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Preparation of Lignan-Rich Extract from the Aerial Parts of Phyllanthus niruri Using Nonconventional Methods.

ABSTRACT: Phyllanthin and related lignans were found to be responsible, at least in part, for most of the activity of Phyllanthus species. This observation encouraged the authors to develop methods for the preparation of an extract rich in phyllanthin and related lignans from the aerial parts of P. niruri L. Direct extraction with solvents produced extracts with variable yields and contents of lignans. Lignans were identified by LC-ESI-MS analysis as phyllanthin (used as marker substance), hypophyllanthin, phylltetralin, nirtetralin, and niranthin. Extraction with boiling water produced 18.10 g% (w/w) extract with a trace amount of lignans (phyllanthin content of 0.33 ± 0.10 mg/g extract), while extraction with MeOH gave 3.6 g% w/w extract with a low phyllanthin content (3.1 mg/g extract), as determined by HPLC. However, Soxhlet extraction with hexane, CH₂Cl₂, or acetone gave extracts with low yields (0.82, 1.12, and 3.40 g% w/w, respectively) and a higher phyllanthin contents (36.2 ± 2.6, 11.7 ± 1.68, and 11.7 ± 1.10 mg/g extract, respectively). Extraction quality and efficiency were optimized by adopting the following three different approaches: (1) Alkaline digestion of the plant material with 30% potassium hydroxide yielded 3.1 g% w/w of purified extract with high phyllanthin content (22.34 ± 0.13 mg/g); (2) microwave-assisted extraction using 80% MeOH gave an extract with a better yield (8.13 g% w/w) and phyllanthin content (21.2 ± 1.30 mg/g) (after filtration through a Diaion HP-20 column); and (3) treatment of the ground plant material at 50 °C with two hydrolytic enzymes, cellulase (9 U/g for 12 h) and then, protease (4 U/g up to 72 h) optimized the yield of extract (13.92 g% w/w) and phyllanthin content (25.9 mg/g extract and total lignans content of 85.87 mg/g extract). In conclusion, the nonconventional methods presented here are superior for optimizing the yield of extract and its lignan contents from the aerial parts of P. niruri.

SUBMITTER: Meselhy MR

PROVIDER: S-EPMC7179407 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Preparation of Lignan-Rich Extract from the Aerial Parts of Phyllanthus niruri Using Nonconventional Methods.

Meselhy Meselhy R MR Abdel-Sattar Ola E OE El-Mekkawy Sahar S El-Desoky Ahmed M AM Mohamed Shanaz O SO Mohsen Sobhy M SM Abdel-Sattar Essam E El-Halawany Ali A

Molecules (Basel, Switzerland) 20200305 5

Phyllanthin and related lignans were found to be responsible, at least in part, for most of the activity of Phyllanthus species. This observation encouraged the authors to develop methods for the preparation of an extract rich in phyllanthin and related lignans from the aerial parts of P. niruri L. Direct extraction with solvents produced extracts with variable yields and contents of lignans. Lignans were identified by LC-ESI-MS analysis as phyllanthin (used as marker substance), h ...[more]

PMID: 32151037

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Project description:BackgroundIndigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents.MethodsCytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach.ResultsWe showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine.ConclusionsISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Project description:In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep AHK-AHP-ARR phosphorelay signaling circuitry, which is initiated by the cytokinin-receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 arr12 double null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. Arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant exhibits a characteristic phenotype with regard to the cytokinin-mediated His-Asp phosphorelay. Here we, therefore, compared response to cytokinin in wild type with that in arr10 arr12 double mutant. In this study, wild type and the arr10 arr12 double mutant grown vertically on MS agar plates for 2 weeks were treated with 20uM t-zeatin or 0.02% DMSO (solvent for t-zetion solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from aerial parts of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The Quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These dataset will provide us with bases for understanding the early response to cytokinin on aerial parts of seedlings in Arabidopsis thaliana. Experimenter name: Hitoshi SAKAKIBARA Experimenter phone: 81455039576 Experimenter fax: 81455039609 Experimenter address: Biodynamics Research Team Experimenter address: RIKEN Plant Science Center Experimenter address: 1-7-22 Suehiro, Tsurumi Experimenter address: Yokohama Experimenter zip/postal_code: 230-0045 Experimenter country: JAPAN Keywords: compound_treatment_design;

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Preparation of Lignan-Rich Extract from the Aerial Parts of Phyllanthus niruri Using Nonconventional Methods.

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Preparation of Lignan-Rich Extract from the Aerial Parts of <i>Phyllanthus niruri</i> Using Nonconventional Methods.

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