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ORIGINAL PAPER

Isolation, Characterization and Quantification of Tricin

and Flavonolignans in the Medicinal Rice Njavara

(Oryza sativa L.), as Compared to Staple Varieties

Smitha Mohanlal & Rathnam Parvathy &

Vasantha Shalini & Antony Helen &

Ananthasankaran Jayalekshmy

Published online: 5 March 2011# Springer Science+Business Media, LLC 2011

Abstract Njavara is an important medicinal rice variety ofKerala, India, widely used in Ayurveda as a health food

and in the treatment of rheumatoid arthritis, paralysis,

neurodegenerative diseases and in rejuvenation therapy.

Phytochemical investigations and spectroscopic studies of

the diethyl ether fraction of methanolic extract of Njavara

Black (NB) rice bran gave three important compounds

namely, tricin and two rare flavonolignans- tricin 4-O-

(erythro--guaiacylglyceryl) ether and tricin 4-O-(threo--

guaiacylglyceryl) ether. The EC50 values of these com-

pounds in DPPH system were 90.39, 352.04 and 208.1 g/

ml, respectively. Quantification of the compounds by

HPLC in NB and staple, non-medicinal rice varieties

Sujatha (SJ) and Palakkadan Matta (PM) showed that tricin

is present 39.64 and 16.12 fold higher in NB, compared to

SJ and PM, respectively. This is the first report on the

occurrence of tricin at significantly higher levels in Njavaraand occurrence of the two flavonolignans in Oryza sativa

species. Of the three compounds, tricin and the threo- form

of flavonolignan showed anti-inflammatory effect of >65%

after 5 h, at 2 mg/kg, in carrageenan-induced, paw edema

experiments in rats. The results of the study corroborate

with the preferential use of Njavara in indigenous medicine,

over staple varieties.

Keywords Flavonolignans .Njavara . Rice bran . Tricin .

Tricin 4-O-(erythro-- guaiacylglyceryl) ether.

Tricin 4-O-(threo--guaiacylglyceryl) ether

Abbreviations

COX cyclooxygenase

DPPH 2, 2- diphenyl-1-picrylhydrazyl

EC50 amount of extract or compound needed to

decrease the initial DPPH concentration by

50%

HPLC-PDA high performance liquid chromatograph-

photodiode array

IR infra red

MS mass spectrometry

NB Njavara Black

ND not detected

NMR nuclear magnetic resonance

NO nitric oxide

NSAIDs non-steroidal anti-inflammatory drugs

PM Palakkadan Matta

RAW 264.7 cell line of mouse macrophages

ROS reactive oxygen species

SD standard deviation

SEM standard error mean

Electronic supplementary material The online version of this article

(doi:10.1007/s11130-011-0217-5) contains supplementary material,

which is available to authorized users.

S. Mohanlal : R. Parvathy : A. Jayalekshmy (*)

Chemical Sciences & Technology Division, National Institute for

Interdisciplinary Science and Technology (NIIST), CSIR,

Industrial Estate P.O.,

Thiruvananthapuram 695019 Kerala, India

e-mail: jayalekshmy9@gmail.com

A. Jayalekshmy

e-mail: jaya@niist.res.in

V. Shalini : A. Helen

Department of Biochemistry, University of Kerala,

Kariavattom Campus,

Thiruvananthapuram 695 581, India

Plant Foods Hum Nutr (2011) 66:9196

DOI 10.1007/s11130-011-0217-5

http://dx.doi.org/10.1007/s11130-011-0217-5http://dx.doi.org/10.1007/s11130-011-0217-5

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SJ Sujatha

SPSS/PC+ statistical programme software

UVVIS ultra violet-visible

Introduction

Contemporary importance of free radicals and reactiveoxygen species (ROS) as causative agents in degenerative

diseases and conditions like atherosclerosis, cancer, inflam-

mation, rheumatism, aging, etc., can not be overlooked [1].

Foods are a safe, natural source of potent antioxidants [2].

India, especially the southern state of Kerala, is well known

for its rich biodiversity, medicinal plants, spices and

practice of Ayurveda. Recently, there is greater acceptance

of holistic treatment regimes to fight degenerative disease

conditions using herbal extracts that are rich in antioxidant

phytochemicals [3, 4].

The ancient texts of Ayurveda report special rice described

as Shaashtikam-a variety of red rice reaped in 60 days time-that is medicinal (http://www.njavara.com/aboutnjavara ).

Njavara is a unique pigmented rice cultivar of extra short

duration (6090 days), endemic to Kerala. It is widely used

in ayurvedic system of medicine and experts in the field

consider Njavara to be the medicinal variety cited in ancient

literature [http://www.njavara.com/aboutnjavara , 5]. This is

the only cultivar traditionally used in specific treatments like

Panchakarma that includes protocols like Njavara kizhi

and Njavara theppu for rheumatoid arthritis, neurological

problems, paralysis and rejuvenation therapy. Njavara is also

recommended as health food for people of all ages and its

brown rice is the main ingredient in the nutraceutical kit

(Karkitaka Kanji) for making Medicinal Porridge, rec-

ommended by physicians to boost immunity against dis-

eases, during monsoon season, in this region (http://www.

njavara.com/aboutnjavara ).

There are two major types of Njavara grown in Kerala

for medicinal use viz. the black glumed and golden

yellow glumed types, depending on the colour of the outer

husk of paddy, both having red-pigmented rice grains

(http://www.njavara.com/aboutnjavara). There are no pre-

vious scientific reports on the bioactive compounds of

Njavara and available reports are on agronomic aspects,

genetic characteristics, proximate composition, lipid profile

and starch characteristics [57]. As our group has been

working on natural antioxidants and bioactives of indige-

nous sources, we undertook a study of the methanolic

extract of Njavara rice bran and rice that showed higher

antioxidant activity for Njavara compared to staple varie-

ties. These interesting results made us to investigate the

nutraceutical relevance of Njavara as a health food. Here,

we present the first report on this important aspect. Our

study is an investigation on the anti-inflammatory com-

pounds of black glumed Njavara grown in a certified

Njavara farm, as compared with a popular white rice variety

Sujatha (SJ) and a preferred pigmented variety Palakkadan

Matta (PM) grown in the same region.

Materials and Methods

Chemicals

2, 2- diphenyl-1-picrylhydrazyl (DPPH) and quercetin were

procured from Sigma-Aldrich Co., USA. Deuterated solvents

for nuclear magnetic resonance (NMR), high performance

liquid chromatography (HPLC) grade acetonitrile and water

were purchased from Merck, Mumbai, India. Sephadex LH-

20 (particle size 25100 m) was purchased from Pharmacia

Fine chemicals AB, Uppsala, Sweden. All chemicals and

solvents were of analytical grade. The inflammatory agent

used for the study was Type IV Lamda carrageenan fromSpectrochem Ltd., India. Diclofenac (Voveran) was pro-

cured from Novartis India Ltd., Mumbai, India.

Plant Material and Extraction

Authentic Njavara black (NB) samples were collected

directly from a certified farm namely, the ECO FARM

Karukamanikalam at Chittoor, Palakkad, Kerala. Samples of

staple varieties SJ and PM were also collected from the same

farmer, for comparison. Plant specimens were verified by Dr.

Maya C. Nair, Department of Botany, Government Victoria

College, Palakkad- 678 001, Kerala, India and voucher

specimens are available at the herbarium of above mentioned

Department. Freshly milled bran samples were stabilized by

heating 100 g lots of bran, spread on a petri dish, at 100 C for

30 min in air oven (Sri Rudran Instruments Co., Chennai,

India). 100 g lots of stabilized rice bran (NB, SJ, PM) were

extracted using 800 ml of petroleum ether solvent for about

16 h in a Soxhlet extractor. The residual rice bran was further

extracted with 800 ml of methanol as described above. These

extractions were repeated for 250 g of bran of NB, SJ and PM.

Methanolic extract residue was suspended in 200 ml water

and partitioned with (5100 ml) of diethyl ether. The diethyl

ether extracts were evaporated to dryness on a rotary

evaporator (Laborota 4000-Heidolph, Germany) and the dried

residue was made up to definite volume in methanol and

stored in the refrigerator until further work up.

DPPH Radical Scavenging Activity

DPPH solution of 0.1 mM was prepared in methanol.

Different concentrations (1001,000 g/ml) of the dif-

92 Plant Foods Hum Nutr (2011) 66:9196

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ferent extracts and compounds were prepared keeping the

sample volume to be 0.5 ml and 5 ml of DPPH solution

was added to each test tube and shaken well. The test

tubes were kept at ambient temperature (302 C) in the

dark for 30 min. A control was prepared with 0.5 ml

methanol and DPPH solution (5 ml). Percentage of

radical scavenging activity and EC50 (amount of extract

or compound needed to decrease the initial DPPHconcentration by 50%) were determined spectrophotomet-

rically at 517 nm, using Shimadzu UV-1601, according to

Sanchez-Moreno et al. [8].

Isolation of Compounds 1, 2 and 3 and their Quantification

by HPLC-PDA Analysis

Three compounds were isolated from 2 g of diethyl ether

residue chromatographed over (a) silica gel column with

peteroleum ether:ethyl acetate gradient, (b) Sephadex

LH-20, and (c) analytical and preparative HPLC, fol-

lowed by crystallization to afford pure compounds 1(24 mg), 2 (3.1 mg) and 3 (3 mg), respectively. Isolated

compounds 1, 2 and 3 were quantitated in NB, SJ and PM

by analytical, reverse phase high performance liquid

chromatography-photodiode array (HPLC-PDA) (details

given in supplementary).

Anti-inflammatory Activity

For the experiment, the rats (details given in supplementary)

were divided into six groups (IVI) with six rats in each

group (n=6). Group I received saline. Acute inflammation

was produced by the sub-plantar administration of 0.1 ml of

carrageenan (1% in normal saline) from group IIVI into the

right hind paw of the rats. The animals were pre-treated

intra-peritoneally in groups IIV, with the compound 1, 2, 3

(2 mg/kg each) and diclofenac (20 mg/kg) in saline

respectively, 30 min before the administration of carrageen-

an. The volume of each paw was measured by means of a

plethysmograph at 0, 3rd, 5th h after carrageenan injection

[9]. The percentage of paw edema was calculated according

to Winter et al. [10].

Statistical Analysis

Values were represented as meanstandard deviation (SD)

and standard error mean (SEM) of two analyses from three

replications (n=6). Analysis of variance was performed for

quantification by HPLC and in vivo anti-inflammatory

experiments using the statistical program software (SPSS/

PC+), version 11.0 (SPSS, Chicago, IL, USA). Duncans

multiple range test was conducted for comparison of means

at P

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extract of NB bran showed

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respectively. HPLC profiles of SJ and PM are magnified in

Fig. 1 to facilitate locating the peaks of 2 and 3.

Anti-inflammatory Activity

The methanolic extract and diethyl ether fraction of NB

showed anti-inflammatory effect of about 80% at 5 mg/kg

dose. Hence, the compounds isolated were evaluated forthis biological effect. The anti-inflammatory activity of

compounds 1, 2 and 3 against acute edema (induced by

carrageenan) is shown in Table 2 and the results are

comparable to that of the standard drug diclofenac. At 3rd h,

compounds 1 and 3 showed 50% edema inhibition whereas

compound 2 showed only 20%. Of the three compounds,

compound 1 inhibited edema formation maximum, to an

extent of 70%, followed by compound 3 with 66.6% and 2

with 44.4% at 5th h. Compared to standard drug diclofenac,

the three compounds showed better inhibition at lower

concentration.

Carrageenan-induced local inflammation (paw edema orpleurisy) is a commonly used method to evaluate and

compare the efficacy of non-steroidal anti-inflammatory

drugs (NSAIDs) and also in determining the role of

mediators involved in vascular changes associated with

acute inflammation [14]. The initial phase of inflammation

(edema, 01 h) has been attributed to the release of

histamine, 5-hydroxytryptamine and bradykinin, followed

by a late phase (16 h) mainly sustained by prostaglandin-

release, attributed to the induction of cyclooxygenase-2 in

the tissue [15]. In the present study, all the compounds

showed edema inhibition as compared to diclofenac. The

edema inhibition by compound 1 and 3 was maximum and

it was more pronounced at the second phase, suggesting its

inhibitory effect on prostaglandin production as a major

mechanism by which compounds exert anti-inflammatory

effect. Moscatelli et al. [16] have studied the in vitro anti-

inflammatory activity of tricin, based on prostaglandin E2levels, cyclooxygenase (COX) and phospholipase activity.

Chang et al. [17] mention about the anti-inflammatory

effect of salcolin B (identical with tricin 4-O-(erythro--

guaiacylglyceryl) ether) only, based on inhibition of nitric

oxide (NO) in a cell line of mouse macrophages (RAW

264.7). Our study gives a comparison of tricin and the two

flavonolignans in carrageenan-induced rat paw edema

model (in vivo).

Tricin is previously reported in rice bran but the rare

flavonolignans (2 and 3) are first time identified and

quantified in Njavara and in the Oryza sativa species [11].

Compounds 2 and 3 are found to be stereoisomers due to

the presence of two adjacent chiral centers. These com-pounds were originally reported in the aerial parts of

Salsola collina (Chenopodiaceae) and also in Avena sativa

L. of the Poaceae (Graminae) family [13, 18]. Tricin is

widely distributed in Gramineae plants. A recent review by

Zhou & Ibrahim [19] on tricin highlights the potential of

the compound as a multifunctional nutraceutical. In this

review, the beneficial health effects of tricin such as

antioxidant effect, inhibition of lipid peroxidation, sparing

effect on vitamin E in erythrocyte membrane, antiviral,

immunomodulatory, antitubercular, antiulcerogenic, anti-

mutagenic, mildly estrogenic, anti-inflammtory and potent

anticancer effects are cited. Hudson et al. [11] have proventricin as a potential chemopreventive constituent of rice

bran and their preliminary (in vitro) study showed it to

interfere potently with the survival of human derived breast

and colon cancer cells. Later, Verschoyle et al. [20]

suggested tricin may be considered safe enough for clinical

development as a cancer chemopreventive agent. Our

studies show that Njavara bran contains tricin, in signifi-

cantly higher concentration compared to staple rice varie-

ties, which give scientific support to its medicinal use.

Njavara is generally used as brown rice, retaining about

80% of bran on it. We can see that Compounds 2 and 3 are

present only in minute quantities in SJ compared to NB

(Table 1). In PM, the peaks 2 and 3 (Fig. 1c) showed maxat 328 nm which is different from the max of 338 nm of the

peaks 2 and 3 (Fig. 1a) representing compounds 2 and 3 of

NB. Hence, the compounds 2 and 3 are absent in PM. The

peaks with max at 328 nm in the HPLC of PM were not

further investigated owing to their very low concentration.

The diethyl ether fraction of the methanolic extract of NB is

having greater radical scavenging activity and the quanti-

fication of the compounds 1, 2 and 3 in the extracts showed

that they are at higher concentration in NB. The higher

Table 2 Anti-inflammatory effect of tricin (compound 1), tricin 4-O-(erythro--guaiacylglyceryl) ether (compound 2), tricin 4 O-(threo--

guaiacylglyceryl) ether (compound 3) and diclofenac on carrageenan induced paw edema in rats

Time Inhibition (%) of different treatment groups (dose 2 mg/kg) Drug diclofenac (dose 20 mg/kg)

Compound 1 Compound 2 Compound 3

3rd h 50.01.44b 20.00.57c 50.01.44b 72.02.08a

5th h 70.02.02b 44.41.27c 66.61.91b 86.02.48a

ac Meanstandard error mean of six determinations followed by different letters in a row are significantly different in the Duncans test at P

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activity of diethyl ether extract of NB can be attributed to

the higher concentration of these compounds compared to

SJ and PM. Hence, the anti-inflammatory property of

Njavara can be correlated with the presence of the three

compounds in higher quantity, especially tricin, in Njavara

compared to staple varieties.

Conclusions

In summary, the study establishes the isolation of three

compounds tricin (compound 1), tricin 4-O-(erythro--

guaiacylglyceryl) ether (compound 2) and tricin 4-O-

(threo--guaiacylglyceryl) ether (compound 3) in Njavara

bran and the presence of rare flavonolignans (compounds 2

and 3) in rice bran for the first time. The in vivo study

confirmed and compared the anti-inflammatory action of

compounds 1, 2 and 3. The bioactive compound tricin, a

promising multifunctional nutraceutical, is present in higher

concentration in Njavara compared to the staple varieties.The rare flavonolignans, which are antioxidant and anti-

inflammatory, also occur in higher quantity in Njavara

compared to staple varieties. These findings corroborate

with the preferential use of Njavara in Ayurveda, over

staple varieties.

Acknowledgements The authors are grateful for the funding

assistance provided by Kerala State Council for Science, Technology

and Environment (KSCSTE), Government of Kerala. The author

Smitha Mohanlal wishes to thank Council of Scientific and Industrial

Research (CSIR), India for the financial support as Senior Research

Fellowship (SRF). Thanks are also due to Director, NIIST, CSIR and

Head, Department of Biochemistry for constant encouragement andsupport.

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