Vol.2, No.8, 968-972 (2010)
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
Openly accessible at
S-adenosyl-L-methionine, trehalose and oleanolic acid
in few plants
Dipankar Malakar, Paramita Chaudhuri, Trina Dutta, Anil K. Ghosh*
Biotechnology Department, Indian Institute of Chemical Biology, Kolkata, India; *Corresponding Author:
Received 22 February 2010; revised 30 March 2010; accepted 2 April 2010.
S-Adenosyl-L-Methionine (AdoMet), S-Adenosyl-
L-Homocysteine (AdoHcy), adenosine, trehalose
and oleanolic acid were measured in six me-
dicinal herbs and three spices. The findings
showed that AdoMet content was forty six fold
higher in the leaves of Catharanthus roseus as
compared with average AdoMet content of rest
of the plants. In comparison to other plants,
Withania somnifera had very high AdoHcy: Ado-
Met and adenosine: AdoMet ratios indicating it
may have contained high AdoMet. Trehalose
was found to be twenty fold and nine fold higher
in bulb of Allium cepa and root of Withania
somnifera respectively with respect to average
trehalose content of rest of the plants. Ocimum
sanctum appeared to be a rich source of olea-
nolic acid. It appeared from our study that Ca-
tharanthus roseus, Allium cepa and Ocimum
sanctum could be utilized as natural sources of
AdoMet, trehalose and oleanolic acid respec-
Keywords: AdoMet; AdoHcy; Adenosine;
Trehalose; Oleanolic Acid
S-Adenosyl-L-Methionine (AdoMet) is the only natural
sulfonium compound present in different living organ-
isms. It is an important metabolic intermediate partici-
pating in different biochemical events and acts as a uni-
versal methyl group donor in transmethylation reactions,
transsulfuration reactions and polyamine synthesis [1].
The molecule has therapeutic importance in the treat-
ment of alcoholic liver disease [2], cirrhosis of liver [3],
depressive syndrome [4], Alzheimer disease [5], Os-
teoarthritis [6] etc. and may be classified as an “anti-
aging compound”.
Trehalose (-D-glucopyranosyl--D-glucopyranoside)
is a non reducing disaccharide having high glass transi-
tion temperature (Tg = 80ºC) is well known for effective
stabilization of macromolecules like proteins [7], small
molecules like AdoMet [8], etc. It also acts as a cryo-
preservative of cellular membranes [9].
Oleanolic acid is a triterpenoid compound that exists
widely in food, medicinal herbs and other plants. It is
effective in protecting against chemically induced liver
injury in laboratory animals where the mechanism of
hepatoprotection may involve the inhibition of toxicant
activation and the enhancement of the body defense sys-
tems [10]. Oleanolic acid has also been long recognized
to have antiinflammatory and antihyperlipidemic proper-
ties in laboratory animals [11]. But more research is
warranted to develop a therapy for patients. Recently,
oleanolic acid has been noted for its anti-tumor activity
Extracts from the leaf of Ocimum sanctum [13], stem
of Tinospora cordifolia [14], rhizosphere of Picrorrhiza
kurroa [15], root of Withania somnifera[16], whole body
(except root) of Eclipta erecta [17], bulbs of Allium sa-
tivum [18] and Zingiber officinale [19] are already pro-
ven for their hepatoprotective activities beside other
therapeutic potentialities.
In the present communication, we have made com-
parative studies on AdoMet, trehalose and oleanolic acid
content among the above mentioned medicinally impor-
tant plants. For our experiments we have taken root of W.
somnifera, leaf of O. sanctum, stem of T. cordifolia,
rhizosphere of P. kurroa, whole body (except root) of E.
erecta and bulbs of A. sativum and Z. officinale. The
bulb of Allium cepa, which is well known for its food
preservation activities and leaf of Catharanthus roseus
[20] which have anti-tumor, anti-oxidative activities
were also chosen for analysis.
The experiments were performed in triplicate by col-
D. Malakar et al. / HEALTH 2 (2010) 969-972
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
lecting different parts of young herbs and spices from a
particular area of eastern India in winter season so that
within different experimental sets geological, environ-
mental and chronological variations could be avoided.
The intracellular matter from the specific parts of the
different herbs and spices were simultaneously extracted
and deproteinized [21] prior to the estimation of treha-
lose, AdoMet and its irreversibly transformed metabolic
products AdoHcy and adenosine. Briefly, each of the
herbs and spices were cut into small pieces and washed
thoroughly with tap water.
The washed pieces were then air-dried and 0.25 g (dry
weight) of each of the plant parts was homogenized with
0.5 ml of cold 0.5 N perchloric acid for deproteinization
purpose. The mixture was centrifuged at 5,500 g for 10
min and the pellet was extracted further with 0.5 ml of
cold 0.2 N perchloric acid and again centrifuged. The
process was repeated and the supernatants were pooled.
The pH value of the pool was adjusted to 4.5 by careful
addition of 3 N KOH. This caused precipitation of po-
tassium per chlorate and the precipitate was removed by
low speed centrifugation for 10 minutes at 4ºC. Careful
reduction of volume under reduced pressure at relatively
low temperature resulted in further precipitation of po-
tassium per-chlorate and this was again removed by low
speed centrifugation. The supernatants were dried and
reconstituted with 1ml of 0.01 N HCl.
AdoMet, AdoHcy and adenosine content of different
herbs and spices were quantitatively estimated by cation
exchange High Performance Liquid Chromatography
(HPLC) [22]. The HPLC column used was Partisil 10SCX,
4.6 250 mm, Whatman Inc., England, fitted to an
HPLC system consisting of two pumps (Model 515), a
Rheodyne injector and a programmer controlled by
pump control module (PCM). Elution of AdoMet was
monitored in-line by measurement of absorbance at 259
nm (A259), using a dual wavelength UV-Visible detector
(Model 2487). Elution times as well as peak quantifica-
tion were obtained from the Millenium32 software. All
these HPLC equipments were from Waters, USA.
AdoMet, AdoHcy and adenosine were eluted near
31.617 min, 10.998 min and 8.993 min. The amounts of
standard compounds AdoMet, AdoHcy and adenosine
(expressed in g) were plotted along the x-axes and re-
spective peak areas were plotted along the y-axes and
compared with the samples (in triplicate) from different
plant extracts. The standard solutions of AdoMet (Sigma,
USA), AdoHcy (Sigma, USA) and adenosine (Sigma,
USA) were prepared by dissolving in 0.01 N HCl.
Different volumes (5, 10, 20 l) of the standard com-
pounds of known concentrations (0.435 mM AdoMet,
2.17 mM AdoHcy and 0.9 mM adenosine) were injected
into the HPLC system to get standard curves. Values
obtained for test sample (in triplicate) were derived from
the standard curve when r2 (square of correlation coeffi-
cient) values were near 0.99.
The amount of trehalose present in the samples was
estimated using acid trehalase (AT) enzyme, purified in
our laboratory according to published method [23,24].
The standard aqueous trehalose (Sigma USA) solution
(1 mg/ml) was prepared. Values represented here are
average of experiments done in triplicate.
Oleanolic acid was estimated by Thin Layer Chroma-
tographic (TLC) method [25] with some modifications.
Different volumes (2.5, 5.0, 10.0 l) of standard oleano-
lic acid (0.9 mg/ml chloroform; Sigma, USA) were
spotted on a precoated alumina TLC plate along with
different plant extracts (1 g dried powder/ml chloroform),
10 l were spotted on different lanes. The optimized
solvent system for best separation of oleanolic acid from
the other chemical constituents present in chloroform ex-
tract of the different plants was the combination of ben-
zene, chloroform and ethyl acetate in the ratio of 6: 3: 1.
After complete air-drying of the TLC plate, it was
sprayed by the anisaldehyde-sulfuric acid reagent for
derivatization and the plate was immediately taken to a
heating chamber (100ºC) for 5.0 min and scanned in a
HP scanjet (Model # 4570c) scanner at a resolution of
600 dpi and densitometrically quantified by a NIH (Na-
tional Institute of Health, USA) make software, namely
“ImageJ”. Different amounts of oleanolic acid were
plotted along the x-axis and the respective total pixel
counts (pixel density x area of the spot (Rf = 0.22)) were
plotted along the y-axis to get a standard curve. Putting
the values of total pixel counts on the standard curve the
amounts of oleanolic acid present in different plant parts
were estimated. The identification of oleanolic acid was
done by mass spectrometry after scraping the spot in the
TLC plate co-linear with the spots of standard oleanolic
acid using ESI-MS analysis in a LC-QTOF system, Mi-
cromass, UK and 1H NMR (300 MHz) study was per-
formed on a DPX 300 NMR instrument, Bruker, Ger-
many using tetra methyl silane (TMS; Sigma, USA) as
internal standard.
Among the herbs and spices studied, C. roseus leaves
contained highest quantity of AdoMet (1.63 mg/g) (Ta-
D. Malakar et al. / HEALTH 2 (2010) 969-972
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
ble 1). Moderate to low AdoMet has been found in the
descending order of O. sanctum> A. sativum> T. cordifo-
lia> W. somnifera> A. cepa> P. kurroa> Z. officinale> E.
erecta. C. roseus was not only rich in AdoMet but this
plant also had highest AdoHcy (0.347 mg/g leaf) and
adenosine (0.084 mg/g leaf), the metabolic products of
AdoMet. The rest of the plants had either low or moder-
ate quantities of AdoHcy which decreased in the order of
W. somnifera > A. sativum > O. sanctum > A. cepa > A.
sativum > E. erecta > T. cordifolia > Z. officinale. No
AdoHcy and adenosine were detected in P. kurroa and
no adenosine was detected in E. erecta under the assay
conditions. Incidentally, these plants had little AdoMet
as well. Besides C. roseus the decreasing order of
adenosine content was W. somnifera > A. sativum > A.
cepa > O. sanctum > T. cordifolia > Z. officinale.
Among the nine plants both A. cepa and W. somnifera
had relatively higher trehalose content (101.94 mg/g
bulb and 45.08 mg/g root respectively). Moderate to low
amount of trehalose was present in the rest of the plants
and decreased in the order of P. kurroa > C. roseus > T.
cordifolia > Z. officinale > A. sativum > O. sanctum > E.
O. sanctum appeared to be the only plant which con-
tained oleanolic acid (10.2 mg/g leaf) (blue spot at Rf =
0.22) among all the herbs and spices studied.
The leaf of C. roseus is well known for the storage of
alkaloids like vinblastine and vincristine, which have
anti-tumor activities [26]. Through this study another
bioactive product of C. roseus leaf viz. AdoMet has been
detected. Leaves of C. roseus contained nearly forty-six
fold higher AdoMet in comparison to average AdoMet
content of the rest of the plants. Genetically modified
Kluyveromyces lactis mutants have been recognized as a
good source of AdoMet [27]. Similar genetic manipula-
tions could be performed to get mutant C. roseus plants
rich in AdoMet. Although W. somnifera contained very
little amount of AdoMet (0.037 mg/g leaf) but it had
both higher AdoHcy (0.148 mg/g root) and adenosine
(0.0508 mg/g root) content and the AdoHcy: AdoMet
and adenosine: AdoMet ratios were remarkably high
compared to other plants. So, it could be said that in W.
somnifera, although AdoMet was synthesized in appre-
ciable quantities but was mostly utilized for its own
metabolic purposes. Both A. cepa and W. somnifera had
twenty fold and nine fold higher trehalose content re-
spectively compared to average trehalose content of the
rest of the seven plants and these two plants could be
exploited as alternative sources of trehalose as their tre-
halose content is comparable with other well known
trehalose rich sources like yeast (10-15% wt/dry wt) [28],
fungi (7-10% wt/dry wt) [29,30], etc. Occurrence of tre-
halose was reported to form a glassy state in the cell, in
which degradative reactions and intracellular ice forma-
tion could sufficiently be arrested during prolonged
storage. In India, bulb of A. cepa is one of the daily used
food ingredients. This is the first report of A. cepa bulbs
containing such a huge percentage of trehalose. Since it
is an economically favorable crop and its production rate
is also very high, more trehalose enriched genetically
improved Allium cepa could be cultivated in near future.
The authors express their hearty thanks to Prof. S. Roy, Director, In-
dian Institute of Chemical Biology, Kolkata for providing infrastruc-
tural facilities. Authors thank D & P program of Department of Science
& Technology, Govt. of India and Dey’s Medical Stores (Mfg.) Ltd.,
Table 1. Estimation of AdoMet, AdoHcy, Adenosine, Trehalose and Oleanolic acid.
Names of the Plants AdoMet (%,
wt/wt) × 10-4
(%, wt/wt)
× 10-4
(%, wt/wt)
× 10-4
(%, wt/wt)
× 10-2
Oleanolic acid
(%, wt/wt)
Tinospora cordifolia 51.37 0.21 0.90 0.014 5.50 0.0076 59.10 0.06 n.d.
Picrorrhiza kurroa 9.18 0.03 n.d. n.d.
99.0 0.06 n.d.
Eclipta erecta 6.36 0.05 1.03 0.006 n.d. 1.01 0.01 n.d.
Ocimum sanctum 79.68 0.30 7.73 0.01 6.83 0.01 17.05 0.03 1.02 0.02
Allium cepa 21.52 0.17 4.78 0.08 7.08 0.02 1019.43 0.13 n.d.
Zingiber officinale 6.58 0.05 0.36 0.01 2.22 0.01 59.03 0.06 n.d.
Allium sativum 69.38 0.20 1.76 0.01 25.34 0.15 38.11 0.03 n.d.
Withania somnifera 37.29 0.23 148.47 0.35 50.82 0.12 450.77 0.19 n.d.
Catharanthus roseus 1630.42 0.23 346.77 0.43 448.29 0.19 84.40 0.29 n.d.
n.d.not detected
D. Malakar et al. / HEALTH 2 (2010) 969-972
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
Openly accessible at
Kolkata for their financial assistance. Thanks also given to Network
Project 0005, Council of Scientific and Industrial Research, Govt. of
India for providing financial assistance to PC and TD.
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