 4.6 mm x 2.7 \u0026micro;m) using 0.25 ml/minute flow rate, 5 \u0026micro;l injection volume and 40\u0026ordm;C, oven temperature. The target isoquinoline alkaloids were successfully separated with a step-wise gradient using (A) 100 mM ammonium acetate with 0.2% triethylamine (adjusted to pH 5.2 with a glacial acetic acid) and (B) 100% acetonitrile as follow: 0\u0026ndash;3 minutes, 3% B; 3\u0026ndash;7 minutes, 3\u0026rarr;7% B; 7\u0026ndash;20 minutes, 7\u0026rarr;18% B; 20\u0026ndash;35 minutes, 18\u0026rarr;25% B; 35\u0026ndash;40 minutes, 25\u0026rarr;30% B; 40\u0026ndash;50 minutes, 30\u0026rarr;33% B; 50\u0026ndash;55 minutes, 33\u0026rarr;55% B; 55\u0026ndash;65 minutes, 55% B; 65\u0026ndash;70 minutes, 3% B (B\u0026thinsp;=\u0026thinsp;3% for 10 minutes.). The absorbance of UV spectrum was read in the range of 200\u0026thinsp;\u0026divide;\u0026thinsp;400 nm and the peak area at 280 nm was taken for the calculations. All determinations were performed in triplicate. The concentrations were expressed in mg/g dry weight.\u003c/p\u003e \u003cp\u003eThe method was validated by following parameters \u0026ndash; system suitability, linearity, range, limit of detection (LOD), limit of quantification (LOQ) and precision in compliance with the International Conference on Harmonization (ICH) guidelines Q2 R1 (\u003cb\u003eSuppl. Material S1\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003ePreparation of stock solutions of the extract, fractions and compounds\u003c/h3\u003e\n\u003cp\u003eDry methanolic extract (St-extr.), fractions (St-1, St-2 and St-3) and reticuline (\u003cb\u003e7\u003c/b\u003e) were dissolved to stock concentration of 10 mg/ml in endotoxin-free dimethyl sulfoxide (DMSO, #D4540, Sigma-Aldrich, Munich, Germany). The initial stocks were further diluted to 1 mg/ml in endotoxin-free phosphate buffered saline (PBS; 8 g NaCl; 0.2 g KCl; 2.81 g Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.12H\u003csub\u003e2\u003c/sub\u003eO; 0.24 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, all from Sigma-Aldrich, Munich, Germany) to reach DMSO concentration below 0.01%. Etoposide was purchased from Sigma-Aldrich (#341205, Munich, Germany), dissolved in 0.2% DMSO/PBS to 1 mM. The p38 MAPK Inhibitor SB203580 was purchased from Invivogene (#inh-sb20-5, InvitroFit\u0026trade;, Thermo Fisher Scientific, Waltham, MA, USA) and dissolved in DMSO/PBS to 1 mg/ml. All stock solutions were aliquoted and stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eCell line and culturing\u003c/h3\u003e\n\u003cp\u003ePromyelocyte HL-60 cell line (#CCL-240, ATCC; USA; gift from Prof. Giovanni Bernardini from University of La Sapienza, Rome, Italy), 3th cell passage, was stored into the nitrogen cryopreservation tank (Taylor-Wharton, Livermore, CA, USA) in cryotubes (#430659, Corning, Sigma-Aldrich, Germany) with 1 ml cryopreservation media containing 20% DMSO, 10% fetal bovine serum (FBS; #P40-37100, PAN, India) and culture medium Roswell Park Memorial Institute 1640 (RPMI) with L-Glutamine and NaHCO\u003csub\u003e3\u003c/sub\u003e (#RPMI-A, Capricorn Scientific, Germany).\u003c/p\u003e \u003cp\u003eHL-60 cells were thawed, centrifuged at 200 x g, and resuspended in pre-warmed 10% FBS/RPMI medium supplemented with amino acids (#R7131, Sigma-Aldrich, Germany) and antibiotics (#R8758, Sigma-Aldrich, Germany). The cells were counted, resuspended at concentration 1x10\u003csup\u003e6\u003c/sup\u003e/ml, cultured in T25 flasks (#83.3910.002, Sarstedt, Germany) in cell incubator (Memmert, Schwabach, Germany) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were let to growth exponentially for 48 hours and then used in the experiments.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay for cell growth\u003c/h2\u003e \u003cp\u003eColorimetric MTT kit (#CT01, Merck KGaA, Darmstadt, Germany) was used to evaluate the cleavage of the yellow tetrazolium salt \u0026minus;\u0026thinsp;(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) to purple formazan crystals by metabolically active cells. The method was performed according the manufacturer\u0026rsquo;s protocol. Briefly, HL-60 cells (1x10\u003csup\u003e5\u003c/sup\u003e cells/ml, 100 \u0026micro;l/well) were seeded at 96-well plates (#3596, Corning, Sigma-Aldrich, Germany) and cultured in the presence the extract, fractions and reticuline (\u003cb\u003e7\u003c/b\u003e). They were diluted at concentrations from 2 to 2000 \u0026micro;g/ml, 100 \u0026micro;l/well. After 48 hours of culture, 0.01 ml MTT/PBS solution was added to each well, incubated for 4 h at 37\u0026deg;C for cleavage of MTT and production of the formazan black crystals in live cells. Then 100 \u0026micro;l/well isopropanol with 0.04 N HCl was added to each well and mixed well to dissolve the crystals. The absorbance was measured within 20 minutes using ELISA plate reader (BioTek EL 800, Winooski, Vermont, USA) with a wavelength of 595 nm and a reference wavelength of 630 nm. The data were acquired using GEN 5.0 software. The cell growth was calculated as a percentage (%) of the control cultures incubated with the vehicle \u0026ndash; 0.01% DMSO/PBS.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCrystal Violet staining for cell viability\u003c/h3\u003e\n\u003cp\u003eCrystal Violet staining is used for assessing cell density and viability, as the dye binds to the basic amino acid residues in cellular proteins, enabling the cell quantification. The Crystal violet Assay Kit of Abcam (#ab232855, Abcam, UK) was used. The cells (1x10\u003csup\u003e5\u003c/sup\u003e cells/ml, 100 \u0026micro;l/well) were seeded in 96-well plate and then incubated with increasing concentrations of St-extr., St-1, St-2 and St-3 (diluted in PBS) for 24 hours, at 37\u0026deg;С, 5% СО\u003csub\u003e2\u003c/sub\u003e. The control of vehicle \u0026minus;\u0026thinsp;0.01% DMSO/PBS, and a background control with growth media was performed. After 24 hours the plate was centrifuged at 200 x g for 10 minutes, the cells were washed with 200 \u0026micro;l/well PBS and then fixed with 100 \u0026micro;l/well of fixation buffer containing 4% paraformaldehyde (PFA)/PBS (#420801, Biolegend, UK) for 10 minutes, at room temperature. The cells were washed 2 times with PBS and the Crystal Violet/MEOH solution was added in each well (50 \u0026micro;l/well) and incubated by orbital shaking for 20 minutes. The plate was washed 3 times with PBS, then 1 time with dH\u003csub\u003e2\u003c/sub\u003eO to remove any excess dye and let dry at room temperature. Then 100 \u0026micro;l/well of the 1% Sodium dodecyl sulphate (SDS) solution was added for 1 hour to dissolve the dye. The adsorption of the samples was measured at 595 nm using microplate reader (INNO, LTEK, China) with INNO-X software.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry to evaluate cell proliferation\u003c/h2\u003e \u003cp\u003eCell proliferation was evaluated by flow cytometry using CellTrace\u0026trade; Violet proliferation kit (#C34571, Invitrogen\u0026trade;, Thermo Fisher Scientific, Waltham, MA, USA). The dye binds permanently to cellular proteins rendering the cells fluorescent but after cell division, each daughter cell contains half of the parent cell\u0026rsquo;s fluorescence, enabling quantification of proliferation and number of cell generations (G). The cells (1х10\u003csup\u003e6\u003c/sup\u003e/ml) were resuspended in 1 ml PBS in FACS tubes (#352052, Falcon; Belgium) and were labelled with 5 \u0026micro;М CellTrace\u0026trade; for 20 minutes, at 37\u0026deg;С, in the dark. The unbound dye was eliminated by addition of 5% FBS and incubation for 5 minutes, at room temperature. The cells were centrifuged at 150 x g for 10 minutes, at 10\u0026deg;С and then were resuspended in 5 ml 10% FBS/RPMI and were let to rest for 10 minutes more. Part of the cells, 10 \u0026micro;l, were used for flow cytometry to prove cell labelling with the dye while the rest were seeded at 48-well plate in the presence of the extract (St-extr.), fractions (St-1, St-2 and St-3) or reticuline (\u003cb\u003e7\u003c/b\u003e). The cells were collected every day for 3 days, washed 3 times with 2 ml PBS, resuspended in 300 \u0026micro;l/tube PBS and subjected to flow cytometry using BSR II flow cytometer (BD Biosiences, San Jose, CA, USA) with DIVA 6.0 software. The analyses were performed after collecting 30 000 events/tube.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial staining by flow cytometry\u003c/h2\u003e \u003cp\u003eThe cells (1х10\u003csup\u003e6\u003c/sup\u003e/ml) treated with 0.2% DMSO/PBS (Vehicle), the extract (St-extr.), fractions (St-1, St-2 and St-3)(4 or 10 \u0026micro;g/ml) and reticuline (\u003cb\u003e7\u003c/b\u003e) at appropriate concentrations for 24 hours, were collected in FACS tubes (#352052, Falcon, Belgium), washed with 1 ml warm PBS and resuspended at concentration 1х10\u003csup\u003e5\u003c/sup\u003e/ml in warm 2% Bovine serum albumin (BSA, #A2153, Sigma-Aldrich, Germany)/PBS. The MitoSpy\u0026trade; NIR DiIC1 (#424807, BioLegend, London, UK) stock solution of 10 \u0026micro;M was prepared in DMSO and was further diluted in warmed 2% BSA/PBS and added to a final concentration of 20 nM. The cells were incubated for 30 minutes, at 37\u0026deg;C, 5%CO\u003csub\u003e2\u003c/sub\u003e. The cells were then washed twice with warm PBS, finally resuspended in 300 \u0026micro;l/tube PBS and analysed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSurface expression of TRAIL and CD11b by flow cytometry\u003c/h2\u003e \u003cp\u003eCultured HL-60 cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml) were collected, washed 3 times with 2 ml PBS, resuspended in 2% BSA/PBS, 100 \u0026micro;l/tube and stained with 0.1 mg/ml antibody against human CD11b (clone QA20A58, #379905, Biolegend, London, UK) labelled with allophycocyanin (APC) or antibody against human TRAIL (clone RIK-2, # 308205, Biolegend, London, UK) labelled with phycoeritrin (PE) or isotype controls of mouse IgG1 labelled with PE (clone MOPC-21, # 400111, Biolegend, London, UK) or mouse IgG1 labelled with APC (clone MOPC-21, #400119, Biolegend, London, UK) (10 \u0026micro;l/tube). After incubation for 20 minutes, the cells were washed with 2%BSA/PBS, resuspended in 300 \u0026micro;l/tube PBS and then subjected to flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhosphorylation of p38 MAPK evaluated by flow cytometry\u003c/h2\u003e \u003cp\u003eHL-60 cells (1x10\u003csup\u003e6\u003c/sup\u003e/ml), at exponential phase of growth, were incubated with extract (St-extr.), fractions (St-1, St-2 and St-3) or reticuline (\u003cb\u003e7\u003c/b\u003e) as described above and activated or not with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, #Р185, Sigma-Aldrich, Germany). After 30 minutes, the cells were washed with 2 ml cold PBS freshly supplemented with phosphatase and proteins inhibitors (#PPC1010, Sigma-Aldrich, Minich, Germany), then fixed with fixation buffer containing 4%PFA/PBS (#420801, Biolegend; London, UK) for 10 minutes, room temperature and washed 3 times with 2% BSA/PBS. The cells were permeabilized with 0.5% Triton X-100/2% BSA/PBS for 10 minutes and or purified antibodies against p38 (clone MK14, #ab170099, anti-p38 alpha/MAPK14 antibody [E229]), the phosphorylated proteins, pp38 (clone MK14 against phospho Y182\u0026thinsp;+\u0026thinsp;T180, #ab278674), all from Abcam (Cambridg, UK) and corresponding isotype controls. After incubation for 1 hour, in the dark, 4\u0026deg;C, the cells were washed with 2% BSA/PBS buffer containing 0.05% Tween-20 and then stained with anti-rabbit IgG (clone Poly4064, #406403, Biolegend, London, UK) or anti-mouse IgG antibodies labelled with FITC (clone Poly4060, #406001, Biolegend, London, UK) for 1 hour, 4\u0026deg;C, in the dark. After washing 4 times with PBS, 300 \u0026micro;l/tube of PBS was added and cells were subjected to flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDifferentiation of HL-60 cells\u003c/h2\u003e \u003cp\u003eHL-60 cells (1x10\u003csup\u003e6\u003c/sup\u003e/ml), at exponential phase of growth, were cultured for 3 days in the presence of medium or 1.5% DMSO/medium to initiate differentiation towards granulocyte lineage. Each day within 3-day culturing, the cells were collected, washed with 1 ml/tube PBS and used in flow cytometry analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStandard deviations (SD) were calculated for the parameters where applicable. ANOVA test with Bonferroni post-hoc correction was used to compare the data. Box-and-whisker plot chart was used to show the data distribution in the groups. The p-values calculated for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (5%) or p\u0026thinsp;\u0026lt;\u0026thinsp;0.083 (Bonferroni corrections) were considered as statistically significant.\u003c/p\u003e \u003cp\u003eThe quantitative HPLC results are expressed as the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;relative standard deviation (% RSD, n\u0026thinsp;=\u0026thinsp;3). Statistical analyses, including one-way ANOVA, Tukey's HSD post-hoc tests, were conducted using StatPlus 7.7.00 (StatPlus Inc., Taipei, Taiwan). Results showing differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (95% confidence level).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhytochemical analysis of the\u003c/b\u003e \u003cb\u003eS. dielsiana\u003c/b\u003e \u003cb\u003eroot extract\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe carried out phytochemical analysis of the \u003cem\u003eS. dielsiana\u003c/em\u003e root tuber extract led to the isolation of 11 isoquinoline-type alkaloids: oxostephanine (\u003cb\u003e1\u003c/b\u003e), O-Me-bulbocapnine (\u003cb\u003e2\u003c/b\u003e), crebanine (\u003cb\u003e3\u003c/b\u003e), tetrahydropalmatine (\u003cb\u003e4\u003c/b\u003e), stephanine (\u003cb\u003e5\u003c/b\u003e), dehydrocrebanine (\u003cb\u003e6\u003c/b\u003e), reticuline (\u003cb\u003e7\u003c/b\u003e), ushinsunine (\u003cb\u003e8\u003c/b\u003e), ayuthianine (\u003cb\u003e9\u003c/b\u003e), liriodenine (\u003cb\u003e10\u003c/b\u003e) and stesakine (\u003cb\u003e11\u003c/b\u003e). The structure characterization of the alkaloids was determined based on NMR spectral data and comparison with the literature [3,10, 11]. All isolated alkaloids have been previously identified in the genus \u003cem\u003eStephania\u003c/em\u003e (Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among them, alkaloids \u003cb\u003e7\u003c/b\u003e and \u003cb\u003e8\u003c/b\u003e were identified in this study as novel compounds in the root of the \u003cem\u003eS. dielsiana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the content and the yield of the alkaloids in the crude extract of \u003cem\u003eS. dielsiana\u003c/em\u003e roots and the alkaloid enriched fractions St-1, St-2 and St-3 was determined. The quantitative data for alkaloids in extract and fractions are expressed as mg per g dry weight (mg/g DW) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Based on the quantitative analysis, the alkaloids found in the largest amount in the root extract were crebanine (\u003cb\u003e3\u003c/b\u003e) (41.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 mg/g DW) and ayuthianine (\u003cb\u003e9\u003c/b\u003e) (35.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mg/g DW), followed by ushinsunine (\u003cb\u003e8\u003c/b\u003e) (24.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22) and reticuline (\u003cb\u003e7\u003c/b\u003e) (19.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44).\u003c/p\u003e \u003cp\u003eAlkaloid \u003cb\u003e3\u003c/b\u003e was determined as a main compound in fraction St-1 with a quantity of 503.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/g DW. Reticuline (\u003cb\u003e7\u003c/b\u003e) was found as the predominant isoquinoline alkaloid in a fraction St-2 (512.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 mg/g DW), followed by the fraction St-3 (184.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 mg/g DW).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuality assessment of the S. dielsiana Y. C. Wu crude root extract (St-extr.) and its fractions St-1, St-2 and St-3.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoquinoline alkaloids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSt-extr.\u003c/p\u003e \u003cp\u003e(mg/g DW\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSt-1\u003c/p\u003e \u003cp\u003e(mg/g DW\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSt-2\u003c/p\u003e \u003cp\u003e(mg/g DW\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSt-3\u003c/p\u003e \u003cp\u003e(mg/g DW\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxostephanine (\u003cb\u003e1\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.845\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO-me-bulbocapnine (\u003cb\u003e2\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.137\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1350\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrebanine (\u003cb\u003e3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e503.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBDL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBDL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetrahydropalmatine (\u003cb\u003e4\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ek\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.554\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStephanine (\u003cb\u003e5\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e135.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBDL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBDL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDehydrocrebanine (\u003cb\u003e6\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1242\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReticuline (\u003cb\u003e7\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e512.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e184.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUshinsunine (\u003cb\u003e8\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.269\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6120\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAyuthianine (\u003cb\u003e9\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiriodenine (\u003cb\u003e10\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.283\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.660\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.934\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStesakine (\u003cb\u003e11\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.861\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4431\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBQL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eResults were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD; BQL \u0026ndash; below quantification limit; BDL \u0026ndash; below detection limit; DW - dry weight; RSD \u0026ndash; relative standard deviation\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eDifferent letters in the same column indicated significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (α\u0026thinsp;=\u0026thinsp;0.05) according to the ANOVA, Tukey\u0026rsquo;s HSD test\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eAntiproliferative effects of\u003c/b\u003e \u003cb\u003eS. dielsiana\u003c/b\u003e \u003cb\u003eroot extract, alkaloid-enriched fractions and reticuline\u003c/b\u003e (\u003cb\u003e7\u003c/b\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn order to evaluate the biological activity of \u003cem\u003eS. dielsiana\u003c/em\u003e crude extract (St-extr), we designed an experiment with promyelocyte HL-60 cell line. The cells in the exponential growth phase were cultured for 48 hours in the presence/absence of St-extr and fractions (St-1, St-2 and St-3) at concentrations varied from 2 to 2000 \u0026micro;g/ml. Cell growth was inhibited by the St-extr and the fractions at concentrations higher than 50 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), most likely due to the presence of various isoquinoline alkaloids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; \u003cb\u003eSuppl. S2.1.\u003c/b\u003e). While St-extr and St-1 inhibited cell growth at lower concentrations, St-2 and St-3 increased cell growth at concentrations below 50 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Crebanine (\u003cb\u003e3\u003c/b\u003e), tetrahydropalmatine (\u003cb\u003e4\u003c/b\u003e) and stephanine (\u003cb\u003e5\u003c/b\u003e) were under the detection minimum in the St-2 and St-3 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and highly abundant in the St-extr and St-1. A high relative content of 25.5% and 58.4% for crebanine (\u003cb\u003e3\u003c/b\u003e) and 8.5% and 15.7% of stephanine (\u003cb\u003e5\u003c/b\u003e) in St-extr and St-1, respectively, resulted in cytotoxic activity. The relative content of reticuline (\u003cb\u003e7\u003c/b\u003e) was enriched from 12.1% in St-extr to 64.9% in St-2 and 97.5% in St-3 and most likely contributed to a cytotoxic effect of the highest concentrations and a stimulatory action of the lower concentrations between 2 and 20 \u0026micro;g/ml of St-2 and St-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Indeed, we evaluated a dose-dependent activity of the isolated compounds on cell growth and we confirmed that reticuline (\u003cb\u003e7\u003c/b\u003e) at low concentrations (less than 50 \u0026micro;g/ml and between 2 and 10 \u0026micro;g/ml) failed to inhibit markedly cell growth (\u003cb\u003eSuppl. S2.1.\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo provide additional evidence that the St-extr and St-1 were not toxic at lower concentrations, the crude extracts and fractions were further dissolved in endotoxin-free PBS to concentrations varied from 0.0002 to 200 \u0026micro;g/ml. The cells were cultured for 24 hours in the presence/absence of the root extract and the fractions and then the cell viability was evaluated by Crystal Violet Assay Kit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The dye staining is directly proportional to the cell biomass and thus can be used to detect cell cytotoxicity. The \u003cem\u003eS. dielsiana\u003c/em\u003e root extract and St-1 fraction showed similar dose-dependent pattern with a cytotoxic effect at concentrations higher than 20 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and an increased vitality at concentration between 0.02-2 \u0026micro;g/ml. Both, St-2 and St-3 increased cell viability reaching peaks at concentrations 0.2 and 20 \u0026micro;g/ml. They showed a lack of significant cytotoxic activity at 20 \u0026micro;g/ml by opposite to St-extr and St-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The results showed that during the first 24 hours of incubation, St-2 and St-3 did not have a cytotoxic effect at doses lower than 20 \u0026micro;g/ml. Although St-extr and St-1 concentrations between 2 and 20 \u0026micro;g/ml did not significantly reduce cell vitality (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), those concentrations may have an impact on cell growth for a longer duration of culture 48 hours as determined by the MTT experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe anti-proliferative effect of the alkaloids isolated from the \u003cem\u003eStephania\u003c/em\u003e species have been well documented (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; 4. Knockleby et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; 5. Thi Thu Hien et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However less is known about the specific actions of some alkaloids on cell proliferation, especially when used at low concentrations. Based on the data from the alkaloid pattern in the extracts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and vitality (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) we focused on studying the biological effect of both, St-2 and St-3 which contain mainly reticuline (\u003cb\u003e7\u003c/b\u003e). We compared that activity with the crude extract and isolated reticuline (\u003cb\u003e7\u003c/b\u003e). We calculated the corresponding relative amount of reticuline (\u003cb\u003e7\u003c/b\u003e) in St-extr, St-2 and St-3 used at two concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConcentrations of St-2 and St-3 corrected to the corresponding relative amount of reticuline (7)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFractions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration of fraction\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration of reticuline in the fraction (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSt-extr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSt-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSt-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the experimental setting, cells were treated with the extract and fractions at a concentration of 10 \u0026micro;g/ml (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). That concentration did not inhibit cell growth and failed to have a significant cytotoxic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The cells were labelled with a fluorescent dye, then cultured in the presence of St-extr, St-2, St-3 and reticuline (\u003cb\u003e7\u003c/b\u003e). The proliferation was daily tracked for 3 days using a flow cytometer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFour generations were detected in a 3-day-culture of the Vehicle-treated group and were assigned as generation G1 to G4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). On day 1, most of the cells were distributed in G1 gate and less in G2 and G3 gates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). On day 2 and 3, the cells shifted to G3 or G3/G4 gates, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, vehicle group). The St-2 and St-3 fractions and mostly St-extr restricted the cells in G1 and G2 on day 1, but this effect was reversed on day 2 and 3, when the cells moved to G3 and G4, similarly to the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe intensity of the staining with the dye was daily monitored and showed a reduction of the staining intensity by each generation, as expected for the daughter\u0026rsquo;s populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Additional analyses using the Cyflogic software (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) was performed to study the cell distribution in each generation for 3 days. The effect of the St-2 and St-3 fractions was compared to that of reticuline (\u003cb\u003e7\u003c/b\u003e) at corresponding concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe etoposide (1 \u0026micro;M) was used as a negative control, because it inhibits G2/M phases of HL-60 cell division (Żuryń A et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While, the cells reached 55\u0026ndash;65% in G1 gate in the Vehicle, St-extr, St-3 and reticuline-treated group on day 1, they were at the highest frequency of 81% in the St-2-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Reticuline (\u003cb\u003e7\u003c/b\u003e) derived of \u003cem\u003eS. dielsiana\u003c/em\u003e at concentration of 6.8 \u0026micro;g/ml, that corresponds to its amount in St-2, was unable to increase G1 indicating that the interaction of \u003cb\u003e7\u003c/b\u003e with other alkaloids in St-2 can shape St-2 biological activity. However, the cells in G1 were reduced almost twice on day 2 and only 3\u0026ndash;9% were at G1 gate on day 3 in all groups proposing that they still underwent divisions and proliferated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eOn day 1, the distribution of the cells in G2 remained the highest in the St-2 group reaching 91% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). As in this group only 4% of the cells shifted from G1 to G2 in comparison to around 30% in other groups (except the negative control of etoposide) on day 2, we concluded that St-2 fraction but not reticuline (\u003cb\u003e7\u003c/b\u003e), itself, inhibited cell proliferation by increasing the G1 and G2 generations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eOn day 1, very few cells were in G3 in all groups but they proliferated and 55\u0026ndash;60% were found in G3 on day 2, and 28\u0026ndash;33% on day 3. The G4 generation was mainly detected on day 3. The only exception was observed in the St-2-treated group, where 45% were distributed in G3 and 50% in G4. However, St-2 was unable to suppress severely cell proliferation unlike etoposide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Notably, the percentages of cells in G4 reached 95% in the St-3 group and the group treated with 9.5 \u0026micro;g/ml reticuline (\u003cb\u003e7\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) indicating that the growth and proliferation of HL-60 cells can be maintained by reticuline (\u003cb\u003e7\u003c/b\u003e). Similar data we observed in the MTT assay, where St-3 increased cell growth at concentrations between 2 and 10 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn conclusion, the data showed that St-2 enhanced the G1 and G2 cell pools. St-2 may affect the initial phases of cell division, thus changing the synchronicity of cell proliferation. The fraction can slow the shift from G1/G2 towards G3/G4 generations in some cells, lowering the percentages of stained cells in G4 gate. By opposite, St-3 and reticuline (\u003cb\u003e7\u003c/b\u003e) failed to have such inhibitory effect.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of the\u003c/b\u003e \u003cb\u003eS. dielsiana\u003c/b\u003e \u003cb\u003eroot extract, fractions and reticuline (7) on cell apoptosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biological effects of the root extract, fractions and isolated reticuline (\u003cb\u003e7\u003c/b\u003e) might affect not only cell growth and proliferation but they might possess pro- or anti-apoptotic properties. The mitochondria activity declines in apoptotic cells and dyes binding the mitochondria are used to discriminate the live from apoptotic cells (Glover et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Such dye is the MitoSpy\u0026trade; NIR DiIC1, which localized in mitochondria depending on the membrane potential and cell vitality. The mitochondrial activity of HL-60 cells was evaluated after 24 hours of culturing in the absence/presence of the \u003cem\u003eS. dielsiana\u003c/em\u003e crude root extract, fractions St-2 and St-3 at concentration of 4 and 10 \u0026micro;g/ml and reticuline at concentrations indicated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eSt-extr at concentration 10 \u0026micro;g/ml decreased the mitochondrial staining on day 1 (24 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cb\u003eSuppl. Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.1.\u003c/b\u003e and \u003cb\u003eS2.2.\u003c/b\u003e). St-2 showed significantly reduced mean fluorescence intensity (MFI) at concentration of 4 \u0026micro;g/ml. However, reticuline (\u003cb\u003e7\u003c/b\u003e) at concentration corresponding to its relative amount in St-2 (2.6 \u0026micro;g/ml) was unable to diminish markedly the mitochondrial activity indicating that other alkaloids can also contribute to the pro-apoptotic effect of St-2. Correspondingly, St-2 increased G1 and G2 generations at day 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) probably triggering initial apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). St-3 (10 \u0026micro;g/ml) and reticuline (\u003cb\u003e7\u003c/b\u003e) (9.5 \u0026micro;g/ml) demonstrated similar ability to decrease the staining intensity proposing that the cells might loss the mitochondrial membrane potential. Reticuline (\u003cb\u003e7\u003c/b\u003e) at low concentration (3.9 \u0026micro;g/ml) sustained mitochondrial activity, but it can initiate the mitochondrial dysfunction at concentrations higher than 6.8 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). However, the compound was unable to diminish significantly the frequency of live cells (positive for MitoSpy\u0026trade; NIR DilC1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eApoptosis can be also activated via the death receptor-mediated pathways. TNF-related apoptosis-inducing ligand (TRAIL; Apo2 ligand (Apo2L)) is expressed on cell surface as a monomer or is secreted in the environment forming homotrimers to crosslink the receptor ligands on responder cells by which can induce and accelerate cell apoptosis (Shiiki et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pimentel et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to our data, TRAIL\u0026thinsp;+\u0026thinsp;cells in HL-60 culture were around 14% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) supporting the notion that HL-60 cells can express TRAIL (Shiiki et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). These cells were probably apoptotic as they were negative for MitoSpy\u0026trade; NIR DilC1 staining live cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The frequency of TRAIL\u0026thinsp;+\u0026thinsp;cells were slightly increased by St-extr and St-2 or were maintained by St-3 and reticuline (\u003cb\u003e7\u003c/b\u003e) (3.9 \u0026micro;g/ml) at low concentration in comparison to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). However, reticuline (\u003cb\u003e7\u003c/b\u003e) at high concentration and the fractions diminished TRAIL\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) probably affecting apoptosis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eEffect of the fractions St-2 and St-3, and reticuline\u003c/b\u003e (\u003cb\u003e7\u003c/b\u003e) \u003cb\u003eon the expression of phosphorylated form of p38 MAPK\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe observed that reticuline (\u003cb\u003e7\u003c/b\u003e) can affect TRAIL\u0026thinsp;+\u0026thinsp;cells but it can maintain the cell survival. TRAIL and its receptors can be involved in several intracellular pathways involving proteins such as MAPKs (Johnstone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, we delineated the cytoplasmic level of non-phosphorylated and phosphorylated p38 MAPK in HL-60 cells cultured in the presence/absence of high concentration of the fractions St-2 and St-3 (10 \u0026micro;g/ml) and reticuline (\u003cb\u003e7\u003c/b\u003e) at corresponding concentrations of 6.8 and 9.5 \u0026micro;g/ml. The cells were incubated for 24 hours, then stimulated with PMA for 30 minutes and the intracellular level of the kinase was evaluated by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe expression level of p38 was similar in non-stimulated and PMA-stimulated cells in control group and changed its pattern in all treated groups in respect to intensity of fluorescence (MFI; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). PMA stimulation induced the phosphorylation of р38 kinase and elevated the level of pp38 in the control group (MFI; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) leading to significantly decreased pp38/p38 ratio. St-2 and St-3 reduced the level of pp38 vs p38 in non-stimulated cells and failed to change that ratio upon PMA-stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eReticuline (\u003cb\u003e7\u003c/b\u003e) at low concentration showed similar effect to St-2 and St-3. The compound \u003cb\u003e7\u003c/b\u003e at higher concentration decreased the phosphorylated vs non-phosphorylated form in non-stimulated cells but elevated significantly that ratio in PMA-stimulated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Indeed, we observed an increased fold of PMA-induced pp38 levels by reticuline (\u003cb\u003e7\u003c/b\u003e) indicating that the compound at high concentration can affect the intrinsic activation pathways leading to enhanced p38 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eEffect of the fractions St-2 and St-3 and reticuline (7) on the expression of CD11b\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eOur data showed that reticuline (\u003cb\u003e7\u003c/b\u003e) (9.5 \u0026micro;g/ml) derived from \u003cem\u003eS. dielsiana\u003c/em\u003e failed to affect significantly the proliferation of HL-60 cells within 3 days but can initiate mitochondrial dysfunction and can decrease p38 phosphorylation in non-stimulated cells at the first 24 h of culture. As proliferation and apoptosis involving p38 signalling are key processes that regulate the lineage commitment of promyelocytes towards granulocytes, we investigated the effect of the fractions and reticuline on the expression of the characteristic differentiation marker - CD11b (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe cells were cultured in medium or differentiation stimulus, 1.5% DMSO, and in the presence of St-2 (10 \u0026micro;g/ml), St-3 (10 \u0026micro;g/ml), reticuline (6.8 and 9.5 \u0026micro;g/ml) and p38 MAPK inhibitor (SB203580; 9.5 \u0026micro;g/ml). Each day the cells were collected and the expression of CD11b was evaluated by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). DMSO initiated granulocyte differentiation and elevated CD11b positive cells in all group on day 1. St-2 modestly inhibited CD11b cells on day 2 of culture in the absence/presence of DMSO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). This effect was not observed when reticuline at corresponding concentration has been used showing that other alkaloids in the St-2 fraction can affect CD11b expression. DMSO increased the CD11b\u0026thinsp;+\u0026thinsp;cell frequency on day 3. Reticuline (\u003cb\u003e7\u003c/b\u003e) at high concentration (9.5 \u0026micro;g/ml) elevated further the percentage of positive cells, similarly to SB203580.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe literature data demonstrates that the aerial parts and root tubers of the genus \u003cem\u003eStephania\u003c/em\u003e are a rich source of isoquinoline alkaloids used to treat infections and relieve inflammatory symptoms (Semwal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since the biological activity of alkaloids isolated from \u003cem\u003eS. dielsana\u003c/em\u003e has been studied to a greater extent in the leaves and stems of the plant, we aimed to investigate the effect on the growth, survival, proliferation, apoptosis and differentiation of the promyelocytic HL-60 cell line of the methanolic extract, the alkaloid-enriched fractions and the alkaloids obtained from root tubers.\u003c/p\u003e \u003cp\u003eEleven isoquinoline alkaloids were identified from the methanolic root tuber extract of \u003cem\u003eS. dielsiana\u003c/em\u003e. Nine of these alkaloids are of the aporphine type (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e3\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e\u0026ndash;\u003cb\u003e6\u003c/b\u003e, \u003cb\u003e8\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e), one is of the protoberberine (\u003cb\u003e4\u003c/b\u003e), and one is of the benzylisoquinoline type (\u003cb\u003e7\u003c/b\u003e). All of the isolated compounds have also been identified in other species of the genus, such as \u003cem\u003eS. venosa\u003c/em\u003e, \u003cem\u003eS. tetrandra, S. cepharantha\u003c/em\u003e ets., which means that there is some genetic stability in the genus in terms of alkaloid biosynthesis (Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previously, alkaloids \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e6\u003c/b\u003e and \u003cb\u003e9\u0026ndash;11\u003c/b\u003e have been also detected in the methanolic leaf extract from \u003cem\u003eS\u003c/em\u003e. \u003cem\u003edielsiana\u003c/em\u003e (Knockleby et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Thi Thu Hien et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The presence of the same metabolites in the leaves and roots indicates that isoquinoline alkaloids are key metabolites in \u003cem\u003eS. dielsiana\u003c/em\u003e and probably, accumulate there during plant growth and adaptation (Yu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eQuantitative analysis indicated the highest abundance of crebanine (\u003cb\u003e3\u003c/b\u003e) and ayuthianine (\u003cb\u003e9\u003c/b\u003e) in the root tuber extract, followed by ushinsunine (\u003cb\u003e8\u003c/b\u003e) and reticuline (\u003cb\u003e7\u003c/b\u003e). The St-extr and St-1 had high relative content of 25.5% and 58.4% for crebanine (\u003cb\u003e3\u003c/b\u003e), respectively and 8.5% and 15.7% of stephanine (\u003cb\u003e5\u003c/b\u003e), respectively. We found that St-extr and St-1 inhibited cell vitality for 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) at concentrations higher than 20 \u0026micro;g/ml and inhibited significantly cell growth for 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) at concentrations higher than 50 \u0026micro;g/ml. The high abundance of crebanine (\u003cb\u003e3\u003c/b\u003e) and the presence of oxostephanine (\u003cb\u003e1\u003c/b\u003e) and stephanine (\u003cb\u003e5\u003c/b\u003e) in St-extr and St-1 was associated with cytotoxic activity (\u003cb\u003esee Suppl. Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.1.\u003c/b\u003e). Similar biological effect of the extract from the stems and leaves of \u003cem\u003eS. dielsiana\u003c/em\u003e has been observed on cancer cell lines (HepG2, MCF7 and OVCAR8) (Tran et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Correspondingly, Knockleby et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) have reported a dose-dependent increase in the anti-proliferative activities of the \u003cem\u003eS. dielsana\u003c/em\u003e derived oxostephanine (\u003cb\u003e1\u003c/b\u003e), crebanine (\u003cb\u003e3\u003c/b\u003e) and stephanine (\u003cb\u003e5\u003c/b\u003e) in several cancer (HeLa, MDA-MB231, MDA-MB-468, MCF-7) and non-cancer cell lines (184B5 and MCF10A) with inhibitory concentration (IC)\u003csub\u003e50\u003c/sub\u003e ranging from 17.82 to 48.17 \u0026micro;M for crebanine (\u003cb\u003e3\u003c/b\u003e); 1.66 to 4.35 \u0026micro;M for oxostephanine (\u003cb\u003e1\u003c/b\u003e) and 3.33 to 7.19 \u0026micro;M for stephanine (\u003cb\u003e5\u003c/b\u003e). The authors have found that oxostephanine (\u003cb\u003e1\u003c/b\u003e) is two-times more potent than stephanine (\u003cb\u003e5\u003c/b\u003e) against cancer cells (Knockleby et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study crebanine (\u003cb\u003e3\u003c/b\u003e), tetrahydropalmatine (\u003cb\u003e4\u003c/b\u003e) and stephanine (\u003cb\u003e5\u003c/b\u003e) were under the detection minimum in the St-2 and St-3 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but fraction St-2 contained oxostephanine (\u003cb\u003e1\u003c/b\u003e), ayuthianine (\u003cb\u003e9\u003c/b\u003e) and stesakine (\u003cb\u003e11\u003c/b\u003e). The biological action of ayuthianine (\u003cb\u003e9\u003c/b\u003e) and stesakine (\u003cb\u003e11\u003c/b\u003e) has not been thoroughly studied while oxostephanine (\u003cb\u003e1\u003c/b\u003e) is known to suppress Aurora kinase activity and to cause aneuploidy in cancer cells (Knockleby et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tran et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We observed that St-2 (10 \u0026micro;g/ml) enhanced the G1 and G2 cell pools and we proposed that St-2 may affect the initial phases of cell division, thus changing the synchronicity of cell proliferation. The fraction also retarded the shift from G1/G2 towards G3/G4 generations in some cells, lowering the percentages of stained cells in G4 gate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We have to note that St-2 was unable to suppress severely cell proliferation unlike our control treated with the topoisomerase inhibitor, etoposide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and despite its inhibitory effects failed to be toxic for the cells. Oxostephanine (\u003cb\u003e1\u003c/b\u003e) may contribute directly to the inhibitory effect of St-2 on cell proliferation as it has been shown to induced G2/M cycle arrest (Knockleby et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), however the synergistic antiproliferative effect of all alkaloids or of reticuline (\u003cb\u003e7\u003c/b\u003e) in the presence of oxostephanine (\u003cb\u003e1\u003c/b\u003e) /ayuthianine (\u003cb\u003e9\u003c/b\u003e) /stesakine (\u003cb\u003e11\u003c/b\u003e) in St-2 cannot be excluded. The anti-proliferative activity of the alkaloids in St-2 (4 and 10 \u0026micro;g/ml) might be also related with a decreased mitochondrial activity indicative for initiation of apoptosis. Indeed, oxostephanine (\u003cb\u003e1\u003c/b\u003e) at concentration of 2.5-5 \u0026micro;M can induce early apoptosis as evaluated by Annexin V staining (Tran et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReticuline (\u003cb\u003e7\u003c/b\u003e) was detected for the first time in the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003edielsiana\u003c/em\u003e. The alkaloid was predominant in a fraction St-2 followed by the fraction St-3. Reticuline (\u003cb\u003e7\u003c/b\u003e) and the alkaloids derived from (S)-reticuline can be found in leaves and stems, but the roots are the predominant site of their biosynthesis and the preferred time-dependent location for alkaloid biosynthetic enzymes (Zeng et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gorpenchenko et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This may explain why reticuline (\u003cb\u003e7\u003c/b\u003e) was highly abundant in the root extract of \u003cem\u003eS. dielsana\u003c/em\u003e and correspondingly in the St-2 and St-3 fractions.\u003c/p\u003e \u003cp\u003eThe compound was enriched from 12.1% in St-extr to 64.9% in St-2 and 97.5% in St-3 and most likely contributed to a cytotoxic effect of the highest concentrations (50 \u0026micro;g/ml) and a stimulatory action on cell growth and vitality at concentrations between 2 and 20 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). While St-2 contained other alkaloids (as we discussed above) which can have cytotoxic activity and interfere with proliferation and apoptosis, reticuline (\u003cb\u003e7\u003c/b\u003e) was at high relative content in St-3. We also compared the biological activity between the crude extract, fractions and isolated reticuline (\u003cb\u003e7\u003c/b\u003e) by using the concentrations of reticuline (\u003cb\u003e7\u003c/b\u003e) corresponding to its relative amount in them (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). St-3 and reticuline (\u003cb\u003e7\u003c/b\u003e) at concentration 9.5 \u0026micro;g/ml failed to show inhibitory effect on cell proliferation as the percentages of cells in G4 on day 3 reached 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Our data indicated that the growth and proliferation of HL-60 cells can be maintained by reticuline and this notion has been supported by the data from the MTT assay, where St-3 increased cell growth at concentrations between 2 and 20 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eInterestingly, St-3 (10 \u0026micro;g/ml) and reticuline (\u003cb\u003e7\u003c/b\u003e) (6.8 and 9.5 \u0026micro;g/ml) demonstrated a decreased mitochondrial staining intensity proposing that the cells might loss the mitochondrial membrane potential and might undergo apoptosis. The mitochondrial-dependent apoptotic pathway includes a disturbed balance between the pro-apoptotic (Bax, Bad, Spike) and the pro-survival (Bcl-2, Bcl-XL, Mcl-1) proteins that was followed by caspase activation. Reticuline (\u003cb\u003e7\u003c/b\u003e) derivative have cytotoxic and pro-apoptotic effects above 30 \u0026micro;M in epithelial like cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) (Kim et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, reticuline (\u003cb\u003e7\u003c/b\u003e) at similar concentration of 9.5 \u0026micro;g/ml (30 \u0026micro;M) in our study showed milder effect on mitochondrial staining without a significant increase in apoptosis and reduction in the frequency of live cells (positive for MitoSpy\u0026trade; NIR DilC1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Indeed, reticuline (\u003cb\u003e7\u003c/b\u003e) at low concentration (3.9 \u0026micro;g/ml) sustained mitochondrial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) proposing that its action on mitochondria is dose-dependent and might be reversible at low concentrations.\u003c/p\u003e \u003cp\u003eApoptosis can be also activated via TRAIL and the death receptor-mediated pathways. TRAIL can crosslink the receptor ligands on responder cells by which can induce and accelerate cell apoptosis (Shiiki et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pimentel et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We observed that reticuline (\u003cb\u003e7\u003c/b\u003e) can affect TRAIL\u0026thinsp;+\u0026thinsp;cells, but we did not show its direct role on TRAIL-induced apoptosis. It has been observed that other alkaloid, tetrandrine, found in \u003cem\u003eStephania\u003c/em\u003e spp, can upregulate the ligands of TRAIL and can elevate the sensitivity of chemotherapy resistant tumor cell lines to TRAIL-induced apoptosis (Shishodia et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is possible that reticuline (\u003cb\u003e7\u003c/b\u003e) may affect TRAIL expression and pathways indirectly via an effect on mitochondrial functions, as the link between both pathways exists (Johnstone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). TRAIL can also have apoptosis-independent functions on myeloid precursors related to the ability of myeloid cells to differentiate to osteoclast or granulocyte lineages (Shiiki et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Dimitrova et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liao et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It will be interesting to delineate the action of reticuline (\u003cb\u003e7\u003c/b\u003e) on TRAIL in respect to osteoclasts differentiation in the future.\u003c/p\u003e \u003cp\u003eTRAIL and its receptors can be involved in several intracellular pathways engaging the activation, phosphorylation and ubiquitination of proteins such as MAPKs and in particular p38 (Johnstone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, we studied the cytoplasmic level of non-phosphorylated and phosphorylated p38 MAPK in HL-60 cells. The cells were pre-cultured in the presence of St-2, St-3 (10 \u0026micro;g/ml) and reticuline (\u003cb\u003e7\u003c/b\u003e) (6.8 and 9.5 \u0026micro;g/ml) for 24 hours, then they were stimulated with PMA for 30 minutes and the intracellular level of the kinase was evaluated by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). St-2 and St-3 reduced the level of pp38 vs p38 in non-stimulated cells and failed to change that ratio upon PMA-stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Reticuline (\u003cb\u003e7\u003c/b\u003e) at low concentration showed similar effect to St-2 and St-3. At higher concentration, it decreased the phosphorylated vs non-phosphorylated form in non-stimulated cells and elevated significantly that ratio in PMA-stimulated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Indeed, we observed an increased fold of PMA-induced pp38 levels by reticuline (\u003cb\u003e7\u003c/b\u003e) indicating that the compound at high concentration can affect the intrinsic activation pathways leading to enhanced p38 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Similarly, other study using the immunoblot has demonstrated the elevated phosphorylated levels of p38 MAPK and of a downstream transcription factor NF-κB p65 \u003cem\u003ein vivo\u003c/em\u003e, in two experimental models of asthma and obesity‐related asthma mice (Lyu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This effect on p38 phosphorylation was also related to the nature of the stimulus applied and was affected by a loop regulating not only the phosphorylation but also the activation of p38 MAPK and entire NF‐κB signaling pathway (Lyu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We also observed a different effect of reticuline (\u003cb\u003e7\u003c/b\u003e) in non-stimulated and PMA-stimulated cells, proposing that effect of reticuline (\u003cb\u003e7\u003c/b\u003e) on p38 MAPK might depend on overall functional cellular state.\u003c/p\u003e \u003cp\u003eAs proliferation and apoptosis involving p38 MAPK are key processes that regulate the lineage commitment of promyelocytes towards granulocytes, we investigated the effect of the fractions and reticuline (\u003cb\u003e7\u003c/b\u003e) on the expression of the characteristic differentiation marker - CD11b (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). We observed that reticuline (\u003cb\u003e7\u003c/b\u003e) at high concentration (9.5 \u0026micro;g/ml) elevated DMSO-induced increase in the percentage of positive cells. This effect was similar to that of p38 MAPK inhibitor, SB203580. Indeed, this inhibitor has been shown to upregulated the expression of the \u003cem\u003eCD11b\u003c/em\u003e gene in HL-60 and NB4 promyelocyte cell lines on day 3 (Suzuki et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and was used to drive myelocyte differentiation in acute leukemia (Takahashi, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, in our study, reticuline (\u003cb\u003e7\u003c/b\u003e) can regulate CD11b expression on differentiating cells. However, its effect might be dependent of its purity as the presence of other alkaloids in the St-3 fraction interfered with the frequency of CD11b positive cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the methanolic root tuber extract of \u003cem\u003eS. dielsiana\u003c/em\u003e Y. C. Wu from Vietnam contained isoquinoline alkaloids inhibiting growth, survival and proliferation of HL-60 cell line. For the first time, reticuline (\u003cb\u003e7\u003c/b\u003e) was identified in the root extract of \u003cem\u003eS. dielsana\u003c/em\u003e. Reticuline-enriched fractions exhibited less cytotoxic effects at concentrations between 0.2 and 20 \u0026micro;g/ml than the individual alkaloid \u003cb\u003e7\u003c/b\u003e. However, the presence of other alkaloids in the fractions masked the biological activity of reticuline (\u003cb\u003e7\u003c/b\u003e). For example, St-2 that consist around 65% reticuline (\u003cb\u003e7\u003c/b\u003e), enhanced the G1 and G2 cell pools and changed the synchronicity of cell proliferation while the compound \u003cb\u003e7\u003c/b\u003e at the same concentration did not show such effect. St-3, containing around 95% reticuline (\u003cb\u003e7\u003c/b\u003e), showed similar effect on mitochondrial function and TRAIL\u0026thinsp;+\u0026thinsp;cells to the individual compound \u003cb\u003e7\u003c/b\u003e, but it was less effective on altering p38 phosphorylation in non-stimulated and stimulated cells. Finaly, reticuline (\u003cb\u003e7\u003c/b\u003e), unlike St-3, elevated the frequency of differentiating CD11b positive cells. This effect was similar to that of the p38 inhibitor but not to St-3. Our study demonstrated that reticuline (\u003cb\u003e7\u003c/b\u003e) derived from \u003cem\u003eS. dielsiana\u003c/em\u003e Y. C. Wu was able to affect growth, proliferation, apoptosis and differentiation of HL-60 cell line.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Vietnam Academy of Science and Technology (QTBG01.01/23-24) and the Bulgarian Academy of Sciences (IC-VT/03/2023-2025) under the International Scientific Cooperation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003cbr\u003e\u003c/strong\u003eNot applicable for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePetya Dimitrova\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Data curation.\u0026nbsp;\u003cstrong\u003eTsvetelina Doncheva\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Resources, Project administration, Data curation.\u0026nbsp;\u003cstrong\u003eNadezhda Kostova\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing,\u0026nbsp;Methodology, Data curation,\u0026nbsp;Formal analysis.\u0026nbsp;\u003cstrong\u003eIveta Uzunova:\u0026nbsp;\u003c/strong\u003eMethodology, Data curation.\u003cstrong\u003eNikol Latinova:\u003c/strong\u003e Methodology, Data curation.\u0026nbsp;\u003cstrong\u003eVanya Gerasimova\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Data curation,\u0026nbsp;Formal analysis.\u0026nbsp;\u003cstrong\u003eNguyen Tien Dat\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Project administration, Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eDo Hoang Giang\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Formal analysis, Data curation.\u0026nbsp;\u003cstrong\u003eNguyen Thi Luyen\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDeng Y, Yu Y, Luo H, Zhang M, Qin X, Li L (2011) Antimicrobial activity of extract and two alkaloids from traditional Chinese medicinal plant \u003cem\u003eStephania dielsiana\u003c/em\u003e. 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Acta Histochem 118(5):537\u0026ndash;543. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.acthis.2016.05.010\u003c/span\u003e\u003cspan address=\"10.1016/j.acthis.2016.05.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Stephania dielsiana, isoquinoline alkaloids, reticuline, p38 kinase, CD11b, HL-60 cell line","lastPublishedDoi":"10.21203/rs.3.rs-5585865/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5585865/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStephania dielsiana\u003c/em\u003e Y. C. Wu is a traditional medicinal plant that, like the other species of the genus \u003cem\u003eStephania\u003c/em\u003e, is used in some Asian countries for the treatment of various conditions and diseases. Phytochemical studies have revealed that the species\u0026rsquo; therapeutic activity is mainly due to isoquinoline alkaloids. This study focused on the biological effects of \u003cem\u003eS. dielsiana\u003c/em\u003e root tuber extract, alkaloid enriched fractions and the isolated alkaloid reticuline on promyelocytic cell line HL-60. Systematic chromatographic techniques were applied to obtain three alkaloid-enriched fractions and eleven pure alkaloids from the methanolic extract of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003edielsiana\u003c/em\u003e root tubers. The quantities of alkaloids in the plant extract and its fractions were determined using HPLC method. The biological effects of the extract, alkaloid fractions and the reticuline were evaluated using colorimetric and flow cytometric assays. The crude extract and alkaloid enriched fractions inhibited cell growth at concentrations higher than 50 \u0026micro;g/ml. One of the alkaloid enriched fractions (St-2) affected the synchronicity of proliferation till day 3. Based on the quantification analysis, reticuline was found as the predominant alkaloid in this fraction with the relative content of 64.9%. Reticuline (9.5 \u0026micro;g/ml) modestly affected mitochondrial functions, decreased the level of phosphorylated p38 kinase in non-stimulated cells, but increased stimulus-induced p38 phosphorylation and elevated the CD11b\u0026thinsp;+\u0026thinsp;cells, indicative for myeloid differentiation. In conclusion, we provide evidence that reticuline derived from \u003cem\u003eS. dielsiana\u003c/em\u003e root tubers affected growth, proliferation, apoptosis and differentiation of HL-60 cell line.\u003c/p\u003e","manuscriptTitle":"Biological effect of alkaloid enriched fractions and reticuline from the Stephania dielsianaY. C. Wu on promyelocyte HL-60 cell line","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 08:11:33","doi":"10.21203/rs.3.rs-5585865/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-02-03T15:13:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-01-08T15:53:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-27T13:01:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2024-12-23T08:05:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-20T09:36:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2024-12-19T09:20:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"239c1026-ba86-4029-8aaa-3f3c71d6c7a7","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:04:36+00:00","versionOfRecord":{"articleIdentity":"rs-5585865","link":"https://doi.org/10.1007/s43450-025-00657-5","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2025-05-28 15:57:17","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2024-12-30 08:11:33","video":"","vorDoi":"10.1007/s43450-025-00657-5","vorDoiUrl":"https://doi.org/10.1007/s43450-025-00657-5","workflowStages":[]},"version":"v1","identity":"rs-5585865","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5585865","identity":"rs-5585865","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}