Salycilic Acid Induces Exudation of Crocin and Phenolics in Saffron Suspension-Cultured Cells

The production of crocin, an uncommon and valuable apocarotenoid with strong biological activity, was obtained in a cell suspension culture of saffron (Crocus sativus L.) established from style-derived calli to obtain an in-vitro system for metabolite production. Salycilic acid (SA) was used at different concentrations to elicit metabolite production, and its effect was analyzed after a 4 days of treatment. HPLC-DAD analysis was used for total crocin quantification while the Folin-Ciocâlteu method was applied for phenolic compounds (PC) content. Interestingly, despite cell growth inhibition, a considerable exudation was observed when the highest SA concentration was applied, leading to a 7-fold enhanced production of crocin and a 4-fold increase of phenolics compared to mock cells. The maximum antioxidant activity of cell extracts was evidenced after SA 0.1 mM elicitation. Water-soluble extracts of saffron cells at concentrations of 1, 0.5, and 0.1 µg mL−1 showed significant inhibitory effects on MDA-MB-231 cancer cell viability. The heterologous vacuolar markers RFP-SYP51, GFPgl133Chi, and AleuRFP, were transiently expressed in protoplasts derived from the saffron cell suspensions, revealing that SA application caused a rapid stress effect, leading to cell death. Cell suspension elicitation with SA on the 7th day of the cell growth cycle and 24 h harvest time was optimized to exploit these cells for the highest increase of metabolite production in saffron cells.

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Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts

Molecules ◽

10.3390/molecules26040791 ◽

2021 ◽

Vol 26 (4) ◽

pp. 791

Author(s):

Muzamil Shah ◽

Hasnain Jan ◽

Samantha Drouet ◽

Duangjai Tungmunnithum ◽

Jafir Hussain Shirazi ◽

...

Keyword(s):

Cell Suspension ◽

Biomass Production ◽

Suspension Cultures ◽

Total Phenolic ◽

Naphthalene Acetic Acid ◽

Silybum Marianum ◽

Enhanced Production ◽

Cell Extracts ◽

Anti Inflammatory

Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S. marianum were exploited for their in vitro potential to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension cultures were maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 ± 1.7 g/L fresh weight (FW) and 17.7 ± 0.5 g/L dry weight (DW) productions. All chitosan treatments resulted in an overall increase in the accumulation of total flavonoids (5.0 ± 0.1 mg/g DW for 5.0 mg/L chitosan), total phenolic compounds (11.0 ± 0.2 mg/g DW for 0.5 mg/L chitosan) and silymarin (9.9 ± 0.5 mg/g DW for 0.5 mg/L chitosan). In particular, higher accumulation levels of silybin B (6.3 ± 0.2 mg/g DW), silybin A (1.2 ± 0.1 mg/g DW) and silydianin (1.0 ± 0.0 mg/g DW) were recorded for 0.5 mg/L chitosan. The corresponding extracts displayed enhanced antioxidant and anti-inflammatory capacities: in particular, high ABTS antioxidant activity (741.5 ± 4.4 μM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan, whereas highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 ± 1.3 %), secretory phospholipase A2 (sPLA2, 33.9 ± 1.3 %) and 15-lipoxygenase (15-LOX-2, 31.6 ± 1.2 %) enzymes involved in inflammation process were measured in extracts obtained in the presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation in the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.

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Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-inflammatory Activities of Cell Extracts

10.20944/preprints202101.0204.v1 ◽

2021 ◽

Author(s):

Muzamil Shah ◽

Hasnain Jan ◽

Samantha Drouet ◽

Duangjai Tungmunnithum ◽

Jafir Hussain Shirazi ◽

...

Keyword(s):

Cell Suspension ◽

Biomass Production ◽

Naphthalene Acetic Acid ◽

Silybum Marianum ◽

Biotechnological Production ◽

High Accumulation ◽

Enhanced Production ◽

Cell Extracts ◽

Anti Inflammatory

Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S marianum were exploited for their in vitro potency to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension culture was maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 g/L fresh weight (FW) and 17.7 g/L dry weight (DW) productions. Chitosan treatment resulted in an overall increase in the accumulation of flavonoids, phenolic compounds and silymarin. High accumulation levels of silybin B, silydianin and silybin A were recorded by HPLC analysis. The corresponding extracts displayed interesting antioxidant and anti-inflammatory capacities. In particular, high ABTS antioxidant activity (741.5 μM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan. On the opposite, highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 %), secretory phospholipase A2 (sPLA2, 33.9 %) and 15-lipoxygenase (15-LOX-2, 31.6 %) enzymes involved in inflammation process were measured in extracts obtained in presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation of the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.

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Effect of microenvironment and cell-line type on carbohydrate-binding proteins of macrophage-like cells

Biochemistry and Cell Biology ◽

10.1139/o88-134 ◽

1988 ◽

Vol 66 (11) ◽

pp. 1169-1176 ◽

Cited By ~ 7

Author(s):

Hans-Joachim Gabius ◽

Katalin Vehmeyer

Keyword(s):

Binding Proteins ◽

Cultured Cells ◽

Sodium Dodecyl ◽

Carbohydrate Binding ◽

Intercellular Interaction ◽

Cell Extracts ◽

Carbohydrate Binding Proteins ◽

Sugar Receptors

The pattern of sugar inhibition of rosette formation, a model for intercellular interaction between cultured cells and glutaraldehyde-fixed, trypsinated rabbit erythrocytes, served to infer the presence of carbohydrate-binding proteins. This profile from cell extracts for the two murine macrophage-like cell lines, P388D1 and J774A.1, was comparatively analyzed by affinity chromatography on supports with immobilized carbohydrates (lactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and maltose) or with the immobilized mannose-rich yeast glycoprotein mannan or fetuin-derived glycopeptides containing sialic acid residues. After elution with specific sugar in the absence of Ca2+ ions, the proteins were separated by sodium dodecyl sulfate – polyacrylamide slab gel electrophoresis. The composition of carbohydrate-binding proteins of the two lines clearly exhibited quantitative and qualitative differences. Moreover, the pattern of P388D1 cells was also demonstrated to change significantly in response to alterations in the conditions of the physiological environment. These alterations were imposed by in vitro growth, by subsequent in vivo growth in nude mice, and by re-adaptation of cells to culture after in vivo passage. Collectively, our observations and other physiological and biochemical reports on macrophage lectins indicate that the presence of sugar receptors with different specificities may be an indicator of macrophage differentiation, being reversibly modulated to a considerable extent by external factors, e.g., microenvironment. Extensive but selective alterations in this respect could play an important role in the control of recognition and effector mechanisms within diverse functions of macrophage subpopulations.

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Plasminogen Activator (PA) Activity In Metastatic Cells: An Amidolytic Approach

10.1055/s-0038-1652138 ◽

1981 ◽

Author(s):

D Coen ◽

A Bini ◽

G Balconi ◽

F Delaini ◽

L Mussoni ◽

...

Keyword(s):

Primary Tumor ◽

Fibrinolytic Activity ◽

Tumor Tissue ◽

Cultured Cells ◽

Cell Extracts ◽

Mechanical Disruption ◽

Metastatic Cells ◽

Triton X ◽

Selection Of

It has been proposed that fibrinolytic activity can play an important role in the process of metastasis formation. Nevertheless, it is not yet clear in which phase of the tumor growth and dissemination this activity is involved. We measured the fibrinolytic activity of cells from primary tumor and metastatic nodules of 3LL, an i.m. implanted murine tumor which selectively metastasizes to the lungs. Tumor cells have been studied both immediately after mechanical disruption of tumor tissue and after in vitro culturing to confluence. Their P.A. activity was tested by an amidolytic assay in which cells were incubated with purified plasminogen (3CU/ml) and 4mM S-2251 (Kabi Diagnostica, Stockholm, Sweden), a plasmin specific chromogenic substrate. After 3 hour incubation at 37°C, the reaction was stopped with acetic acid and absorbance read at 405 nm.Cells from the primary tumor and metastatic nodules showed a similar fibrinolytic activity, which was in both cases in- increased 3 to 4 fold in cell extracts obtained after preincubation with TRITON X-100. A dose-response curve plotted with increasing urokinase concentrations showed a parallel course. This data suggests that, in the 3LL model, PA activity is not one of the properties characterizing the selection of metastatic cells.On the other hand,cultured cells presented consistently higher levels of PA than their native counterparts, suggesting that adhesion of cells in culture may stimulate PA production or, alternatively, that cultured cells are a selected population in comparison to the overall number of native cells.

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Mechanical forces and their second messengers in stimulating cell growth in vitro

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1992.262.3.r350 ◽

1992 ◽

Vol 262 (3) ◽

pp. R350-R355 ◽

Cited By ~ 23

Author(s):

H. H. Vandenburgh

Keyword(s):

Cell Growth ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Second Messengers ◽

Second Messenger ◽

Model Systems ◽

Mechanical Forces ◽

Unicellular Organisms ◽

Multicellular Organisms

Mechanical forces play an important role in modulating the growth of a number of different tissues including skeletal muscle, smooth muscle, cardiac muscle, bone, endothelium, epithelium, and lung. As interest increases in the molecular mechanisms by which mechanical forces are transduced into growth alterations, model systems are being developed to study these processes in tissue culture. This paper reviews the current methods available for mechanically stimulating tissue cultured cells. It then outlines some of the putative “mechanogenic” second messengers involved in altering cell growth. Not surprisingly, many mechanogenic second messengers are the same as those involved in growth factor-induced cell growth. It is hypothesized that from an evolutionary standpoint, some second messenger systems may have initially evolved for unicellular organisms to respond to physical forces such as gravity and mechanical perturbation in their environment. As multicellular organisms came into existence, they appropriated these mechanogenic second messenger cascades for cellular regulation by growth factors.

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A Novel Fluorescence-Labeled Curcumin Conjugate: Synthesis, Evaluation and Imaging on Human Cell Lines

Current Pharmaceutical Design ◽

10.2174/1381612824666180406103317 ◽

2018 ◽

Vol 24 (16) ◽

pp. 1821-1826 ◽

Cited By ~ 2

Author(s):

Sumbla Sheikh ◽

Alexander Sturzu ◽

Hubert Kalbacher ◽

Thomas Nagele ◽

Christopher Weidenmaier ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Human Cell ◽

Cellular Localization ◽

Membrane Damage ◽

Antibacterial Properties ◽

Water Soluble ◽

Osteosarcoma Cell ◽

Human Cell Lines

Curcumin, as the main ingredient of the curcuma spice, has increasingly become the target of scientific research. The turmeric root where the spice is obtained from has been widely used in the traditional medicine. Moreover, scientific studies have found that curcumin has anti-inflammatory, anti-cancer, anti-angiogenic effects as well as antibacterial properties. Recently, curcumin has gathered interest as a potential therapeutic agent in the research on Alzheimer’s disease. A consistent problem in the investigative and therapeutic applications of curcumin is its poor solubility in aqueous solutions. In the present study, we synthesized a conjugate of curcumin, the amino acid lysine and the fluorescent dye fluorescein. This conjugate was soluble in cell culture medium and facilitated the examination of curcumin with fluorescence imaging methods. We studied the cell growth impact of unmodified curcumin on seven different human cell lines and then analyzed the uptake and cellular localization of our curcumin conjugate with confocal laser scanning imaging and flow cytometry on the seven cell lines. We found that unbound curcumin inhibited cell growth in vitro and was not taken up into the cells. The curcumin conjugate was internalized into the cell cytoplasm in a dot-like pattern and cellular uptake correlated with the cell membrane damage which was measured using propidium iodide. The CAL-72 osteosarcoma cell exhibited 3-4fold increased conjugate uptake and a strong uniform fluorescein staining in addition to the dot-like pattern observed in all cell lines. In conclusion, we successfully synthesized a novel water-soluble fluorescent curcumin conjugate which showed a strong preference for CAL-72 osteosarcoma cells in vitro.

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IN VITRO PROPAGATION AND SECONDARY METABOLITE PRODUCTION IN CROCUS SATIVUS L.

Saffron ◽

10.1201/9780203303665-19 ◽

1999 ◽

pp. 131-142

Keyword(s):

Secondary Metabolite ◽

In Vitro Propagation ◽

Crocus Sativus ◽

Secondary Metabolite Production ◽

Metabolite Production ◽

Crocus Sativus L

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Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-017-1237-0 ◽

2017 ◽

Vol 130 (2) ◽

pp. 417-426 ◽

Cited By ~ 2

Author(s):

Peeyush Kumar ◽

Vartika Srivastava ◽

Rakhi Chaturvedi ◽

Durai Sundar ◽

V. S. Bisaria

Keyword(s):

Cell Suspension ◽

Cell Suspension Cultures ◽

Suspension Cultures ◽

Tinospora Cordifolia ◽

Protoberberine Alkaloids ◽

Enhanced Production

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Production of cartilage link protein by human granulosa-lutein cells

Journal of Endocrinology ◽

10.1677/joe.0.1750505 ◽

2002 ◽

Vol 175 (2) ◽

pp. 505-515 ◽

Cited By ~ 3

Author(s):

GW Sun ◽

H Kobayashi ◽

M Suzuki ◽

N Kanayama ◽

T Terao

Keyword(s):

Time Course ◽

Matrix Protein ◽

Cultured Cells ◽

Extracellular Matrix Protein ◽

Regulatory Function ◽

Human Ovary ◽

Vitro Fertilization ◽

Link Protein ◽

Cell Extracts

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

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