Elsevier

Phytochemistry

Volume 68, Issue 14, July 2007, Pages 1992-2003
Phytochemistry

Production and secretion of resveratrol in hairy root cultures of peanut

https://doi.org/10.1016/j.phytochem.2007.04.039 Get rights and content

Abstract

Resveratrol and its derivatives are natural stilbenes associated with many health benefits that include those conferred by their antioxidant and anticancer properties. While stilbenes can be recovered as an extract from a selected number of plants, these products are not suitable for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its derivatives in the extract. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut (Arachis hypogaea) were established and tested as a bioproduction system for resveratrol and associated derivatives. Analyses by HPTLC and GC–MS of ethyl acetate extracts showed that a single 24 h sodium acetate elicitation resulted in a 60-fold induction and secretion of trans-resveratrol into the medium of peanut hairy root cultures. trans-Resveratrol accumulated to levels of 98 μg/mg of the dried extract from the medium representing 99% of the total resveratrol produced. Other stilbenes, including trans-pterostilbene, were also detected in the medium. Our results demonstrate the capacity of hairy root cultures as an effective bioprocessing system for valued nutraceuticals like resveratrol and resveratrol derivatives. In being able to effectively induce and recover high levels of resveratrol and associated derivatives from the media fraction, hairy roots may offer a scalable and continuous product recovery platform for naturally-derived, high quality, enriched nutraceuticals.

Graphical abstract

Hairy root cultures of peanut were established and elicited to produce trans-resveratrol. Sodium acetate increased the levels of trans-resveratrol in the medium by almost 60-fold in comparison to non-elicited cultures.

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Introduction

trans-Resveratrol 1 (trans-3,5,4′-trihydroxystilbene, Fig. 1), and its many derivatives (Larronde et al., 2005, Rimando and Barney, 2005), are naturally occurring phytoalexins produced in a select number of plant species. These plant polyphenols have received considerable interest based upon a number of associated health benefits (Baur and Sinclair, 2006, Delmas et al., 2006). Most notably, the significant levels of resveratrol 1 in red wine have been credited to the phenomenon known as “the French Paradox”, wherein low incidence of heart disease is observed among a population with a relatively high saturated fat diet and moderate wine consumption (Frankel et al., 1993, Siemann and Creasy, 1992). Over the past two decades, numerous health benefits impacting cardiovascular disease, various cancers, atherosclerosis and aging have been linked with resveratrol 1 (reviewed; Baur and Sinclair, 2006, Roupe et al., 2006). While resveratrol 1 is one of the better known and well studied phytochemicals, several other resveratrol derivatives have also been shown to have similar and/or additional health benefits. Included among these derivatives is the methylated resveratrol compound, pterostilbene 2 (Fig. 1), that has demonstrated in vivo effects for reducing cholesterol levels (Rimando et al., 2005).

A number of taxonomically unrelated plant families have been reported to produce marked levels of resveratrol 1 including: grapes, peanuts, several types of berries, some pine trees and most recently tomato fruit skin (Ragab et al., 2006). Resveratrol 1 is a phytoalexin, a class of antibiotic compounds produced as part of the plant’s defense system and believed to be the major active stilbene that confers pathogen resistance in these plants (Dixon, 2001). The terminal enzyme in the production of resveratrol 1 is resveratrol synthase, which condenses p-coumaroyl-coenzyme A and three malonyl-coenzyme A molecules to form resveratrol 1 (Schöppner and Kindl, 1984). This enzyme is highly regulated by elicitors and general plant defense compounds in an effort to protect the plant. Resveratrol 1 exists as both the trans- and cis-isomers with numerous reports suggesting trans-resveratrol to be the most bioactive form of this molecule (Roupe et al., 2006). trans-Resveratrol 1 can readily be converted to cis-resveratrol when exposed to UV light and is unstable when exposed to high pH conditions.

The use of complementary or alternative beneficial products for human health is increasing worldwide with their continued popularity in Europe and Asia and a dramatic upward trend of their use in the United States (Frost and Sullivan, 2005). Currently resveratrol 1 is primarily marketed as an herbal or dietary supplement in the form of pills, capsules, powders, and extracts from raw botanical sources (i.e. grape seeds/skins; Japanese knotweed Polygonum cuspidatum), with more recent applications beginning to incorporate this popular phytochemical into fortified food/beverage products (i.e. Old Orchard Beverage Company, Sparta, MI). While stilbenes are cost-effectively recovered from these raw materials and will continue to serve this market, these relatively crude sources of resveratrol 1 often lack the consistency and purity required for many applications in the food/pharmaceutical sectors. Furthermore, more natural product consumers and nutrition practitioners are demanding higher quality supplements that are scientifically tested and better defined in their product content (validated by third party quality assurance testing) in a desire to mitigate ineffective and/or erratic responses with these supplements.

The majority of resveratrol-containing dietary supplements are composed of unknown/unidentified botanical components wherein resveratrol 1 and resveratrol derivatives only make up a small fraction of the product. While chemically-synthesized resveratrol 1 may address this issue, natural sources often contain derivatives, co-factors and other phytonutrients that provide added or synergistic benefits to the nutraceutical product and are often preferred by the consumer (Wallace, 1998). Recent studies showing anti-aging benefits of resveratrol 1 (Baur et al., 2006) further accelerate interest in a natural, food-grade, source of enriched resveratrol/resveratrol derivatives that delivers a more defined and consistent product composition and ensures a stable supply chain, several biotic production strategies targeting recombinant plants, yeast and bacteria have been advanced (Becker et al., 2003, Paiva and Hipskind, 2005, Watts et al., 2006). While these approaches potentially offer a more consistent, concentrated resveratrol 1 source, widespread use of these strategies have not been adopted due in part to natural product consumers’ negative perception of genetically modified organisms and issues with associated production efficiency/costs. Grape cell suspension cultures for resveratrol 1 production avoid some of these recombinant issues and provide potential production of a suite of resveratrol compounds (Bru et al., 2006, Liu et al., 2003), however, cell suspension production systems have reported issues of genetic instability and losses of secondary metabolite production following elicitation (Gossens et al., 2003) or repetitive subculturing (Chattopadhyay et al., 2002).

To this end, plant hairy roots offer a novel and sustainable tissue-based system that preserves the multiple specialized cell types believed important in maintaining better consistency in the synthesis of bioactive secondary molecules. Tissue-based systems more accurately reflect the metabolic phenotype and performance of the host plant in comparison to plant cell cultures and further the potential of producing various combinations of valued products from a single production line (Guillon et al., 2006a, Guillon et al., 2006b, Sevon and Oksman-Caldentey, 2002). Recent advances with large scale production have successfully produced ginseng roots in a 10,000 l bioreactor establishing the feasibility of the root system to accommodate industrial processes (Sivakumar et al., 2006).

Here we report the development of peanut hairy root lines for sustained and reproducible production of a naturally-derived source of resveratrol and resveratrol derivatives. These phytochemicals, can be readily recovered from the culture medium in relatively enriched form, detected and quantitated. The use of plant elicitors to enhance secretion of resveratrol 1 and other resveratrol derivatives support our efforts in exploring the commercial potential of this scalable bioprocessing system for valued botanical compounds with nutraceutical properties such as resveratrol and its associated derivatives.

Section snippets

Establishment of peanut hairy root lines

With numerous studies demonstrating that peanut is among a divergent group of plants with endogenously high levels of resveratrol 1 (Chen et al., 2002, Liu et al., 2003, Sanders et al., 2000, Sobolev and Cole, 1999), we targeted a runner peanut cultivar for establishing hairy root lines as a sustainable bioproduction platform in the delivery of a well-defined and enriched resveratrol 1 product. In initial experiments with 21-day old seedlings of cv. Andru II, a minimum of one explant of each

Concluding remarks

The hairy root culture platform is a unique bioproduction system for generating well-defined, highly-enriched fractions of resveratrol 1 and other beneficial stilbene compounds. In capturing the spatio-temporal organization of the source plant, this tissue-based culture system may better preserve the natural metabolic processes as they occur in nature. Maintenance of tissue integrity likely supports the distinctive genetic and biosynthetic stability of hairy roots and enables their fast growth

Establishment of hairy root cultures

Seeds of peanuts (Arachis hypogaea) cv. Andru II (kindly provided by Dr. Daniel Gorbet, University of Florida) were surface sterilized as follows. Seeds were presoaked for 2 min in sterile water containing 0.003% Ivory™ detergent; immersed for 15 min in sterilization solution (50% Clorox™, 0.003% Ivory™ detergent) and rinsed in sterile water. To minimize Chlorox™ damage to the embryo, the testa was aseptically removed and seeds were further rinsed in two changes of sterile water over a 15 min

Acknowledgements

We acknowledge Dr. Daniel Gorbet (University of Florida) for providing seeds of peanut cv. Andru II for these studies. Funding for this research was provided by the Arkansas Biosciences Institute at Arkansas State University. Additional funding through Nature West Inc. was provided by the Arkansas Science and Technology Authority (ASTA).

References (50)

  • J.L. Slightom et al.

    Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames

    J. Biol. Chem.

    (1986)
  • M. Tolomeo et al.

    Pterostilbene and 3′-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells

    Int. J. Biochem. Cell Biol.

    (2005)
  • Q. Yan et al.

    Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

    J. Biotechnol.

    (2005)
  • A. Aziz et al.

    Laminarin elicits defense reponses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

    Mol. Plant Microbe Interact.

    (2003)
  • J.A. Baur et al.

    Resveratrol improves health and survival of mice on a high-calorie diet

    Nature

    (2006)
  • J. Baur et al.

    Therapeutic potential of resveratrol: the in vivo evidence

    Nat. Rev. Drug Disc.

    (2006)
  • R. Bru et al.

    Modified cyclodextrins are chemically defined glucan inducers of defense responses in grapevine cell cultures

    J. Agric. Food Chem.

    (2006)
  • C. Camilleri et al.

    The TR-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine-type plasmid pRiA4: nucleotide sequence analysis and introduction into tobacco plants

    Mol. Plant Microbe Interact.

    (1991)
  • S. Chattopadhyay et al.

    Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

    Biotechnol. Bioprocess Eng.

    (2002)
  • R.S. Chen et al.

    Peanut roots as a source of resveratrol

    J. Agric. Food Chem.

    (2002)
  • D. Delmas et al.

    Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer

    Curr. Drug Targets

    (2006)
  • R. Dixon

    Natural products and plant disease resistance

    Nature

    (2001)
  • Frost, Sullivan, 2005. Strategic analysis of the US dietary supplement markets. Dialog® File #767; Accession...
  • A. Gossens et al.

    A functional genomics approach toward the understanding of secondary metabolism in plant cells

    PNAS

    (2003)
  • J.H. Haas et al.

    Universal primers for detection of pathogenic Agrobacterium strains

    Appl. Environ. Microbiol.

    (1995)
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