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Introduction
The classical techniques of plant compound extraction
include direct distillation of essential oils, steam distillation of essential oils, and organic solvent extraction of
organic compounds. All of these extraction techniques
usually require some kind of mechanical disruption of
plant tissues, making these techniques inherently destructive.
Replacing and re-growing slow growing plants does not
make these techniques economically desirable when
continuous production is required.
We investigated an innovative alternative method for
nondestructive extraction of products from plant tissues.
Ultrasound was chosen as a means of nonlethal extraction because of its flexibility in the degree of disruptions,
which depends on adjustable acoustic parameters such
as sound intensity and frequency. In addition, recent
studies have indicated that ultrasound-assisted extraction techniques enhance the efficiency of product extraction by shortening the time of the extraction process in
the release of secondary metabolites from various plant
tissues such as excised leaves of tea, mint, sage, chamomile, ginseng, arnica, and gentian (1-5). The review of
ultrasonic isolation of chemicals from plants can be found
in a recent review paper by Vinatoru et al. (6). Although
these ultrasound-assisted techniques are still destructive,
* Corresponding author: Professor of Chemical and Biomedical
Engineering, Rm 3324, Dow Conn. Building, The University of
Michigan, Ann Arbor, Michigan 48109-2136. Tel: 734-763-5659.
Fax: 734-763-0459. E-mail: hywang@engin.umich.edu.
Department of Chemical Engineering.
Department of Biology.
10.1021/bp010074u CCC: $20.00
the results suggested the ability of ultrasound to penetrate more effectively into plant tissues than with
conventional techniques. Furthermore, ultrasound has
been shown to repeatedly release betanin from Beta
vulgaris cells (7). However, no studies, thus far, have
shown the possibility of using this technique to extract
the products from particular tissues attached to intact
whole plants.
In this paper, we show the feasibility of using ultrasound as a technique for nonlethal extraction of menthol
from intact peppermint plants. The peppermint plants
were chosen as our model system because they represent
the mint or Labiatae family, which is of great economic
importance owing to the presence of volatile oils, which
are produced in the glandular trichomes. These glandular
trichomes are located on the epidermal surfaces of
peppermint leaves, and are therefore readily accessible
to the ultrasound treatment. Furthermore, the menthol
and other monoterpene biosynthetic pathways have been
extensively studied (8-10).
In this feasibility study, we used an ultrasonic cleaning
bath, which has a fixed sound intensity and frequency.
We investigated the effects of time of ultrasonic treatment on menthol release and the feasibility of repeated
ultrasonic extractions. Furthermore, scanning electron
micrographs of the ultrastructure of the glandular trichomes provided an insight into structural changes
induced by the ultrasonic treatment.
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Conclusions
This study shows that nonlethal repeated ultrasonic
extraction of menthol from peppermint plants is feasible.
The elevated temperatures were found to have a significant effect on the amount of the product released, as they
increase the solubility of menthol in the medium and high
temperatures cause the menthol crystals to undergo a
phase transition to a liquid state, and thus, be more
easily released from the cells. The amount of the product
released and the degree of damage vary directly with the
duration of ultrasound treatment. The mechanism of
extraction, as shown from scanning electron micrographs,
includes diffusion through unperturbed gland cell walls,
diffusion through the cell walls that are partially collapsed, and exudation from the glandular cells that might
eventually break.
To enhance menthol product release efficiency further,
we must understand the molecular mechanism of the
release. Further study must be performed to determine
the relationships between the product release, the trichome physiological and morphological changes, and the
ultrasonic parameters such as sound intensity and
frequency. Furthermore, it is crucial to determine how
the trichome biosynthetic capacity might have been
altered as a result of ultrasound treatment.
(3) Salisova, M.; Toma, S.; Mason, T. J. Comparison of conventional and ultrasonically assisted extractions of pharmaceutically active compounds from Salvia officinalis. Ultrason.
Sonochem. 1997, 4, 131-134.
(4) Vinatoru, M.; Toma, M., Radu O.; Filip. P. I.; Lazurca, D.;
Mason, T. J. The Use of Ultrasound for the Extraction of
Bioactive Principles form Plant Materials. Ultrason. Sonochem.
1997, 4, 135-139.
(5) Hromadkova, Z.; Ebringerova, A.; Valachovic, P. Comparison
of Classical and Ultrasound-assisted Extraction of Polysaccharides from Salvia officinalis L. Ultrason. Sonochem. 1999,
5, 163-168.
(6) Vinatoru, M.; Toma, M.; Mason, T. J. Ultrasonically Assisted
Extraction of Bioactive Principles from Plants and Their
Constituents, Advances in Sonochemistry, 5th ed.; Mason, T.
J., Ed.; JAI Press: Stamford, CT, 1999; pp 209-248, ISBN
0-7623-09331-X.
(7) Kilby, N. J.; Hunter, C. S. Repeated Harvest of Vacuolelocated Secondary Product from In Vitro Grown Plant Cells
Using 1.02 MHz Ultrasound. Appl. Microbiol. Biotechnol.
1990, 33, 448-451.
(8) Maffei, M.; Chialva, F.; Sacco, T. Glandular Trichomes and
Essential Oils in Developing Peppermint Leaves. I. Variations
of Peltate Trichrome Number and Terpene Distribution
within Leaves. New Phytol. 1989, 111, 707-716.
(9) Spencer, A.; Hamill, J. D.; Rhodes, M. J. C. In vitro
Biosynthesis of Monoterpenes by Agrobaterium Transformed
Shoot Cultures of Two Mentha Species. Phytochemistry 1993,
32, 911-919.
(10) McCaskill, D.; Rodneym C. Monoterpene and Sesquiterpene Biosynthesis in Glandular Trichomes of Peppermint
(Mentha x piperita) Rely Exclusively on Plastid-derived
Isopentenyl Diphosphate. Planta 1995, 197, 49-56.
(11) Hoagland, D. R.; Arnon, D. I. The water culture method of
growing plants without soils. Calif. Agric. Exp. Sta. Circ.
1938, 347, 99. A revised edition was published in 1950.
(12) Burbott, A. J.; Loomis D. W. Effect of light and temperature
on themonoterpenes of peppermint. Plant Physiol. 1967, 42,
20-28.
(13) Kilby, N. J.; Hunter, C. S. Towards Optimization of the
Use of 1.02 MHz Ultrasound to Harvest Vacuole-located
Secondary Product From In Vitro Grown Plant Cells. Appl.
Microbiol. Biotechnol. 1991, 34, 478-480.
(14) Neppiras E. A. Acoustic Cavitation. Phys. Rep. 1980, 61,
159-251.
(15) a. Romdhane, M.; Gourdon, C.; Casamatta, G. Local Investigation of Some Ultrasonic Devices by Means of a Thermal Sensor. Ultrasonics 1995, 33, 221-226. b. Romdhane,
M.; Gourdon, C.; Casamatta, G. Development of a Thermoelectric Sensor for Ultrasonic Intensity Measurement. Ultrasonics 1995, 33, 139-146.
(16) Soudagar S. R.; Samant, S. D. Semiquantitative Characterization of Ultrasonic Cleaner Using a Novel Piezoelectric
Pressure Intensity Measurement Probe. Ultrason. Sonochem.
1995, 2, S49-S52.