Bioguided Fractionation of Phyllanthus spp.: Unveiling Anticancer Potential through Metabolomic Correlation and ADMETox Insights.

Cancer remains a significant global health concern, with mortality rates steadily rising and prompting an urgent search for effective treatments. This study focuses on the medicinal properties of plants from the Phyllanthus genus, specifically Phyllanthus amarus and Phyllanthus niruri, which have shown promise in traditional medicine. Through bioguided fractionation using preparative high-performance liquid chromatography (HPLC), bioactive compounds were isolated and identified using ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-QTOF-MSE) and nuclear magnetic resonance (NMR) spectroscopy. Chemometric analyses such as principal component analysis (PCA) aided in understanding metabolite distribution. Biological assays demonstrated cytotoxic activities of specific fractions against cancer cell lines, notably the PhyN 4n fraction from P. niruri, which induced S-phase cell cycle arrest and apoptosis in HL60 cells. These findings underscore the anticancer potential of Phyllanthus species and lay the groundwork for future drug development efforts. The study's integration of advanced analytical techniques, chemometrics, and biological assays provides valuable insights for harnessing natural products in the fight against cancer.

Free radical scavenging polyphenols isolated from Phyllanthus niruri L. ameliorates hyperglycemia via SIRT1 induction and GLUT4 translocation in in vitro and in vivo models.

Type 2 diabetes milletus (T2DM) is a complex multifaceted disorder characterized by insulin resistance in skeletal muscle. Phyllanthus niruri L. is well reported sub-tropical therapeutically beneficial ayurvedic medicinal plant from Euphorbiaceae family used in various body ailments such as metabolic disorder including diabetes. The present study emphasizes on the therapeutic potential of Phyllanthus niruri L. and its phytochemical(s) against insulin resistance conditions and impaired antioxidant activity thereby aiding as an anti-hyperglycemic agent in targeting T2DM. Three compounds were isolated from the most active ethyl acetate fraction namely compound 1 as 1-O-galloyl-6-O-luteoyl-ß-D-glucoside, compound 2 as brevifolincarboxylic acid and compound 3 as ricinoleic acid. Compounds 1 and 2, the two polyphenols enhanced the uptake of glucose and inhibited ROS levels in palmitate induced C2C12 myotubes. PNEAF showed the potent enhancement of glucose uptake in palmitate-induced insulin resistance condition in C2C12 myotubes and significant ROS inhibition was observed in skeletal muscle cell line. PNEAF treated IR C2C12 myotubes and STZ induced Wistar rats elevated SIRT1, PGC1-α signaling cascade through phosphorylation of AMPK and GLUT4 translocation resulting in insulin sensitization. Our study revealed an insight into the efficacy of marker compounds isolated from P. niruri and its enriched ethyl acetate fraction as ROS scavenging agent and helps in attenuating insulin resistance condition in C2C12 myotubes as well as in STZ induced Wistar rat by restoring glucose metabolism. Overall, this study can provide prospects for the marker-assisted development of P. niruri as a phytopharmaceutical drug for the insulin resistance related diabetic complications.

Screening of Phyllanthus niruri Leaves Phytoconstituents for Antiviral and Antibacterial Activity by Molecular Docking Studies.

The genus Phyllanthus belongs to one of the largest plant families, the Phyllantaceae (L.). Phyllanthus niruri is an annual perennial herb that grows in tropical Asia, America, China, and the islands of the Indian Ocean. Numerous alkaloids, steroids, flavonoids, lignans, coumarins, polyphenols, and lipids are present in Phyllanthus. The effects of plants have been studied for a variety of purposes, including their antioxidant (Giribabu et al., Evid Based Complement Alternat Med, 2014), anti-inflammatory (Porto et al., Revista Brasileira de Pharmacognosy, 2013), antinociceptive (Sathisha et al., Indian Drugs, 2009), analgesic (Mostofa et al., BMC Complement Altern Med, 2017), antiulcer (Mali et al., Biomed Aging Pathol, 2011), antiarthritic (Obidike and Salawu, Planta Medica, 2010), antiplasmodial (Shilpa et al., Environ Dis, 2018), immunomodulatory (Manikkoth et al., Anticonvulsant activity of Phyllanthus amarus in experimental animal models), anticonvulsant (Wasnik et al., Int J Pharm Sci Rev Res, 2014), antidepressant (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antiviral (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antitumor (Sharma et al., Asian Pac J Cancer Prev, 2009), hyperlipidemia (Khanna et al., J Ethnopharmacol, 2002), and antifertility (Ezeonwu, Inquiries J, 2011). For additional docking investigations with distinct proteins, the leaf chemicals are assessed, that is, the crystal structure of serine protease hepsin in complex with inhibitor [PDB ID:5 CE1] for antiviral activity human topoisomerase II beta in complex with DNA and etoposide [PDB ID:3QX3] and crystal structure of E. coli GyraseB 24 kDa in complex with 4-(4-bromo-1H-pyrazol-1-yl)-6-[(ethylcarbamoyl)amino]-N-(pyridin-3-yl) pyridine-3-carboxamide [PDB ID: 6F86] for antibacterial activity and have been selected. To evaluate the in silico results and grading of virtual screening, or molecular docking, ritonavir antiviral activity and ampicillin for antibacterial activity were used as a benchmark.

Screening of Phyllanthus niruri Plant Active Constituents for Anticancer and Antifungal Activity by Insilico Methods.

A large genus of shrubs, trees, and rare plants belonging to the Euphorbiaceae family, Phyllanthus contains 600-700 species. The Phyllanthus niruri (L.) species is a tiny, erect annual herb that can reach heights of 30-40 cm. Its 7-12 cm long, sessile, alternating leaves are native to the Amazon rainforest, but they can also be found in other tropical regions such as South East Asia, Southern India, America, China, and the islands of the Indian Ocean. Phyllanthus contains many classes of alkaloids, steroids, flavonoids, lignin, polyphenols, and lipids. Numerous activities of the plant have been studied, including antidepressant (Wasnik et al., Int J Pharm Sci Rev Res, 6:26-29, 2014), anticancer (Sayuti et al. Studies, 10:17, 2020), anti-inflammatory, antinociceptive (Porto et al., Revista Brasileira de Farmacognosia, 23:138-144, 2013), analgesic (Bhat et al., Pharm Res, 7:378, 2015), antiarthritic (Mali et al., Biomed Aging Pathol, 1:185-190, 2011), immunomodulatory, antibacterial, antifungal (Shilpa et al., Environm Dis, 3:63, 2018), antidiabetic (Kumar et al., Biomed Pharm J, 12:57-63, 2019), antiulcer (Mostofa et al., BMC Complement Altern Med, 17:1-10, 2017), antiviral (Wahyuni et al., Malays Appl Biol, 48:105-111, 2019), antiplasmodial (Ifeoma et al., Asian Pacific J Trop Med, 6:169-175, 2013), anticonvulsant (Amaechina and Omogbai, Nig J Nat Prod Med, 17:61-65, 2013), and hepato human cytochrome P450 CYP17A1 in association with abiraterone [PDB ID: 3RUK] plant extracts. New selective androgen receptor modulators were synthesized, and they were biologically evaluated (SARMs) (Micah et al., J Veter Med Anim Health 5(1):8-15, 2013, Rusmana et al., Indonesian Biomed J 9(2):84-90, 2017, Al Zarzour et al., Nutrients 10(8):1057, 2018, Khanna J Ethnopharmacol 82(1):19-22, 2002). In the present study [PDB ID: 3RUK,5T8E] with anticancer and [PDB ID: 6F0E,1EA1] with antifungal activities were used for docking study. In this study fluconazole's antifungal activity and dacarbazine's anticancer activity were used as benchmarks for molecular docking with Schrodinger 13.0 to compare the activity of Phyllanthus niruri's active constituents.

Molecular Docking Studies of Phyllanthus niruri Root Phytoconstituents for Antibreast Cancer Activity Using Multiple Proteins.

The systematic exploitation of the structural variety of natural products is made possible by docking studies, which have been shown to be a crucial technique. The objective of the current work was to use molecular docking studies with different proteins to identify acceptable and efficient compounds from root phytoconstituents of Phyllanthus niruri plant. Numerous human disorders have been treated using Phyllanthus niruri. Antioxidant, antibacterial, antifungal, anti-inflammatory, antipyretic, antimalarial, antiviral, immunomodulatory, antidepressant, anticonvulsant, antinociceptive, anti-ulcer, anti-arthritic, anticancer, hyperlipidemia, and antifertility were only a few of its many pharmacological effects. One of the most prevalent malignancies in women worldwide, breast cancer is one of the leading causes of mortality worldwide. The most successful breast cancer treatments now on the market have negative side effects and are useless in people. In order to develop drugs, molecules with such a framework have been utilized as the lead. Schrodinger Maestro (v13.0) software was used to conduct a molecular docking analysis of root components with certain proteins linked to the illnesses. In comparison to commercially available conventional medications, molecular docking data also demonstrated greater scores. Dacarbazine's ability to treat cancer was utilized as benchmark to assess the in silico outcomes and grading of virtual screening or molecular docking.

Anti-inflammatory ent-cleistanthane-type diterpenoids from Phyllanthus rheophyticus.

A bioactivity-guided isolation from the aerial parts of Phyllanthus rheophyticus obtained 17 undescribed ent-cleistanthane-type diterpenoids, namely phyllarheophols A-Q, as well as 12 known analogs. Their structures were characterized by a combination of spectroscopic data interpretation, single-crystal X-ray diffraction and ECD analysis. The anti-inflammatory activities of these compounds were evaluated by measuring their inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophages, and their preliminary structure-activity relationships were also discussed. Further study showed that promising compounds phyllarheophol D and phyacioid B significantly suppressed the expressions of cytokines and nitric oxide synthase through the NF-κB signaling pathway.

Polyphenolic compounds of Phyllanthus amarus Schum & Thonn. (1827) and diabetes-related activity of an aqueous extract as affected by in vitro gastrointestinal digestion.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the aerial part of Phyllanthus amarus have been extensively used in several countries to cure diabetes. No data is available on the impact of gastrointestinal digestion of such crude extracts on their antidiabetic activity. AIM OF THE STUDY: The aim of this study was to identify active fractions and compounds of fresh aerial parts of P. amarus extracted by an infusion method that are responsible for antidiabetic effects occurring at the level of glucose homeostasis. MATERIALS AND METHODS: An aqueous extract was obtained by an infusion method and its polyphenolic composition was analysed by reverse phase UPLC-DAD-MS. The influence of in vitro gastrointestinal digestion was evaluated both on the chemical composition and on the antidiabetic effect of P. amarus infusion extract using glucose-6-phosphatase enzyme inhibition and stimulation of glucose uptake. RESULTS: Analysis of the chemical composition of the crude extract revealed the presence of polysaccharides and various families of polyphenols such as phenolic acids, tannins, flavonoids and lignans. After simulated digestion, the total content of polyphenols decreased by about 95%. Caffeoylglucaric acid derivates and lignans exhibited strong stimulation of glucose uptake similar to metformin with an increase of 35.62 ± 6.14% and 34.74 ± 5.33% respectively. Moreover, corilagin, geraniin, the enriched polysaccharides fraction and the bioaccessible fraction showed strong anti-hyperglycemic activity with about 39-62% of glucose-6-phosphatase inhibition. CONCLUSION: Caffeoylglucaric acid isomers, tannin acalyphidin M1 and lignan demethyleneniranthin were reported for the first time in the species. After in vitro gastroinstestinal digestion, the composition of the extract changed. The dialyzed fraction showed strong glucose-6-phosphatase inhibition.

Combination Curcuma longa and Phyllanthus niruri Extract Potentiate Antiproliferative in Triple Negative Breast Cancer MDAMB-231 Cells.

BACKGROUND: Triple negative breast cancer cells (TNBC) are a small part of cancer-inducing cells in breast cancer, which are characterized by high metastatic and self-renewal. Self-renewal has the ability to renew itself and loses control of proliferation. Curcuma longa extract (CL) and Phyllanthus niruri extract (PN) known to have anti-proliferative effects on cancer cells. However, the effects of combination CL and PN on TNBC proliferation still unclear. AIMS: This study aimed to evaluate the antiproliferative effects of the combination CL and PN on TNBC MDAMB-231 and attempted to elucidate the underlying molecular mechanisms. SUBJECTS AND METHODS: The dried rhizomes of Curcuma longa and the herbs of Phyllanthus niruri were macerated with ethanol for 72 h.The antiproliferative and synergistic effects of combination CL and PN were investigated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Combination index values were calculated using CompuSyn (ComboSyn, Inc, Paramus, NJ). The cell cycle and apoptosis assay were determined by propidium iodide (PI) and PI-AnnexinV assay under flow cytometer, respectively. The intracellular ROS levels were evaluated using 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) assay. The mRNA expressions of proliferation-related genes in the cells were determined using bioinformatic assay. RESULTS: The CL and PN single treatment caused a potent and dose-dependent decrease in the percentage of viable cells with IC50 value of 13 µg/mL and 45 µg/mL for 24 h, respectively. The combination index values of the different combinations ranged from 0.08 - 0.90, indicating slightly strong to very strong synergistic effects. The combination of CL and PN also remarkably induced the S- and G2/M-phases cell cycle arrest that leading to apoptosis induction. Furthermore, the combination of CL and PN treatment induced the intracellular reactive oxygen species (ROS) levels. Mechanistically, the AKT1, EP300, STAT3 and EGFR signaling as potential targets of combination CL and PN in antiproliferation and antimetastatic of TNBC. CONCLUSIONS: The combination of CL and PN exerted promising antiproliferative effects in TNBC. Therefore, CL and PN may be considered a potential source for the development of potent anticancer drugs for breast cancer treatment.

Cytotoxic and chemomodulatory effects of Phyllanthus niruri in MCF-7 and MCF-7ADR breast cancer cells.

The members of the genus Phyllanthus have long been used in the treatment of a broad spectrum of diseases. They exhibited antiproliferative activity against various human cancer cell lines. Breast cancer is the most diagnosed cancer and a leading cause of cancer death among women. Doxorubicin (DOX) is an anticancer agent used to treat breast cancer despite its significant cardiotoxicity along with resistance development. Therefore, this study was designed to assess the potential cytotoxicity of P. niruri extracts (and fractions) alone and in combination with DOX against naïve (MCF-7) and doxorubicin-resistant breast cancer cell lines (MCF-7ADR). The methylene chloride fraction (CH2Cl2) showed the most cytotoxic activity among all tested fractions. Interestingly, the CH2Cl2-fraction was more cytotoxic against MCF-7ADR than MCF-7 at 100 µg/mL. At sub-cytotoxic concentrations, this fraction enhanced the cytotoxic effect of DOX against the both cell lines under investigation (IC50 values of 0.054 µg/mL and 0.14 µg/mL vs. 0.2 µg/mL for DOX alone against MCF-7) and (1.2 µg/mL and 0.23 µg/mL vs. 9.9 µg/mL for DOX alone against MCF-7ADR), respectively. Further, TLC fractionation showed that B2 subfraction in equitoxic combination with DOX exerted a powerful synergism (IC50 values of 0.03 µg/mL vs. 9.9 µg/mL for DOX alone) within MCF-7ADR. Untargeted metabolite profiling of the crude methanolic extract (MeOH) and CH2Cl2 fraction exhibiting potential cytotoxicity was conducted using liquid chromatography diode array detector-quadrupole time-of-flight mass spectrometry (LC-DAD-QTOF). Further studies are needed to separate the active compounds from the CH2Cl2 fraction and elucidate their mechanism(s) of action.

Agent in Urgent Need of Clinical Practice: Corilagin.

Corilagin is a naturally occurring water-soluble retrogallic acid tannin, which can be extracted from many kinds of plants. Known at present, it is the main effective ingredient of Phyllanthus urinaria L., Geranium wilfordii Maxim., Phyllanthus matsumurae Hayata, and Trifolium repens L. It also exists in Phyllanthus emblica L., Dimocarpus longan Lour., Canarium album (Lour.) Raeusch., and Terminalia chebula Retz. It can participate in a variety of signaling pathways in vivo and has multiple biological activities, including antitumor, anti-microbial, anti-oxidation, anti-inflammation, hepatoprotective, anti-allergy, anti-proliferation and so on. Given the limited efficacy of first-line treatments for many diseases such as oncology, chronic liver disease, and rheumatic immune system diseases, and the potential for adverse effects to outweigh the therapeutic effects, attention is being focused on alternative treatments, and natural plant extracts are a natural target for alternative treatments, as natural substances tend to have low toxicity to normal tissues. Some proprietary Chinese medicines containing corilagin have been used in clinical applications, being clinically applied to treat chronic liver disease, viral hepatitis B, rheumatoid arthritis and other diseases. This paper reviews the extraction, determination, distribution and harvesting, pharmacokinetics, biological activity, safety assessment of corilagin and its application in clinical practice.

Cultivo in vitro de raíces pilosas del arbusto Phyllanthus acuminatus (Phyllanthaceae) / In vitro culture of hairy roots of the shrub, Phyllanthus acuminatus (Phyllanthaceae)

Introducción: En condiciones naturales, las raíces del arbusto, Phyllanthus acuminatus, producen bajas concentraciones de metabolitos secundarios de interés medicinal. Esto abre una oportunidad para el cultivo in vitro, para aumentar la concentración de metabolitos. Objetivo: Determinar las condiciones óptimas de cultivo líquido para raíces pilosas de P. acuminatus. Métodos: Se utilizó la evaluación del crecimiento de la biomasa según porcentaje de inóculo inicial (0.50 y 0.10 %), porcentaje de nutrientes de los medios (100, 50 y 25 %) y tasa de agitación (90, 100 y 110 min-1) (N= 15 repeticiones). Resultados: Las mejores condiciones de cultivo líquido fueron: 0.10 % de inóculo inicial, nutrientes al 25 % y 90 min-1 para la tasa de agitación. Hay diferencias entre las raíces pilosas y las raíces no transformadas. Conclusiones: es factible producir raíces pilosas de P. acuminatus a gran escala, aplicando e implementando las condiciones evaluadas de porcentaje de inóculo, nutrientes en el medio y tasas de agitación utilizadas en este estudio.

Introduction: Under natural conditions, the roots of the shrub, Phyllanthus acuminatus, produce low concentrations of secondary metabolites of medicinal interest. This opens an opportunity for in vitro culture, to increase metabolite concentration. Objective: To determine the optimal liquid culture conditions for hairy roots of P. acuminatus. Methods: We used biomass growth evaluation according to initial inoculum percentage (0.50 and 0.10 %), percentage of medium nutrients (100, 50 and 25 %) and agitation rate (90, 100 and 110 min-1) (N=15 replications). Results: The best liquid culture conditions were: 0.10 % of initial inoculum, nutrients at 25 % and 90 min-1 for the agitation rate. There are differences among hairy roots and non-transformed roots. Conclusions: It is feasible to produce P. acuminatus hairy roots at a large scale, applying and implementing the evaluated conditions of inoculum percentage, nutrients in the medium and agitation rates.

Genotoxicity, Cytotoxicity and Phenolic Compounds from Aqueous Extracts of Phyllanthus tenellus Roxb. Cultivated Under Different Light Conditions.

<b>Background and Objective:</b> <i>Phyllanthus tenellus</i> Roxb. is a medicinal species widely used in Brazil for diseases of the urinary tract, kidney stones, infections and as a diuretic. The therapeutic property of this species is due to the production of phenolic compounds by secondary metabolism. However, cultivation conditions can alter the production of phenolic compounds and compromise the medicinal use of the species. The aim of this research was the evaluation of the genotoxic and cytotoxic activity of aqueous extracts of the species <i>Phyllanthus tenellus</i> Roxb. grown with and without shading, on the <i>Allium cepa</i> cell cycle and also, determine the phenolic compounds present in the aqueous extracts in each of the cultivation conditions. <b>Materials and Methods:</b> For the <i>Allium cepa</i> test three concentrations of 5, 10 and 15 g L¹ of the aerial part of the plant were used for the preparation of aqueous extracts, referring to the two forms of cultivation, with and without shading. <b>Results:</b> The aqueous extracts of <i>Phyllanthus tenellus</i> Roxb. have cytotoxic activity, except for the 5 g L¹ concentration of the cultivation with shading, which is the only concentration that has a genotoxic effect. <b>Conclusion:</b> The incidence of light stimulates the increase in the concentration of phenolic compounds (total polyphenols, flavonoids and tannins) in the species when cultivated in full sun.

Restoration of Hepatic and Intestinal Integrity by Phyllanthus amarus Is Dependent on Bax/Caspase 3 Modulation in Intestinal Ischemia-/Reperfusion-Induced Injury.

Ethnopharmacological relevance: Oxidative stress is a key player in intestinal ischemia/reperfusion (I/R) injury (IIRI) with a tendency to trigger systemic inflammatory response, resulting in progressive distal organ injury. To date, the role of Bax/caspase 3 signaling in IIRI has not been reported. Furthermore, the discovery of a safe and effective drug remains pertinent in improving the outcome of IIRI. Therefore, this study investigated the role of Bax/caspase 3 signaling in intestinal I/R-induced intestinal and hepatic injury. In addition, the protective effect and possible associated mechanism of action of methanolic Phyllanthus amarus leaf extract (PA) against intestinal I/R-induced intestinal and hepatic injury were evaluated. Materials and methods: Fifty male Wistar rats were randomized into five groups (n = 10). The sham-operated group was received 0.5 mL of distilled water for seven days prior to the sham surgery, while the IIRI, febuxostat (FEB) + IIRI, low-dose PA (LDPA) + IIRI, and high-dose PA (HDPA) + IIRI groups underwent the I/R procedure. In addition to the procedure, IIRI, FEB + IIRI, LDPA + IIRI, and HDPA + IIRI received 0.5 mL of distilled water, 10 mg/kg of febuxostat, 200 mg/kg of PA, and 400 mg/kg of PA, respectively, for seven days prior to the I/R procedure. Results: Administration of methanolic Phyllanthus amarus leaf extracts attenuated the intestinal I/R-induced rise in intestinal and hepatic injury markers, malondialdehyde, nitric oxide, TNF-α, IL-6, and myeloperoxidase activities. In addition, Phyllanthus amarus ameliorated I/R-induced suppression of reduced glutathione, thiol and non-thiol proteins, and superoxide dismutase, catalase, and glutathione peroxidase activities in intestinal and hepatic tissues. These were coupled with the suppression of I/R-induced bacterial translocation, downregulation of I/R-induced activation of Bax/caspase 3 signaling, and improvement of I/R-induced distortion of intestinal and hepatic histoarchitecture by Phyllanthus amarus. Conclusion: Methanolic Phyllanthus amarus leaf extract protects against intestinal and hepatic injuries associated with intestinal I/R by suppressing oxidative-stress-mediated activation of Bax/caspase 3 signaling. The beneficial effects of Phyllanthus amarus may be ascribed to its constituent bioactive molecules, especially tannins, anthocyanin, alkaloids, and phenolics.

Network pharmacology identification and in Vivo validation of key pharmacological pathways of Phyllanthus reticulatus (Euphorbiaceae) leaf extract in liver cancer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus reticulatus (Euphorbiaceae) is a medicinal plant that has been used in Zhuang medicine since ancient times. Traditionally, it has the effect of removing toxins and detumescence and can be used to treat hepatitis in China and India. Our previous studies have proved that the ethyl acetate extract of its leaves (PRPE) has an anti-hepatoma effect. AIM OF THE STUDY: To predict targets of an ethyl acetate extract of Phyllanthus reticulatus leaves (PRPE) in hepatoma treatment via network pharmacology and verify the predictions in a mouse model of liver cancer. MATERIALS AND METHODS: Chemical constituents and therapeutic targets of P. reticulatus (PRP) were searched and predicted via public databases. A protein-protein interaction network comprising common targets was constructed, and the key gene targets were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for biological function and pathway enrichment analyses. The effects of PRP on BEL-7404 and HepG2 cells were determined by MTT assay, apoptosis was measured by flow cytometry and hoechst44432/PI. And a nude mouse xenograft model was established to verify the anti-tumour effect in vivo. The histopathology of tumours was observed by staining with haematoxylin and eosin (H&E). Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to determine the gene and protein expression levels of phosphoinositide 3-kinase (PI3K), Akt1, p53, caspase-3, Bcl-2 and Bax, respectively. RESULTS: Twenty-seven chemical components and 567 potential therapeutic targets of PRP were identified. GO analysis indicated that these targets are mainly associated with peptidyl-tyrosine phosphorylation and steroid metabolic process. KEGG analysis showed that the targets are mainly located in the PI3K/Akt, apoptosis, mitogen-activated protein kinase (MAPK), Ras and vascular endothelial growth factor (VEGF) signalling pathways. According to the p-adjust value, the PI3K/Akt pathway is the core pathway. In vitro, PRPE could inhibit proliferation and induce apoptosis in hepatoma cells. IC50 values of PRPE were 2.48 and 6.34 mg/mL for BEL-7404 and hepG2 cells, respectively. PRPE significantly reduced tumour volume and weight. H&E results showed that PRPE repaired necrotic areas in hepatoma cells. PRPE reduced the protein expression of PI3K, Akt1 and Bcl-2 and increased the protein expression of p53 and Bax. Meanwhile, PRPE reduced the mRNA expression of PI3K, AKT1 and BCL2 and increased the mRNA expression of TP53, CASP3 and BAX. CONCLUSION: The targets of PRPE are the PI3K/Akt, apoptosis, MAPK, Ras and VEGF signalling pathways. Passing through the PI3K/Akt pathway to induce apoptosis is the main mechanism of PRPE.

Phyllanthus taxodiifolius Beille Disrupted N-cadherin, Vimentin, Paxillin and Actin Stress Fibers in Glioblastoma.

OBJECTIVE: Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus taxodiifolius Beille extract on molecules known to be hallmarks of aggressive glioblastoma including N-cadherin and vimentin, mesenchymal markers, as well as paxillin, a major adaptor protein that regulates the linking of focal adhesions to the actin cytoskeleton. METHODS: P. taxodiifolius were air-dried, powdered and percolated with methanol, filtered, concentrated and lyophilized to yield a crude methanol extract. C6 glioblastoma cell line was used in this study. The expression of N-cadherin and vimentin, as well as the activation of paxillin was determined using Western blot analysis. The effect of the extract on focal adhesions and actin cytoskeleton were investigated using immunofluorescence staining and confocal imaging. RESULTS: In the presence of 40 µg/ml Phyllanthus taxodiifolius Beille extract, the expression of N-cadherin and vimentin were significantly decreased (p<0.001 and p<0.05, respectively). Activation of paxillin was also diminished as indicated by a reduction of phosphorylated-paxillin (p<0.01). Consequently, actin stress fibers in glioblastoma cells were abolished as evidenced by the decrease in focal adhesion (p<0.001) and stress fibers numbers (p<0.001). CONCLUSION: Our study demonstrates for the first time that P. taxodiifolius interferes with multiple key molecules related to pathological hallmarks of glioblastoma. These molecules are involved with cell contacts, focal adhesions, and the formation and stabilization of actin stress fibers, which are required for glioblastoma metastatic behavior. These results provide further evidence supporting the potential of P. taxodiifolius and its bioactive compounds as anti-cancer agents.

Contrasting patterns of nickel distribution in the hyperaccumulators Phyllanthus balgooyi and Phyllanthus rufuschaneyi from Malaysian Borneo.

Globally, the majority of Ni hyperaccumulator plants occur on ultramafic soils in tropical regions, and the genus Phyllanthus, from the Phyllanthaceae family, is globally the most represented taxonomical group. Two species from Sabah (Malaysia) are remarkable because Phyllanthus balgooyi can attain >16 wt% of Ni in its phloem exudate, while Phyllanthus rufuschaneyi reaches foliar concentrations of up to 3.5 wt% Ni, which are amongst the most extreme concentrations of Ni in any plant tissue. Synchrotron X-ray fluorescence microscopy, nuclear microbe (micro-PIXE+BS) and (cryo) scanning electron microscopy with energy dispersive spectroscopy were used to spatially resolve the elemental distribution in the plant organs of P. balgooyi and P. rufuschaneyi. The results show that P. balgooyi has extraordinary enrichment of Ni in the (secondary) veins of the leaves, whereas in contrast, in P. rufuschaneyi Ni occurs in interveinal areas. In the roots and stems, Ni is localized mainly in the cortex and phloem but is much lower in the xylem. The findings of this study show that, even within the same genus, the distribution of nickel and other elements, and inferred processes involved with metal hyperaccumulation, can differ substantially between species.

In silico, in vitro screening of antioxidant and anticancer potentials of bioactive secondary metabolites from an endophytic fungus (Curvularia sp.) from Phyllanthus niruri L.

The main objective of this research work is to discover novel and efficient phytochemical substances from endophytic fungus found in medicinal plants. Curvularia geniculata L. (C. geniculata L.), an endophytic fungus isolated from Phyllanthus niruri L. (P. niruri L.), was tested against hepatoma cell lines (HepG2) in order to screen their antioxidant and anticancer potentials. The profiling of phytochemicals from the fungal extract was characterized using gas chromatography-mass spectrometry (GC-MS), and molecular docking was done for the identified compounds against one of the potential receptors predominantly present in the hepatocellular carcinoma cell lines. Among the phytochemicals found, 2-methyl-7-phenylindole had the highest binding affinity (- 8.8 kcal mol-1) for the epidermal growth factor receptor (EGFR). The stability of 2-methyl-7-phenylindole in the EGFR-binding pockets was tested using in silico molecular dynamics simulation. The fungal extract showed the highest antioxidant activity as measured by DPPH, ABTS radical scavenging, and FRAP assays. In vitro cytotoxicity assay of fungal extract demonstrated the concentration-dependent cytotoxicity against HepG2 cells after 24 h, and the IC50 (50% cell death) value was estimated to be 62.23 µg mL-1. Typical morphological changes such as condensation of nuclei and deformed membrane structures are indicative of ongoing apoptosis. The mitochondria of HepG2 cells were also targeted by the endophytic fungal extract, which resulted in substantial generation of reactive oxygen species (ROS) leading to the destruction of mitochondrial transmembrane potential integrity. These outcomes suggest that the ethyl acetate extract of C. geniculata L. has the potential to be an antioxidant agent and further to be exploited in developing potential anticancer agents.

New ent-Kaurane and cleistanthane diterpenoids with potential cytotoxicity from Phyllanthus acidus (L.) Skeels.

Six diterpenoids including three ent-kauranes (1-2, 4) and three cleistanthanes (3, 5-6) were isolated from the roots and stems of Phyllanthus acidus (L.) Skeels. Of them, (16S)-ent-16,17,18-tri-hydroxy-19-nor-kaur-4-en-3-one (1), phyllanthone A (2), and 6-hydroxycleistanthol (3) are new compounds, while the ent-kaurane diterpenoids were reported from the titled plant for the first time. Their structures were elucidated on the basis of the extensive spectroscopic analyses. Compounds 2 and 4-6 displayed cytotoxic potential with IC50 values ranging from 1.96 to 29.15 µM. They also showed moderate anti-inflammatory activities (IC50 = 6.30-12.05 µM). Particularly, the new ent-kaurane 2 displayed cytotoxic potential against HL-60 (IC50 = 2.00 µM) and MCF-7 (IC50 = 3.55 µM) cells, and anti-inflammatory activity (IC50 = 6.47 µM).

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer.

PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.

Modulatory effects of stonebreaker (Phyllanthus amarus) and bitter gourd (Momordica charantia) on enzymes linked with cardiac function in heart tissue of doxorubicin-stressed rats.

Doxorubicin (DOX) has been linked with impairment in cardiovascular function and redox balance. In the present study, the effect of Phyllanthus amarus (PA) and Momordica charantia (MC) leaves on some biomolecules [Angiotensin-I converting enzyme (ACE), arginase, acetylcholinesterase (AChE), adenosine deaminase (ADA), lactate dehydrogenase (LDH)] and antioxidant [catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA)] linked with cardiac function in DOX-stressed rats was evaluated. Animals were grouped and pretreated with PA and MC leaf extracts at different doses (200 and 400 mg/kg/bwt orally), while DOX (15 mg/kg/bwt) was administered intraperitoneally on the last day of the experiment. Result revealed an increase of ACE, arginase, AChE, ADA, LDH activities and MDA level as well as a significant reduction in CAT and SOD activities, and GSH level in the rats treated with DOX compared to the control. However, these were significantly mitigated in the rats pretreated with PA and MC dose dependently. Chemical characterization of the leaf extracts via high performance liquid chromatography revealed the presence of some phenolic compounds which included kaempferol, catechin, epicatechin, ellagic acid, gallic acid quercetin, isoquercitrin and rutin. These findings revealed a significant improvement in redox imbalance and other biomolecules associated with cardiac function, which was altered by DOX. This improvement could be linked to the presence of cardioprotective agents present in PA and MC, thereby making these plants therapeutic agents for the treatment of cardiovascular complications associated with drugs such as DOX.