924

Biotechnol. Prog. 2001, 17, 924928

Feasibility Study of Repeated Harvesting of Menthol from

Biologically Viable Mentha x piperata Using Ultrasonic Extraction
Artiwan Shotipruk, Peter B. Kaufman, and Henry Y. Wang*,
Department of Chemical Engineering and Department of Biology, The University of Michigan,
Ann Arbor, Michigan 48109

To potentially replace the conventional destructive extraction process, we have shown

the feasibility of devising a novel technique that uses ultrasound to nonlethally and
repeatedly extract menthol from biologically viable peppermint plants (Mentha x
piperita). Our results show that plants ultrasonicated for 1 h at 22 C in a standard
40 kHz ultrasonic bath could release approximately 17.8 g of menthol per gram of
leaf tissue (2% of total product). The amount of menthol release increases with the
time of treatment and is greatly affected by the temperature of the ultrasonic bath
water. An increase from 2% to 12% of total product was observed when the temperature
was increased from 22 C to 39 C. When the temperature effects were isolated, the
mechanism of the product release was found to be that of cavitation. The treated plants
remained viable and were ready for the subsequent ultrasound extraction after
approximately 4 days of recuperation. However, the amount of product released is
reduced in subsequent extractions. Scanning electron micrographs indicate that there
are two mechanisms involved in extraction: (1) the diffusion of product through the
cuticle of peppermint glandular trichomes and (2) the exudation of the product from
broken and damaged trichomes. This study has shown the possibility of using an online ultrasonic, nondestructive extraction method to continuously release intracellular
plant metabolites from the plants while maintaining the plants viability.

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.

Materials and Methods

Plants and Product. Peppermint plants (Mentha x
piperita) used in this study were obtained from the

2001 American Chemical Society and American Institute of Chemical Engineers

Published on Web 09/19/2001

Biotechnol. Prog., 2001, Vol. 17, No. 5

925

Figure 1. Schematic diagram of experimental procedure.

Figure 2. Effect of time of ultrasonic treatment on product release.

University of Michigan Matthaei Botanical Gardens, Ann

Arbor, MI. A number of mint cuttings, each consisting
of the tuft of youngest leaves at the growing tip plus the
next three leaf pairs, were rooted for approximately 14
days before being transferred to a hydroponic bath
containing Hoaglands complete nutrient solution (11).
The hydroponic system allows the plants to be easily

inverted into the ultrasonic bath, and returned for the

period of recuperation before repeated ultrasonic extraction treatments.
Ultrasound Extraction Experiment. The ultrasonic
bath used (FS 60, Fisher Scientific, Pittsburgh, PA) was
basically a rectangular container (15 cm 28 cm 15
cm), to which series of 40 kHz transducers were annealed

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Biotechnol. Prog., 2001, Vol. 17, No. 5

at the bottom. In it, a glass beaker containing 500 mL of

deionized water (where the plants were inverted and
immersed) was placed directly in the center of the bath.
The water surface in the beaker was kept at the level of
that in the ultrasonic bath. The bath contained 3.5 L of
water. Water in the bath was circulated and regulated
at a constant temperature (22 ( 3 C) to avoid the water
temperature rise that is caused by ultrasonic exposure.
The extraction was performed according to the scheme
shown in Figure 1.
Effect of Duration of Ultrasonic Exposure on
Product Release. Identical sampled plants were rinsed
with deionized water to rid plants of rooting medium
residues (Sunshine mix no. 1). To determine the effect
of different durations of ultrasonic exposure on the
menthol release, five or six washed plants were then
inverted and immersed in the 500 L beaker of water in
the ultrasonic bath (described earlier). The plants were
subjected to ultrasonic treatment for various durations
(0 to 3 h). After the sonication, the product is recovered
from the water medium by solvent extraction with
hexane. The 500 mL of water medium was extracted with
three 30 mL portions of hexane (gas chromatography
[GC] grade). A small amount of anhydrous sodium sulfate
was added to extracts to absorb all the excess water. The
recovered solvents were then concentrated by evaporation
under a stream of air to 1 mL before being subjected to
menthol analysis by GC. At the end of the experiment,
the plant leaves were harvested and the fresh and dried
weight measurements were determined. These measurements were used as the basis for the product extraction
calculation. The experiments were done in triplicates.
The scanning electron micrographs of sonicated leaves
were taken with a Hitachi S3200N Environmental Scanning Electron Microscope for a study of the mechanisms
involved in product release.
Repeated Extraction Experiment. To determine
the feasibility of repeated ultrasonic extraction, plants
were sonicated for 1 h for each ultrasonic exposure. After
each sonication, the plants were returned to the hydroponic system to recuperate for 4 days before the subsequent extractions. A total of 3 ultrasonic exposures were
given to the plants in this experiment. The treated plants
were observed visually and the plant biomass was
measured to determine the vitality and health of treated
plants after repeated extractions.
Grinding Extraction and GC Analysis. The menthol product determined by grinding extraction was used
as the basis for assessment of the total amount of
menthol in the leaves (100% release). The protocol for
extraction of ground leaves follows that reported by
Burbott and Loomis (12). The extracts were analyzed by
gas chromatogrphy (GC) with helium as the carrier gas.
A temperature program is used in which the column is
heated from 75 C to 180 C at a rate of 3 C min-1. The
monoterpene components are detected with a flame
ionization detector (FID). The injector and detector
temperatures were set at 180 C and 250 C, respectively.

Results and Discussion

Effects of Ultrasonic Extraction on Product Release. The results of the effects of ultrasonic extraction
on product release is shown in Figure 2. The first bar in
Figure 2 represents a control or the menthol release from
plants immersed in water medium for 1 h without
ultrasound exposure. This shows that there is a nonzero
release of extract, which indicates that a small amount
of menthol can readily permeate through the cuticular

Figure 3. Scanning electron micrographs of leaf tissues of

ultrasound-treated leaf: (a) control (untreated), (b) 30 min
treatment, (c) 60 min treatment, (d) 60 min treatment (high
magnification).

layer of peppermint glandular trichomes into the water

medium. From Figure 2, it is evident that plants ultrasonicated in an ultrasound bath for 1 h are capable of
further releasing menthol product to approximately 17
g of menthol per gram fresh weight of peppermint (2%
of the total product that can be obtained from ground
tissue extraction). The product release increases with the
time of ultrasound treatment between 0 and 3 h. It is
well established that the mechanism that causes biological effects are that of cavitation (13). Kilby and Hunter
(1991), reported no betanin release from sonicated Beta
vulgaris cells when the medium was previously degassed,
and therefore, concluded that cavitation is the main cause

Biotechnol. Prog., 2001, Vol. 17, No. 5

927

Figure 4. Menthol release by repeated extraction of peppermint plants.

of the release of betanin metabolite from Beta vulgaris

cells. However, in our study, when degassed (only vaporous cavities remain) water was used as the sonication
medium, product release is doubled (Figure 2). Although
the result is opposite to that reported by Kilby and
Hunter, it corresponds to the theoretical and experimental work that shows that vaporous transient cavitation
collapses are more violent than those of both gaseous and
vaporous cavitation, because in the latter case, the
motion is cushioned, in the final stages, by the compression of the residual gas (14).
Another interesting observation is the effect of temperature on product release. Even though the results in
Figure 2 were from the experimental runs that were
performed at a controlled constant temperature in order
to isolate the thermal effect of ultrasound, a few runs
were performed where the temperature was not controlled so as to test the effect of temperature on product
release. In this case, the temperature was found to rise
from 22 C to as high as 39 C over a 1-h period due to
absorption of sound energy by the water in the bath
during the treatment period. This increase in the temperature causes an increase in product release as compared to the controlled temperature ultrasonic extraction;
an increase from 2% to 12% of total product was found
under conditions where the water bath temperature
increased, as noted above. A possible reason for this is
that the solubility of menthol in water increases with the
temperature. Furthermore, menthol has a melting point
of 41 to 43 C. This indicates that the solid menthol might
start to undergo phase transition to liquid phase and is
then more readily released out of the leaf tissues to the
medium. However, high water bath temperatures resulted in a smaller amount of product release in the
subsequent extractions, possibly due to the inactivation
of enzyme activities in the cells by higher temperatures.
Unlike the cavitation effect, the thermal effect is only
indirectly related to the sound parameters. Therefore, in
this paper, we focus on the cavitation effects, whose theoretical considerations are discussed in the next section.
Scanning Electron Micrography (SEM). To examine the effects of ultrasound on the structure of the

glandular tissues, the scanning electron micrographs

were taken of peppermint leaves of the same leaf pair,
one after 30 min and the other after 60 min of ultrasonic
extraction. The results differ markedly. In the former
case (Figure 3b), the glands were still intact and remain
a normal spherical shape similar to that of the control
plants (Figure 3a). In the case in which plants were
sonicated for 60 min, not only had the glands appeared
to be completely disintegrated from the surface of the
leaf, but the surface structure was also altered. This can
be clearly seen in Figure 3c and 3d. It was also observed
that the damage seen in different leaf pairs was nonuniform. Even within the confines of a localized area on the
same leaf, the response of the glands to period of
extraction varied (Figure 3c). One possible explanation
for this is that in the ultrasonic bath, standing waves
are set up within the enclosure as a result of interference
of waves reflected off the liquid-air boundary. Therefore,
the acoustic pressures (or acoustic intensities) vary with
the level of medium in the cleaning bath. Furthermore,
the standing-wave pattern produced by an array of
transducers in a cleaning bath can produce local variations in the acoustical parameters (15a, b). This is
confirmed by previous studies in which ultrasonic fields
were measured inside the ultrasonic bath (16).
In general, the SEM observations pointed out two
distinct extraction mechanisms for peppermint leaves.
One involves diffusion of the essential oil components
across the unbroken gland cell walls, and the other,
involves the exudation of the essential oil from ruptured
glands into the surrounding solution. The longer the
extraction exposure, the greater the frequency of damaged glands occurs. These broken and damaged glands
should, in general, give rise to an instantaneous release
of the essential oil components into the surrounding
medium. However, due to the low intensity of ultrasound
in such an ultrasonic bath as used here, the product
release in general is still small, and the majority of the
glands are still intact even after several hours of ultrasound extraction.
Repeated Menthol Extraction. For continuous production of the plant compounds, it is required that the

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Biotechnol. Prog., 2001, Vol. 17, No. 5

plants can be repeatedly treated with ultrasound. Figure

4 a shows that the plants can be repeatedly extracted
after 4 days of recuperation. The first set of results was
obtained from sequential extractions, whereas the second
and the third extractions immediately followed the first
extraction. The last two bars represent the amount of
product released for the second and the third extraction
after a 4-day period of recuperation. The amount product
released in the second and the third extractions decreases
in both cases, and there is no significant difference is
these values. This decrease may be caused by the loss of
product from the disrupted tissues or the loss of biosynthetic capability of glandular cells after experiencing
extreme conditions caused by transient cavitation. The
overall vitality of the plants was determined from visual
observation of treated plants and from the measurement
of plant fresh weights during the whole repeated extraction period (treatment and recuperation period). After
ultrasonic exposure, there were some localized cell death
and tissue disruptions. However, the overall plant vitality
was retained. There was no significant difference in plant
growth rate for plants repeatedly extracted for up to three
times from the controlled plants that were not subjected
to ultrasonic exposures.

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.

References and Notes

(1) Mason, T. J.; Zhao, Y. Enhanced Extraction of Tea Solid
Using Ultrasound. Ultrasonics 1994, 32, 375-377.
(2) Li, H.; Ohdaira, E.; Ide, M. Effects of Ultrasound on
Extraction of Saponin from Ginseng. Jpn. J. Appl. Phys. 1994,
33, 3085-3087.

(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.

Accepted for publication July 16, 2001.

BP010074U