Biotransformation of menthol and geraniol by hairy root cultures of Anethum graveolens.pdf

Biotechnol Lett (2009) 31:897–903

DOI 10.1007/s10529-009-9934-3

ORIGINAL RESEARCH PAPER

Biotransformation of menthol and geraniol by hairy root

cultures of Anethum graveolens: effect on growth

and volatile components

Jorge M. S. Faria Æ In ˆ s S. Nunes Æ

A. Cristina Figueiredo Æ Luis G. Pedro Æ

Helena Trindade Æ Jos ´ G. Barroso

Received: 13 January 2009 / Revised: 20 January 2009 / Accepted: 23 January 2009 / Published online: 11 February 2009

Springer Science+Business Media B.V. 2009

Abstract Two oxygen-containing monoterpene sub-

strates, menthol or geraniol (25 mg l -1 ), were added

to Anethum graveolens hairy root cultures to evaluate

the inﬂuence of the biotransformation capacity on

growth and production of volatile compounds. Growth

was assessed by the dissimilation method and by fresh

and dry weight measurement. The volatiles were

analyzed by GC and GC–MS. The total constitutive

volatile component was composed, in more than 50%,

by falcarinol (17–52%), apiole (11–24%), palmitic

acid (7–16%), linoleic acid (4–9%), myristicin (4-8%)

and n-octanal (2-5%). Substrate addition had no

negative inﬂuence on growth. The relative amount of

menthol quickly decreased 48 h after addition, and the

biotransformation product menthyl acetate was con-

comitantly formed. Likewise, the added geraniol

quickly decreased over 48 h alongside with the pro-

duction of the biotransformation products. The added

geraniol was biotransformed in 10 new products,

the alcohols linalool, a-terpineol and citronellol, the

aldehydes neral and geranial, the esters citronellyl,

neryl and geranyl acetates and linalool and nerol oxides.

Keywords Anethum graveolens

Biotransformation Geraniol Hairy roots

Menthol Volatiles

Introduction

Chemical synthesis is still the major source of

important phytochemicals used in the pharmaceutical

and food industries. Nevertheless, in the past few

years, a renewed interest in the use of natural

products has motivated various industries in attempt-

ing

alternative

production

procedures,

namely

plant biotechnology.

Hairy root cultures offer a ﬂexible and versatile

system that is a promising technology for large scale

production of valuable phytochemicals (Srivastava

and Srivastava 2007 ). Moreover, the production, in

these systems, can be qualitatively and/or quantita-

tively different from the whole plant (Santos et al.

2002 ). Phytochemical production by hairy roots can be

improved by substrate feeding which stimulates the

plant cell enzymatic machinery—mainly enzymes that

can undergo reactions such as reductions, oxidations,

methylations and, particularly, glycosylation, that are

very common in plant cells (Li et al. 2003 ).

Monoterpenes are of great importance in Apiaceae

and, in some cases, represent their dominant volatile

Electronic supplementary material The online version of

this article (doi : 10.1007/s10529-009-9934-3 ) contains

supplementary material, which is available to authorized users.

J. M. S. Faria I. S. Nunes A. C. Figueiredo ( & )

L. G. Pedro H. Trindade J. G. Barroso

Faculdade de Ci ˆ ncias de Lisboa, Departamento de

Biologia Vegetal, Instituto de Biotecnologia e

Bioengenharia, Centro de Biotecnologia Vegetal,

Universidade de Lisboa, C2, Campo Grande,

1749-016 Lisbon, Portugal

e-mail: acsf@fc.ul.pt

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Biotechnol Lett (2009) 31:897–903

components (Croteau 1980 ). In modern society,

terpene alcohols play an essential role in the food

industry, as ﬂavours, and in the perfume industry, as

fragrances. Considering the importance of monoter-

pene alcohols and given the lack of information on the

biotransformation of these compounds by hairy root

cultures, this study evaluates the biotransformation

capacity of Anethum graveolens hairy root cultures by

studying the inﬂuence of the addition of two oxygen-

containing monoterpenes, menthol and of geraniol, on

growth and volatiles composition.

and

fresh

and

dry

weight

determination,

previously described (Santos et al. 2002 ).

Isolation of the volatile components

Volatiles were isolated from dill hairy roots by

distillation–extraction, for 3 h, using a Likens-Nick-

erson type apparatus (Likens and Nickerson 1964 )

using distilled n-pentane as organic solvent. The

volatile oils were stored at -20C in the dark until

analysis.

GC and GC–MS

Materials and methods

The isolated volatiles were analyzed by GC and GC–

MS as previously described (Costa et al. 2008 ).

Hairy root cultures

Anethum graveolens hairy roots, established as pre-

viously described by Santos et al. ( 2002 ), were

maintained in SH medium (Schenk and Hildebrandt

1972 ) with 30 g sucrose l -1 , in darkness at 24Con

orbital shakers (80 rpm). Routinely the cultures were

subcultured every three weeks.

Results and discussion

Hairy root growth

Independently of the growth evaluation procedure

(Fig. 1 ), dill hairy roots showed an initial latent phase

of about seven days, followed by a short exponential

phase, subsequently a linear growth phase that

reached approximately the 30th day and then the

stationary phase. This growth proﬁle is similar to that

obtained

Biotransformation

Erlenmeyer ﬂasks with 100 ml SH medium were

aseptically inoculated with 1 g (fresh wt) of dill hairy

roots and maintained as above. Fifteen days following

subculture, 2% (v/v) or 2% (w/v) for geraniol or

menthol in methanol, respectively was added to each

culture ﬂask, at 25 mg l -1 . Growth and volatiles

production were evaluated after 1, 4, 8, 24, 32 and 48 h

and weekly during the following 4 weeks. Two

independent experiments were separately run, for each

substrate, and two replicates of each ﬂask were used in

each experiment.

Control cultures (=without substrates) were grown

simultaneously. Substrate evaporation and decompo-

sition control experiments were performed by adding

the same amount of substrate to ﬂasks containing only

basal culture medium, and keeping them in the

same conditions as the culture ﬂasks throughout the

experiment.

previously,

with

dill

hairy

root

cultures

(Santos

al.

2002 ),

indicating

stable

growth

pattern for this in vitro system.

Menthol or geraniol addition, in the linear phase

(15th day), did not greatly affect A. graveolens hairy

roots growth, as shown by the growth curves similar

to those of the control cultures (Fig. 1 a, b). Similar

results were obtained with Achillea millefolium cell

suspension cultures (Figueiredo et al. 1996 ) and with

Levisticum ofﬁcinale hairy root cultures (Nunes et al.

2009 ).

Constitutive volatile components

Forty-two components were identiﬁed in the constitu-

tive volatile fraction of dill hairy roots, for 6 weeks,

in a total relative amount [82% (Table 1 ). Falcarinol

(17–52%), apiole (11–24%) and palmitic acid (7–16%)

were the dominant compounds. Other major compo-

nents were linoleic acid (4–9%), myristicin (4–8%) and

n-octanal (2–5%). Polyacetylenes constituted the major

Determination of hairy root growth

Hairy roots growth was measured, both in control and

substrate added cultures, by the dissimilation method,

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Biotechnol Lett (2009) 31:897–903

899

same in vitro system, suggesting that A. graveolens

hairy roots have a relatively stable production of

volatile compounds, as the cultures have been main-

tained for over twelve years with a routine subculture

every three weeks. The higher relative amount of fatty

acids, found in the present study, when compared to

that obtained by Santos et al. ( 2002 ), may be due to the

different culture medium used, which may alter the

volatile composition as was found in L. ofﬁcinale hairy

roots (Costa et al. 2008 ).

(a)

Biotransformation products

The detailed relative amounts of the substrates and

their biotransformation products during the time-

course study are given in Supplementary Table 1 and

a biosynthetic scheme showing the probable relation-

ship between these various compounds is given in

Supplementary Fig. 1 .

Menthol addition, 15 days after subculture, dras-

tically altered the constitutive volatiles composition

of A. graveolens hairy root cultures. Menthol was

quickly biotransformed into menthyl acetate, decreas-

ing from a maximum of 51% in the ﬁrst hour, to 11%,

48 h after addition (Fig. 2 a). Concomitantly, menthyl

acetate increased from 4% in the 1st hour to a

maximum of 55%, 48 h after menthol addition. From

this

Time (days)

(b)

point

both

compounds

slowly

decreased

throughout the next 4 weeks.

Menthol has been most thoroughly studied in

species of the genus Mentha, particularly in Mentha

9 piperita. In this species, Martinkus and Croteau

( 1981 ) have identiﬁed an acetyl-CoA:monoterpenol

acetyltransferase that is responsible for the transfor-

mation of l-menthol into menthyl acetate. According

to Lange et al. ( 2000 ), studying the same species, the

enzyme menthol acetyltransferase was responsible for

menthol acetylation. Probably an enzyme of this

acetyltransferase family is active in A. graveolens

hairy root cultures that acetylates menthol, with no

detectable effects in growth. Nevertheless this capac-

ity seems to be species speciﬁc, as in Levisticum

ofﬁcinale hairy root system this capacity was not

detected (Nunes et al. 2009 ) and in A. millefolium

cell

Time (days)

Fig. 1 Growth curves of dill hairy roots evaluated by the

dissimilation method (a) and by fresh and dry weight methods

(b). Dissimilation growth curves: control cultures [=without

substrates, r (standard deviation 0–2 mg l -1 )], menthol [j

(standard deviation 0–3 mg l -1 )] and geraniol [m (standard

deviation 0–1 mg l -1 )] added cultures. Fresh and dry weight

growth curves: control [r (standard deviation 0–5 g), e

(standard deviation 0–1 g), respectively], menthol [j (standard

deviation 1–5 g), h (standard deviation \0.5 g)] and geraniol

[m (standard deviation 0–4 g), D (standard deviation \0.5 g)]

added cultures, respectively

group of volatile components, represented only by

falcarinol, also named panaxynol, which is a powerful

anti-fungal substance (Seigler 1998 ) and a potential

anti-cancer drug (Zheng et al. 1999 ). Phenylpropa-

noids (16-31%) and fatty acids (13-25%) were also

present in considerable amounts. The terpene fraction

was constituted only by monoterpenes and didn’t

exceed 8%. These results are in agreement with those

obtained previously by Santos et al. ( 2002 ) with the

suspension

cultures

(Figueiredo

al.

1996 ),

menthyl

acetate

was

produced,

though

trace

amounts.

The addition of geraniol to A. graveolens hairy

root cultures resulted in the formation of 10 new

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Biotechnol Lett (2009) 31:897–903

Table 1 Percentage composition of the constitutive volatiles isolated from dill hairy roots maintained for 6 weeks in SH medium, in

darkness at 24C and 80 rpm

Components

Anethum graveolens hairy root cultures

Time (days)

Benzaldehyde

927

0.1

0.3

0.9

0.3

0.6

a-Pinene

930

0.5

0.8

0.1

0.3

n-Heptanol

952

0.1

0.2

0.5

0.2

0.1

0.2

Sabinene

958

1-Octen-3-ol

961

b-Pinene

963

0.4

0.9

1.4

0.8

0.7

2-Octanone

967

0.2

0.1

0.2

0.1

0.2

0.1

2-Pentyl furan

973

0.1

0.2

0.7

0.1

n-Octanal

973

2.6

3.8

3.2

1.9

3.5

4.5

Benzene acetaldehyde

1002

0.3

0.2

0.7

0.2

0.3

p-Cymene

1003

0.3

0.2

0.8

Limonene

1009

0.3

0.8

1.1

0.4

0.3

1.1

n-Octanol

1045

0.1

0.5

0.1

2-Nonanone

1058

0.3

0.8

0.6

0.3

0.4

0.5

Terpinolene

1064

0.3

0.1

0.2

2-Hexyl furan

1064

Phenyl ethyl alcohol

1064

Nonanal

1073

0.4

1.3

1.0

0.5

0.9

Vertocitral C*

1077

0.3

0.1

0.2

0.3

trans-Tagetone

1116

0.3

0.5

1.1

0.8

0.9

1.5

cis-Tagetone

1123

0.2

0.4

2- trans -Nonen-1-al

1114

0.9

1.2

1.1

1.2

2.1

Decanal

1180

0.4

0.3

0.2

0.3

0.2

0.4

cis-Ocimenone

1200

0.7

0.1

0.6

0.3

0.5

trans-Ocimenone

1207

0.1

2-trans-Decenal

1224

0.5

1.0

0.7

0.5

0.8

2 trans-4-cis-Decadienal

1242

0.1

0.2

0.1

0.2

2-Undecanone

1271

0.4

0.1

0.3

0.5

Carvacrol

1286

0.7

1.1

1.0

2.2

2.5

2.3

2 trans-4 trans-Decadienal

1286

0.5

0.2

0.5

trans-2-Undecenal

1323

0.8

0.2

0.6

0.2

0.4

trans-4-Undecenal*

1402

0.6

0.3

0.5

1.1

Myristicin

1493

4.2

7.5

7.9

3.5

4.8

3.7

c-Undecalactone*

1535

0.6

2,4-Dimethoxyacetophenone

1544

0.1

0.2

Dill apiole

1587

2.6

1.8

1.3

1.2

1.7

Apiole

1640

23.7

20.5

15.2

11.2

13.0

17.6

Myristic acid

1723

0.5

0.2

0.5

0.1

Pentadecanoic acid*

1778

1.0

0.6

0.8

1.4

Palmitic acid

1908

16.4

14.6

14.7

6.7

8.1

12.3

Falcarinol

2002

17.2

23.8

27.4

51.6

42.6

25.1

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Biotechnol Lett (2009) 31:897–903

901

Table 1 continued

Components

Anethum graveolens hairy root cultures

Time (days)

Linoleic Acid

2125

5.8

8.8

5.8

5.0

4.3

4.2

% Identiﬁcation

81.7

93.5

91.0

92.5

90.7

86.4

Grouped components

Monoterpene hydrocarbons

1.8

2.0

4.3

1.3

1.1

2.1

Oxygen-containing monoterpenes

2.0

1.8

3.2

3.5

3.9

5.1

Polyacetylenes

17.2

23.8

27.4

51.6

42.6

25.1

Phenylpropanoids

30.5

29.8

24.4

15.9

19.5

23.0

Fatty acids

22.7

24.6

21.1

12.5

13.7

18.0

Others

7.5

11.5

10.6

7.7

9.9

13.1

RI Retention index relative to C 9 –C 22 n-alkanes on the DB-1 column, t traces (\0.1%)

* Identiﬁcation based on mass spectra only

geranyl acetates and, in traces, linalool and nerol

oxides. Geranial and neral occurred in similar relative

amounts throughout the time-course study. L. ofﬁcin-

alle (Apiaceae) hairy root cultures, grown under

similar conditions, were able to biotransform geraniol

into nerol and neral but not into geranial (Nunes et al.

2009 ). According to several authors (in Iijima et al.

2004 ), it is not yet established if citral (mixture of

geranial and citral) is formed from geraniol by the

action of an alcohol dehydrogenase or by an oxidase,

not even if geraniol is the only substrate in the

formation of citral, since nerol can also be a precursor.

All the new biotransformation products, with the

exception of linalool and nerol oxides, were detected

1 h after geraniol addition (Fig. 2 b). Geraniol

decreased quickly from 20%, in the ﬁrst hour, to

\3%, 48 h after addition. The new alcohols formed,

didn’t exceed 10%, throughout the time-course study

(Fig. 2 b). Citral and citronellol reached a maximum

8 h after substrate addition and linalool attained a

maximum only 32 h after geraniol addition.

Studying Vitis vinifera L., grape berry mesocarp,

Luan et al. ( 2005 ) were able to identify a stereose-

lective geraniol reductase responsible for citronellol

production after labelled geraniol addition. Never-

theless they were unable to clarify its origin since

geraniol also yielded its isomer neral, which can also

be converted to citronellol. These authors have also

reported the production, although in small amounts,

of geranial and neral which requires the presence of

an oxidase and/or a dehydrogenase.

(a)

Menthyl acetate

Menthol

168

336

504

672

Time after substrate addition (h)

(b)

Esters

Alcohols

Aldehydes

Geraniol

168 336

504

672

Time after substrate addition (h)

Fig. 2 Time-course study of the relative amounts of substrates

and of their biotransformation products. a Menthol and the

biotransformation product, menthyl acetate. b Geraniol and the

biotransformation products, esters, alcohols and aldehydes

biotransformation products. These were the alcohols

linalool, a-terpineol and citronellol, the aldehydes

neral and geranial, the esters citronellyl, neryl and

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