Method Article

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

DOI:

10.3791/58166

⸱

October 2nd, 2018

 ,  ,  ,  ,  , 
1Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, 2CONACYT, Facultad de Ingeniería Química, Campus de Ciencias Exactas e Ingeniería, Universidad Autónoma de Yucatán

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Caffeine is a secondary metabolite that is biosynthesized by plants of the genus Coffea1. This alkaloid belongs to the methylxanthine family and is regarded as a chemical plant defense because it can act against the adverse effects of pathogens and herbivores2,3. In addition, this metabolite is responsible for the stimulating properties of the coffee drink, which is commonly consumed worldwide4,5. Due to its properties, several research groups are interested in studying the biosynthetic pathway and catabolism of caffein....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Caffeine Extraction in Cell Suspensions of C. arabica L.

  1. Use C. arabica cell suspensions9. Maintain the suspensions by biweekly subcultures in Murashige and Skoog medium at pH 4.3 with constant 100 rpm shaking at 25 °C under continuous light (8.3 W/m2).
  2. Harvest the cells under vacuum filtration using 11 µm pore filter paper and a Buchner funnel.
  3. Register the fresh weight of the collected cells using a scale, wrap them in aluminum foil, freeze them in liquid nitrogen and keep them at -80 °C until analysis.
  4. Lyophilize the frozen cellular material for 72 h.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Caffeine extracts obtained via the process presented here were analyzed by TLC-densitometry by subjecting the samples to plate chromatography according to the scheme shown in Figure 1. To quantify the levels of caffeine in the cell extracts, a curve with various concentrations of commercial standard for this compound was used (Figure 2A). The pattern of absorbance for caffeine was analyzed in the visible light spectrum (UV-VIS) u.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We present here the optimal conditions for evaluating the caffeine content, CS activity and transcript levels in an in vitro plant tissue culture, such as cell suspensions of C. arabica. Previous reports have confirmed that maintaining cells under light irradiation and in the presence of theobromine in the culture medium are suitable parameters for increasing the level of caffeine, making it possible to evaluate the caffeine separation methods using reversed-phase high-performance liquid chromatography .......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The work of our laboratory was funded by a grant from the Consejo Nacional de Ciencia y Tecnología (CONACyT 219893) to SMTHS. This research was also supported by a fellowship granted to RJPK (No. 37938) by CONACyT and the Sistema Nacional de Investigadores (4422). The authors thank CIATEJ for the use of its installations during the writing of this manuscript. Special thanks are extended to Dr. Víctor Manuel González Mendoza for all recommendations in the molecular biology section and Valentín Mendoza Rodríguez, IFC, UNAM for the facilities during the filming of this article.

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Murashige & Skoog Basal salt mixturePhytoTechnology LaboratoriesM524Packge Size: 50 L
Reagent (mg/L)
Ammonium Nitrate (1650)
Boric acid (6.2)
Calcium chloride, anhydrous (322.2)
Cobalt Chloride•H2O (0.025)
Cupric Sulfate•5H2O (0.025)
Na2EDTA•2H2O (37.26)
Ferrous Sulfate•7H2O (27.8)
Magnesium Sulfate, Anhydrous (180.7)
Manganese Sulfate•H2O (16.9)
Molybdic Acid (Sodium Salt)• 2H2O (0.25)
Potassium Iodide (0.83)
Potassium Nitrate (1900)
Potassium Phosphate, Monobasic (170)
Zinc Sulfate•7H2O (8.6)
Supplemented with
myo-inositol (100)
thiamine (10)
cysteine (25)
sucrose (30000)
2,4-dichlorophenoxyacetic acid (3)
6-benzylamine purine (1)
CaffeineSIGMAC0750-5GSTANDARD-5g
TheobromineSIGMAT450020 g
CAMAG TLC Scanner-4CAMAG27.62
WinCATS Planar Chromatography Manager softwareCAMAG1.4.10Software
Isoamyl alcohol (24:1)SIGMAC-0549500 mL
CyclohexaneJALMEXC4375-131 L
AcetoneJ.T. BAKER9006434 L
MethanolJ.T. BAKER9093-034 L
ChloroformJALMEXC-4425-153.5 L
TLC silica gel 60 F254Merck1.05554.0001TLC plate
β-mercaptoethanolM6250SIGMA100 mL
(+)-sodium L- ascorbateA4034SIGMA100 g
Trizma baseSIGMAT60661 Kg
Hydrochloric acid 36.5-38%J.T. Baker9535-052.5 L
Pierce BCA Protein Assay KitThermo scientific232227Kit
Methyl [3H]-S-adenosyl methioninePerkin ElmerNET155Specific activity of 15 Ci/mmol
Liquid scintillation vialsSIGMAZ253081
Thermostatic bath/circulatorCole Parmer60714
Micro centrifugue tubeEppendorfTube of 1.5 mL
Cryogenic vialsHeathrow ScientificHS23202A2 mL
Centrifuge 5804Eppendorf5804 000925
VortexThermolyneLR 5947
Porcelain mortarFisherbrandFB961B
Filter paperWhatmanZ274844Porosity medium
PicofugeStratagene4005502000 x g
Analytical balanceANDHR-120Model HR-120
Scintillation counterBeckman Coulter6500
Gel photodocumentation systemBio-RadChemic XRSModel Chemic XRS
Compact UV lampUVP95002112UVGL-25
Scienceware HDPE Buchner funnelSIGMA2419907Type 37600 mixer
TRIzol reagentThermo scientific15596-018200 mL
ReverdAid Reverse transcriptaseThermo scientific#EP044110000 U
Oligo (dT)18 primerThermo scientific#S0131100 µM
DNase I, RNase-freeThermo scientific#EN05251000 U
Magnesium chlorideThermo scientificEN05251.25 mL
Ethylenediaminetetraacetic acidThermo scientificEN05251 mL
dNTP mixThermo scientificR0191R0191
SYBR Green qPCR Master Mix (2X)Thermo scientificK0251For 200 reactions of 25 µL
PikoRealThermo scientific2.2Software
Phenol, pH 8.0, equilibrated, Molecular Biology Grade, UltrapureUSBJ75829100 mL
Isopropyl alcoholKaral20401 L
Ethyl alcoholSIGMA641751 L
Diethyl pyrocarbonateSIGMAD5758100 mL
Lab RotatorLW ScientificMod. LW210

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Ferruzzi, M. G. The influence of beverage composition on delivery of phenolic compounds from coffee and tea. Physiolgy & Behavior. 100 (1), 33-41 (2010).
  2. Majhenič, L., Škerget, M., Knez, Ž Antioxidant and antimicrobial activity of guarana seed extracts. Food Chemistry. 104 (3), 1258-1268 (2007).
  3. Sled....

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Caffeine ExtractionEnzymatic ActivityGene ExpressionCaffeine SynthasePlant Cell SuspensionsTLC DensitometryRNA IsolationReal Time PCRScintillation CounterProtein Assay

Related Articles