Indian Journal of Biochemistry & Biophysics

Vol. 51, February 2014, pp.75-80

Role of γ-oryzanol in drought-tolerant and susceptible cultivars of rice

(Oryza sativa L.)

M S Sujith Kumar, Anil Dahuja, R D Rai, Suresh Walia# and Aruna Tyagi*

Division of Biochemistry, #Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi-110012, India

Received 15 May 2013; revised 08 October 2013

Drought-tolerant cultivars and their phytochemical composition, which has a role in providing drought tolerance are

gaining importance. In this study, rice bran oil and semi-purified oryzanol (SPO) obtained from five rice (Oryza sativa L.)

cultivars, namely P1401 and PB1 (drought-susceptible) and N22, PNR381 and APO (drought-tolerant) were analyzed for

the γ-oryzanol content, an antioxidant present in considerable amount in the rice bran. The higher level of γ-oryzanol and its

antioxidant activity was observed in drought-tolerant cultivars (N22, PNR381 and APO) as compared to drought-susceptible

(PB1 and P1401), suggesting the role of γ-oryzanol in drought tolerance, as antioxidants are known to play an important role

by scavenging free radicals. The total antioxidant activity of γ-oryzanol might be attributed to 24-methylene cycloartanyl

ferulate, a major component of γ-oryzanol. By enhancing the level of active oryzanol components identified in this study by

genetic and molecular means could impart increased drought tolerance.

Keywords: Oryza sativa, γ-Oryzanol, RP-HPLC, Drought tolerance, Rice bran oil

Rice (Oryza sativa L.), one of the most important

crops of the world, has the evolutionary particularity

of being semi-aquatic. As a result, it has relatively

few adaptations to water limited conditions and is

extremely sensitive to drought stress1. With

diminishing water supplies for agriculture worldwide,

the need to improve drought adaptation of rice is

becoming increasingly important2.

The sequence of events in the plant tissue subjected

to drought stress are: (i) increased production of ROS

and of oxidized target molecules, (ii) increase in the

expression of genes for antioxidant functions,

(iii) increase in the levels of anti-oxidative systems

and antioxidants, and (iv) increased scavenging

capacity for ROS, resulting in tolerance against

drought stress3. Lipid peroxidation occurs through a

free radical-mediated chain reaction and can cause a

change in the organoleptic and technological

properties of oils and fats. The initiation phase of this

process can be triggered by inorganic oxygen-derived

initiators or by the presence of trace amounts of

preformed lipid hydroperoxides4. The membrane

lipids are destroyed and they lose their characteristic

properties during stress and this is a potentially

dangerous condition for the rice plant. The plants, as a

part of its defense against oxidative stress produce

antioxidants which help in scavenging these free

radicals and provide tolerance against stress.

The unsaponifiable matter of crude rice bran oil

contain high levels of components with antioxidant

properties: tocopherols/tocotrienols (up to 300 mg/kg)

and γ-oryzanol (up to 3000 mg/kg)5,6

. γ-Oryzanol is

reported to inhibit lipid soluble organic radicals at

concentration of 50-100 µM and has shown

superoxide dismutase like anti-oxidative activity7. Its

protective effect from lipoperoxidation induced by

heating and O2 exposition is interesting, especially in

oils rich in polyunsaturated fatty acids8.

Initially, γ-oryzanol was thought to be a single

compound, but now it is known that it is a mixture of

at least 10 phytosteryl ferulates6. Both sterol and

phenolic hydroxyl groups in its structure may be

responsible for its anti-oxidative function9. The

γ-oryzanol by virtue of its ferulic acid or sterol

component helps in this activity. The γ-oryzanol level

and its components in rice bran oil vary in relation to

environmental conditions. Environment (planting

location or planting year) as well as genetics has been

reported to affect the content of and/or composition of

γ-oryzanol in rice seed10

.

_______________

*Corresponding author:

Tel: 011-25842038.

E-mail: at_bio@iari.res.in

Abbreviations: CUPRAC, cupric ion reducing antioxidant; RBO,

rice bran oil, ROS, reactive oxygen species, SPO, semi-purified

oryzanol.

INDIAN J. BIOCHEM. BIOPHYS., VOL 51, FEBRUARY 2014

76

Although reports are available regarding

nutraceutical properties of γ-oryzanol owing to its anti-

oxidative effects3,8,9,11

, studies are lacking on role of

γ-oryzanol in drought-tolerant vs susceptible cultivars

of rice. Thus, in the present study, we have investigated

γ-oryzanol content in three drought-tolerant (N22 and

APO and PNR381) and two drought susceptible

(P1401 and Pusa Basmati-1) cultivars of rice to find

out cultivar-specific variation in γ-oryzanol content and

individual components of oryzanol in different

cultivars. The total antioxidant activity is also assayed

to ascertain the functional role of γ-oryzanol as an

antioxidant in providing drought tolerance in the rice.

Materials and Methods

Plant material

Two drought-susceptible cultivars — P1401 and

Pusa Basmati-1 and three drought tolerant cultivars —

N22 (an early maturing, deep-rooted adapted to

upland conditions), APO (aerobic suitable for upland

cultivation) and PNR381 (suitable for upland

cultivation) of rice (Oryza sativa L.) were selected.

Seeds (2 kg) of each cultivar were obtained from the

Division of Genetics, Indian Agricultural Research

Institute, New Delhi, India.

Extraction of rice bran oil

Hulling of the whole seeds was carried out using

SATAKE hulling machine. After hulling, the grains

were polished using SATAKE rice polisher for 1 min

to obtain the rice bran, which was used for extracting

the oil by Soxhlet method.

Semi-purification of oryzanol

Semi-purification of oryzanol was done by the method

described earlier6. A glass column (2.5 cm x 25 cm)

packed with 20 g of silica was used to remove the

triglycerides and other lipids. Initially, the crude oil

was solubilized in 50 ml of solvent (hexane/ethyl

acetate = 9:1) for flushing through the column. Then,

50 ml of solvent (hexane/ethyl acetate = 7:3) was

allowed to flow through the column and the eluant

was collected. The column was then washed with

50 ml hexane/ethyl acetate (1:1) and the semi-purified

oryzanol was obtained by evaporating the solvent on a

water bath till the odor of the solvent was lost.

HPLC analysis of crude rice bran oil and semi-purified oryzanol

Analytical reverse-phase HPLC of the crude rice

bran oil was carried out according to method

described earlier6, with slight modification. The

HPLC system consisted of WatersTm

600 controller,

Waters 2996 photodiode array detector and

EMPOWER software. The WatersTm

C18 column was

used and the mobile phase was MeOH/acetonitrile/

CH2Cl2/HOAc (50:44:3:3) and the flow rate was

2 ml/min instead of 1.4 ml/min. CH2Cl2 was used to

dissolve the samples. 40 mg of oil was dissolved in

6 ml CH2Cl2 and 20 µL was injected. Pure γ-oryzanol was gifted by Dr. A G Gopalakrishna

from Central Food Technology Research Institute

(CFTRI, Mysore, India). The standard oryzanol

(200 ppm) was prepared by dissolving 2 mg of pure

oryzanol in 10 ml CH2Cl2. The same procedure was

used for the estimation of oryzanol in semi-purified

oryzanol (SPO) obtained from the RBO.

Estimation of total antioxidant capacity (CUPRAC assay)

For estimation of total antioxidant capacity,

CUPRAC assay was done using the semi-purified

oryzanol obtained from each cultivar as described

earlier12

. CuCl2 solution (10-2

M) was prepared by

dissolving 0.4262 g CuCl2.2H2O in water and diluting

to 250 ml. Ammonium acetate buffer (1 M) was

prepared by dissolving 19.27 g ammonium acetate in

water and diluting to 250 ml. Neocuproine solution

(7.5 mM) was prepared by dissolving 0.078 g

neocuproine in 96% ethanol and diluting to 50 ml.

The reagents are added in the following order: 1 ml

Cu2+

, 1 ml neocuproine, 1 ml ammonium acetate

buffer, 0.5 ml 10-3

M oryzanol solution, 9.5 ml

CH2Cl2 to make final volume to 13 ml. The

absorbance was measured at 450 nm. The antioxidant

capacity was expressed as Trolox equivalent

(mmol TE/g) using the formula:

mmol TE/g = (Af/€TR) (Vf/Vs) r (Vinitial/m)

where Vinitial = initial volume, m = weight of sample,

r = dilution factor, Vf = final volume, Vs = volume of

aliquot, Af = absorbance, €TR = 1.67 * 104 Lmol

-1cm

-1.

Results and Discussion

In this study, we estimated the levels of oryzanol in

drought-tolerant (N22 and APO and PNR381) and

susceptible (P1401 and Pusa Basmati-1) cultivars of rice

in order to explore relationship of γ-oryzanol content

with drought tolerance characteristics of each cultivar.

Bran and oil yield

The whole bran from each cultivar was processed for

extracting the RBO. The whole bran yield from different

cultivars is shown in Table 1 and ranged from 9.94 to

15.94%. Among the five cultivars, maximum bran yield

was obtained from PNR 381, while PB1 gave the least.

KUMAR et al.: ROLE OF γ-ORYZANOL IN RICE CULTIVARS

77

The rice bran generally contains about 12-18% oil11

,

depending upon the cultivar and method used for the

extraction of oil. Generally, crude RBO contains

triglycerides, waxes and other lipids which may be

considered as impurities and are removed using a

solvent extraction method with saponification13

.

However, saponification of RBO is found to hydrolyze

the ester bond between triterpenoids and ferulic acid of

γ-oryzanol6, leading to reduced yield of γ-oryzanol

from RBO. Further, polarity of solvent used for

extraction also significantly affects the extractability of

γ-oryzanol. Hexane/petroleum ether is commonly used

as a solvent to chemically extract oil from rice bran,

however, hexane possesses potential fire, health and

environmental hazards. In the present study, petroleum

ether was used for the extraction of RBO. The oil yield

of different cultivars is given in Table 1 and ranged

between 11.90% in PNR 381 to 14.74% in APO.

Semi-purified oryzanol (SPO) yield in different cultivars

Each oil sample (3 g) was used to obtain the

semi-purified γ-oryzanol. The SPO yield varied from

2.04 to 2.75 g among the cultivars (Table 1). Among

the five cultivars, APO gave the maximum SPO yield

and PB1 the least. Further, the SPO yield of other

drought-tolerant cultivars N22 and PNR 381 was

found to be higher as compared to PB1, a drought-

susceptible cultivar, suggesting the relationship

between oryzanol content with drought tolerance.

Thus, results obtained in this study showed that the

oryzanol content of drought-susceptible cultivars was

considerably lower as compared to drought-tolerant

cultivars, indicating that oryzanol plays an important

role in imparting drought tolerance characteristics.

Reverse phase-HPLC of oryzanol

The reverse phase (RP)-HPLC of standard oryzanol

(200 ppm) gave four peaks at 330 nm (Fig. 1). All the

five cultivars gave their characteristic chromatograms

with four prominent peaks. The experimental

conditions used in our study were based on earlier

report6 and the retention time for the peaks was also

similar. This indicated that the four peaks (1, 2, 3 and 4)

obtained in present study correspond to cycloartenyl

ferulate, 24-methylenecycloartanyl ferulate, campesteryl

ferulate and sitosteryl ferulate, respectively as

identified in earlier report6.

The retention time of peaks of the components of

γ-oryzanol in RP-HPLC mainly depends upon the

number and position of double bonds6. In the present

study, retention time of components containing two

double bonds was found to be less than those having

Table 1—Bran and oil yield of different rice cultivars

Cultivars Bran %

Oil %

SPO

(g ± SE)/g oil

P1401 10.05 12.95 2.38 ± 0.04

N22 10.4 14.65 259 ± 0.04

APO 11.22 14.74 2.75 ± 0.04

PNR381 15.94 11.90 2.47 ± 0.06

PB1 9.94 13.57 2.04 ± 0.13

Fig. 1—Oryzanol content after semi-purification [Four peaks (1, 2, 3 and 4) obtained in present study correspond to cycloartenyl ferulate,

24-methylenecycloartanyl ferulate, campesteryl ferulate and sitosteryl ferulate, respectively]

INDIAN J. BIOCHEM. BIOPHYS., VOL 51, FEBRUARY 2014

78

one or no double bond. Further, components having a

double bond on a side chain eluted prior to those

having a double bond in the triterpene. Thus, the

number and position of double bond might influence

polarity in a molecule, which in turn determined the

retention time of that molecule during RP-HPLC.

Earlier, the above-mentioned components have

been suggested as the major components of oryzanol

by many other workers14-17

. However, the nature of

individual components needs to be reconfirmed using

mass spectroscopy. In most of the chromatograms, the

peak 2 corresponding to 24-methylene cycloartanyl

ferulate was the principal component. In N22 and

PNR381, peak 1, which corresponds to

cycloartenylferulate was major peak. But, in all cases,

peak 4 (sitosteryl ferulate) was the least observed

component. Thus, cycloartenyl ferulate and

24-methylene cycloartanyl ferulate which have been

earlier reported as principal components of oryzanol6,14-16

were observed at higher levels in drought-tolerant

cultivars. Furthermore, 24-methylene cycloartanyl

ferulate has been shown to be a more effective inhibitor

of free radical initiated cholesterol oxidation in vitro

than cycloartenyl or campesteryl ferulate17

.

However, further studies are required to evaluate

the bioactivity of individual components of oryzanol

and the relative importance of free phytosterol versus

esterified ferulic acid. It is possible that the benefit

from oryzanol derives from ferulic acid and that the

major impact of steryl component is to affect the

bioavailability18

. In rice, sterols are esterified,

especially with ferulic acid. In one study, oryzanol

and phytosterols in rice seeds are reported to be

present in approximately equal amounts19

, suggesting

bound form of sterol as major one compared to free

sterols. Phytosterols are found mainly in plant cell

walls and membranes. Abiotic stresses, such as water

deficit stress affect the relative composition of sterols

in ways likely to stabilize membranes20

.

The comparison of oryzanol content in bran, crude

RBO and SPO obtained from all five cultivars with

standard oryzanol showed four similar peaks in each

cultivar. However, area under the peaks was different

in the cultivars, indicating the different levels of the

corresponding components. The oryzanol content in

crude rice bran oil ranged from 1.53% in P1401 to

2.63% in PNR 381 (Fig. 2). RP-HPLC revealed

higher oryzanol content in tolerant cultivars N22,

APO and PNR 381 i.e. 2.39%, 2.53% and 2.63%, as

compared to susceptible ones i.e. PB1 1.76% and

Fig. 2—Oryzanol content of bran and rice bran oil (RBO)

obtained from different rice cultivars

Fig. 3—Oryzanol content of semi-purified oryzanol (SPO)

obtained from different rice cultivars

Fig. 4—Total antioxidant capacity of SPO obtained from different

rice cultivars by CUPRAC assay

KUMAR et al.: ROLE OF γ-ORYZANOL IN RICE CULTIVARS

79

P1401 1.53%, respectively. The average oryzanol

content of five cultivars was 2.17% in crude rice bran

oil and 0.29% in bran. Quantification of oryzanol

from bran also revealed a similar trend. The tolerant

cultivars N22, APO and PNR 381 had higher oryzanol

content per mg bran as compared to susceptible ones

i.e. PB1 and P1401 (Fig. 2). These results were in

accordance with previous reports10,16

. Importantly,

P1401 being a drought-susceptible cultivar had the

least amount of oryzanol and PNR381, a tolerant

cultivar showed maximum oryzanol content. APO,

another tolerant cultivar showed maximum oryzanol

recovery from bran (0.36%), when compared to

P1401 (0.19%).

Further, RP-HPLC also revealed higher levels of

SPO in drought-tolerant cultivars. RP-HPLC of SPO

from the five cultivars under similar experimental

conditions (Fig. 3) showed oryzanol content ranging

from 1.68% in P1401 to 3.14% in PNR381 with an

average of 2.48%. Values in terms of oryzanol mg/g

SPO were higher for drought-tolerant cultivars i.e.

N22, APO and PNR 381, as compared to susceptible

ones i.e. PB1 and P1401 (Fig. 3). Thus, the results

showed that the oryzanol content of drought-

susceptible cultivars was considerably low, compared

to drought-tolerant cultivars.

Functional role of SPO

The functional role of γ-oryzanol as an antioxidant

was studied. The γ-oryzanol content was compared

with total antioxidant capacity of the studied rice

cultivars. The SPO obtained from the five cultivars

was used for the estimation of their total antioxidant

capacity (Fig. 4). The antioxidant activity was

recorded in terms of µmol Trolox equivalent (TE)/g11

.

Trolox equivalent antioxidant capacity (TEAC) was

defined as the mM concentration of a Trolox solution

having the antioxidant capacity equivalent to 1 mM

solution of the substance under investigation. The

antioxidant activity of pure oryzanol was found to be

29.13 µmol TE/g. The highest antioxidant activity

(23.76 µmol TE/g) was observed in N22, while the

lowest (14.01 µmol TE/g) was recorded in P1401.

Other three cultivars, namely APO, PNR381 and PB1

showed 20.42, 20.07, 19.21 µmol TE/g, respectively.

In general, drought-susceptible cultivars (PB1 and

P1401) showed lower antioxidant capacity, compared

to drought-tolerant cultivars (N22, APO and

PNR381). A good correlation was observed between

antioxidant capacity of these rice cultivars and their

γ-oryzanol content, as revealed by RP-HPLC.

The γ-oryzanol, a chain-breaking antioxidant, is

known to scavenge reactive oxygen species generated

due to changes in photosynthetic electron transport,

since the molecular oxygen competes with NADP+ for

reduction at the acceptor side of photosystem l (PSl)8.

The protective effect of oryzanol from

lipoperoxidation induced by O2 exposition is more in

lipids rich in polyunsaturated fatty acids. The

membrane lipids associated with plants are known to

be rich in galactolipids which contain polyunsaturated

fatty acids. Oryzanol can scavenge organic radicals at

lower concentrations20

. In addition to the radical

scavenging activity, both its polarity and

three-dimensional interactions with lipid bilayers

might participate in stabilizing the membranes during

oxidative stress21

. A good correlation between

antioxidant capacity and γ-oryzanol content suggested

a potential linkage between drought tolerance and the

antioxidant activity of the rice cultivars. The good

antioxidant/anti-polymerization activity of rice bran

oil might be due to other minor antioxidant

components, such as avenasterols/tocopherols in

addition to oryzanol22,23

.

To conclude, higher γ-oryzanol content and

antioxidant activity in drought-tolerant cultivars

suggested role of γ-oryzanol in drought tolerance, as it

might be involved in free radical scavenging and

membrane stabilization which are manifestations

associated with drought. By enhancing the level of

active oryzanol components identified in this study by

genetic and molecular means could impart increased

drought tolerance.

Acknowledgements Authors thank Dr. A G Gopalakrishna, Central

Food Technology Research Institute (CFTRI),

Mysore for providing the pure γ-oryzanol. We also

thank Mr. Eldho Verghese, Indian Agricultural

Statistics Research Institute (IASRI) for helping with

the statistical analysis of data and Indian Council of

Agricultural Research (ICAR) for financial assistance.

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