Aktivitas NADP(H) Oksidoreduktase pada Kultur Sel Kina (Cinchona ledgeriana Moens) Terelisitasi

Cinchona ledgeriana Moens is an industrial plant producing secondary metabolite quinoline alkaloids. To maintain and moreover, to increase the quinoline production especially quinine, in vitro culture system through cell culture could be a potential alternative. If the use of elicitor in cell culture can increase the production of a secondary metabolite, the activity of the enzymes involved in the biosynthetic pathway of the secondary metabolite in question might be increasing. This study aimed to examine the activity of NADPH oxidoreductase in the elicitated cell culture of C. ledgeriana and to evaluate the correlation between the activity of this enzyme and the level of quinine production. The cell cultures of Cinchona were treated with abscisic acid (ABA) or paclobutrazol (PBZ), combined with sucrose, sorbitol, or mannitol in Wood Plant (WP) media, for 7 weeks on a shaker. The quinine concentration was determined using high-performance liquid chromatography (HPLC) and the enzyme activity was measured using fluorometry. The results showed that the highest enzyme activity was found in the P7M cells (PBZ 7 mg/L + mannitol 5.3 g/L + sucrose 20 g/L), followed by the A3S cells (ABA 3 mg/L + sorbitol 5.3 g/L + sucrose 20 g/L). These results correspond to their production level of the quinine alkaloids. The lowest enzyme activity was found in the cultures without elicitor. The increase of NADP(H) enzyme activity in the P7M and A3S treatments were 13.5 and 8.5%, respectively, compared to that in the control cells. Keywords: elicitation, fluorometry, NADP(H) oxidoreductase, quinoline alkaloid

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Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2C11 Enzymes in Rat Liver Microsomes: In Vitro Inhibitory Effect of Salicylic Acid on CYP2C11 Enzyme

Molecules ◽

10.3390/molecules24234294 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4294 ◽

Cited By ~ 1

Author(s):

Salhab ◽

Naughton ◽

Barker

Keyword(s):

High Performance Liquid Chromatography ◽

Enzyme Activity ◽

Salicylic Acid ◽

Liquid Chromatography ◽

Rat Liver ◽

High Performance ◽

Inhibitory Effect ◽

Metabolic Reaction ◽

Rat Liver Microsomes

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%–120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug–drug interactions.

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Influence of Iron on Cylindrocarpon Root Rot Development on Ginseng

Phytopathology ◽

10.1094/phyto-96-1179 ◽

2006 ◽

Vol 96 (11) ◽

pp. 1179-1187 ◽

Cited By ~ 14

Author(s):

Mahfuzur Rahman ◽

Zamir K. Punja

Keyword(s):

Enzyme Activity ◽

Phenolic Compounds ◽

Root Rot ◽

High Performance ◽

Germ Tube ◽

Dry Weight ◽

Colony Growth ◽

Tube Length ◽

Germ Tube Length

Cylindrocarpon root rot, caused by Cylindrocarpon destructans, is an important disease on ginseng (Panax quinquefolius) in Canada. We studied the effects of iron (Fe) on disease severity and pathogen growth. When Hoagland's solution was amended with Fe at 56 and 112 μg/ml compared with 0 μg/ml, disease initiation and final severity on hydroponically maintained ginseng roots was significantly (P<0.0001) enhanced. Under field conditions, wounding of roots with a fine needle followed by application of 0.05% FeNaEDTA to the rhizosphere of treated plants significantly enhanced Cylindrocarpon root rot in 2003 and 2004 compared with unwounded roots with Fe or wounded roots without Fe. Foliar applications of Fe (as FeNaEDTA) to ginseng plants three times during the 2002 and 2003 growing seasons significantly increased Fe levels in root tissues. These roots developed larger lesions following inoculation with C. destructans in vitro. When radioactive Fe (59Fe) was applied to the foliage of ginseng plants, it was detected in the secondary phloem and in cortical and epidermal tissues within 1 week. Artificially wounded areas on the roots accumulated more 59Fe than healthy areas. Diseased tissue also had threefold higher levels of phenolic compounds and Fe compared with adjoining healthy tissues. High-performance liquid chromatography analysis revealed enhanced levels of protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, cinnamic acid, phloridizin, and quercetin. Phenolic compounds produced in diseased and wounded tissues sequestered Fe in vitro. The effects of Fe on mycelial growth, conidial germ tube length, and secondary branching of germ tubes of C. destructans were examined in vitro. When grown on Chrome-azurol S medium, Fe also was sequestered by C. destructans through siderophore production, which was visualized as a clearing pigmented zone at the margin of colonies. Mycelial dry weight was significantly increased in glucose/ yeast broth containing Fe at 56 or 112 μg/ml. Conidial germ tube length and secondary branching of hyphae also were enhanced after 8 and 16 h by Fe. Colony growth of C. destructans was not enhanced by Fe, but significantly greater spore production was observed with Fe at 56 and 112 μg/ml compared with no Fe in the medium. Although these levels of Fe had no effect on fungal pectinase enzyme activity, polyphenoloxidase (PPO) activity was significantly (P <0.0001) enhanced. We conclude that Fe enhances Cylindrocarpon root rot through enhanced pathogen growth, sporulation, and PPO enzyme activity. Fe sequestered by phenolic compounds produced in wounded tissues can enhance Fe levels at the site of infection. The pathogen also has the ability to sequester Fe at these sites.

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Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-012-9529-5 ◽

2012 ◽

Vol 48 (8) ◽

pp. 463-468 ◽

Cited By ~ 4

Author(s):

Aastha Chhabra ◽

Astha Jaiswal ◽

Umang Malhotra ◽

Shrey Kohli ◽

Vibha Rani

Keyword(s):

Cell Culture ◽

Enzyme Activity ◽

In Situ Zymography

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Cannabis Sativa L. Flower and Bud Extracts inhibited In vitro Cholinesterases and b-Secretase Enzymes Activities: Possible Mechanisms of Cannabis use in Alzheimer Disease

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530321666210222124349 ◽

2021 ◽

Vol 21 ◽

Author(s):

Teboho Mooko ◽

Asis Bala ◽

Satyajit Tripathy ◽

Chethan S. Kumar ◽

Chandrashekara P. Mahadevappa ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Enzyme Activity ◽

High Performance ◽

Cannabis Sativa ◽

Vero Cells ◽

Scientific Data ◽

Secretase Activity ◽

Inhibitory Potential

Background: There are anecdotal claims on the use of Cannabis sativa L. in the treatment of Alzheimer’s disease, but there is lack of scientific data to support the efficacy and safety of Cannabis sativa L. for Alzheimer’s disease. Aim: The aim of the study was to evaluate the effect of aerial parts of Cannabis sativa L. on the cholinesterases and β-secretase enzyme activity as one of the possible mechanisms of Alzheimer’s disease. Methods: The phytochemical and heavy metal contents were analysed. The extracts were screened for acetylcholinesterase, butyrylcholinesterase and β-secretase activity. Cytotoxicity of extracts was performed in normal vero and pre-adipocytes cell lines. The extracts were characterized using high performance thin layer chromatography and high-performance liquid chromatography for their chemical fingerprints. Alkaloids, flavonoids and glycosides were present amongst the tested phytochemicals. Cannabidiol concentrations were comparatively high in the hexane and dichloromethane than in dichloromethane: methanol (1:1) and methanol extracts. Results: Hexane and dichloromethane extracts showed a better inhibitory potential towards cholinesterase activity, while water, hexane, dichloromethane: methanol (1:1) and methanol showed an inhibitory potential towards β-secretase enzyme activity. All extracts showed no cytotoxic effect on pre-adipocytes and vero cells after 24- and 48-hours of exposure. Conclusion: Therefore, this may explain the mechanism through which AD symptoms may be treated and managed by Cannabis sativa L. extracts.

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1120: Citrate and Vitamin E Blunt the SWL-Induced Free Radical Surge in an In-Vitro MDCK Cell Culture Model

The Journal of Urology ◽

10.1016/s0022-5347(18)38357-5 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 295-295

Author(s):

Fernando C. Delvecchio ◽

Ricardo M. Brizuela ◽

Karen J. Byer ◽

W. Patrick Springhart ◽

Saeed R. Khan ◽

...

Keyword(s):

Cell Culture ◽

Free Radical ◽

Vitamin E ◽

Mdck Cell ◽

Cell Culture Model ◽

Culture Model

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3D in vitro reconstruction of synthetic liver sinusoids in a microfluidic cell culture device

Zeitschrift für Gastroenterologie ◽

10.1055/s-0032-1331999 ◽

2013 ◽

Vol 51 (01) ◽

Author(s):

J Böttger ◽

J Schütte ◽

K Benz ◽

C Freudigmann ◽

B Hagmeyer ◽

...

Keyword(s):

Cell Culture ◽

Liver Sinusoids ◽

Microfluidic Cell Culture

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IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

Acta Endocrinologica ◽

10.1530/acta.0.068s027 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S27-S40 ◽

Cited By ~ 1

Author(s):

T. Kobayashi ◽

T. Kigawa ◽

M. Mizuno ◽

T. Watanabe

Keyword(s):

Cell Culture ◽

Organ Culture ◽

Cultured Cells ◽

Cell Sheet ◽

Short Term ◽

Hypothalamic Extract ◽

Numerous Cell ◽

Single Cell Culture ◽

Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

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Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

Acta Endocrinologica ◽

10.1530/acta.0.1140018 ◽

1987 ◽

Vol 114 (1) ◽

pp. 18-26 ◽

Cited By ~ 5

Author(s):

Chohei Shigeno ◽

Itsuo Yamamoto ◽

Shegiharu Dokoh ◽

Megumu Hino ◽

Jun Aoki ◽

...

Keyword(s):

Bone Resorption ◽

High Performance ◽

Basic Protein ◽

Gel Filtration ◽

Exchange Chromatography ◽

Protein Factor ◽

Human Tumours ◽

Acid Stable ◽

Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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