S-adenosyl-L-methionine, trehalose and oleanolic acid in few plants

doi:10.4236/health.2010.28144

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Vol.2, No.8, 968-972 (2010)

doi:10.4236/health.2010.28144

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:

aghosh@iicb.res.in

Received 22 February 2010; revised 30 March 2010; accepted 2 April 2010.

ABSTRACT

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-

tively.

Keywords: AdoMet; AdoHcy; Adenosine;

Trehalose; Oleanolic Acid

1. INTRODUCTION

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

[12].

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.

2. MATERIALS AND METHODS

The experiments were performed in triplicate by col-

D. Malakar et al. / HEALTH 2 (2010) 969-972

969

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.

3. EXTRACTION AND DEPROTEINIZATION

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.

4. ESTIMATION OF ADOMET, ADOHCY

AND ADENOSINE

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.

5. RESULTS AND DISCUSSION

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

970

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.

erecta.

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.

6. ACKNOWLEDGEMENTS

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

AdoHcy

(%, wt/wt)

× 10-4

Adenosine

(%, wt/wt)

× 10-4

Trehalose

(%, 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

971

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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