Indian Journal of Exprimental Biology
Vol. 52, February 2014, pp. 153-158
Role of Triticum aestivum aqueous extract in glucocorticoid
induced osteoporosis in rats
David Banji*, Otilia J F Banji, Vijaya Laxmi Chiluka & Saidulu Abbagoni
Department of Pharmacology, Nalanda College of Pharmacy, Charlapally,
Nalgonda 508 001, India
Received 3 August 2012; revised 8 November 2013
Administration of aqueous extract of T. aestivum (200 and 400 mg/kg/day, po, for 30 days) and risedronate (20 µg/kg, sc,
five times a week for 30 days) following methyl prednisolone sodium succinate (10 mg/kg, sc, thrice a week for 4 weeks)
induced osteoporosis in Wistar rats showed an increase in the serum levels of bone mineral content markers, decrease in the
serum and urinary levels of bone resorption markers. An incline in strength of femur and tibia was seen particularly with
400 mg/kg of T. aestivum. Maintenance of calcium homeostasis, formation of collagen and scavenging of free radicals can
plausibly be the mode of action of aqueous extract of T. aestivum thereby combating osteoporosis induced by
glucocorticoids.
Keywords: Bone resorption, Free radicals, Glucocorticoids, Neutraceutical, Wheat grass
Glucocorticoids are steroidal hormones possessing
wide applicability and are extensively used for the
treatment of autoimmune disorders, pulmonary
disorders, malignancy, and organ transplantation1.
Glucocorticoids exert their action by binding to
glucocorticoid receptors and regulate tissue
differentiation, control intermediary metabolism and
enable the body to cope with stress. Long-term
treatment or high doses adversely affects bone health
and is considered to be a leading cause of drug
induced osteoporosis2. Osteoporosis is a chronic
bone disorder characterized by low bone mass and
deterioration of the micro architecture of bone tissue2.
Maintenance of bone health requires a healthy diet.
Steroids deplete minerals causing an alteration in the
mechanical and structural integrity of skeletal tissue3.
Neutraceuticals are widely employed to curb the
spread of diseases owing to the presence of several
antioxidant principles. Triticum aestivum (family:
Poaceae) seedlings commonly called as wheat grass
is consumed in the form of juice for healthy growth
of human body4. Wheat grass is used to reduce
ulcerative colitis5, and myelotoxicity6. It is reported
to be beneficial in mitigating thalassemia and
——————
* Correspondent author
Teephone: 91-8682-273910
Fax: 91-8682-248547
E-mail:[email protected]
possesses tremendous anti-oxidant potential7,8.
T. aestivum seedlings are a rich source of minerals
like magnesium, potassium, calcium, phosphorous,
manganese, iron, zinc and vitamins like A, C, D, and E9.
T. aestivum is rich in nutritional constituents and
has not been explored for its potential in maintaining
skeletal health. Therefore, the present study has been
undertaken to investigate the effect of aqueous extract
of T. aestivum on osteoporosis induced by methyl
prednisolone sodium succinate, a glucocorticoid (GC)
in rats by biochemical and biomechanical assessment.
Materials and Methods
Drugs and chemicals—Methyl prednisolone
sodium succinate and risedronate sodium were
procured from Sigma Aldrich, USA. The reagent kits
for the measurement of calcium (Ca), inorganic
phosphate (P), alkaline phosphatase (ALP) and
creatinine (Cr) were procured from ERBA
Diagnostics (Transasia Bio-medicals Ltd., Daman,
India). All other reagents and chemicals were of
analytical grade.
Plant material—Fifteen day old seedlings of
T. aestivum were collected in the month of
December in Nalgonda. They were authenticated by
Prof. A Laxman Reddy, N.G. College, Nalgonda and
a voucher specimen is maintained in the herbarium.
Ariel parts of 15 day old seedlings were dried
in shade and made into fine powder. Dried powder
154
INDIAN J EXP BIOL, FEBRUARY 2014
(100 g) was soaked in 300 mL of water for 30 min
and then shaken on a gyratory shaker for 6 h at
70 gyrations. The solution was filtered under vacuum
and the filtrate was concentrated under vacuum. The
concentrated product was used as aqueous extract of
T. aestivum (AETA) and stored in refrigerator for
future use. The percentage yield of aqueous extract of
T. aestivum was 8% (w/w).
Phytochemical screening—Qualitative analysis of
the aqueous extract of T. aestivum was performed to
detect the presence of phytoconstituents such as
carbohydrates, amino acids, proteins, tannins, and
flavonoids. Thin layer chromatography was
performed to detect the presence of ascorbic acid
using ethanol:acetic acid:water in the ratio of
9:0.1:0.9 as the solvent system and detected in the
short wavelength region of UV. Detection of amino
acids in AETA was performed by using a solvent
system of n-butanol:acetic acid:water in the ratio of
4:1:1 and developed using ninhydrin reagent10.
Determination of chlorophylls a and b—
Methanolic solutions of wheat grass ranging in
1.0-4.0 mg/mL concentration were analyzed for
the presence of chlorophyll. The determination of
chlorophyll was done by recording the absorbance at
653 and 666 nm using a UV/VIS spectrophotometer.
The determinations were carried out in triplicate and
the concentration of chlorophyll α and b in wheat
grass powder were determined from the formula
proposed by Lichtenthaler and Wellburn11:
Chlorophyll a (mg/L) = 15.65 Abs666 – 7.340 Abs653
Chlorophyll b (mg/L) = 27.05 Abs653 – 11.21 Abs666
Determination of total carotenoids—Methanolic
solutions of wheat grass (1.0-4.0 mg/mL) were
analyzed for the presence of total carotenoids.
The absorbance was recorded at 470 nm using a
UV/VIS spectrophotometer. The total carotenoid
content was determined from the sample in triplicate
and calculated based on the formula proposed by
Lichtenthaler and Wellburn11
Total carotenoids (mg/L) =
1000 Abs470 – 2.860 Ca – 129.2 Cb/245
where Ca is chlorophyll a and Cb is chlorophyll b.
Ferric reducing capacity of plasma (FRAP)
assay—Total antioxidant activity of aqueous extract
of T. aestivum was evaluated. By mixing 100 µL of
AETA with 3 mL of working FRAP reagent and
absorbance (593 nm) was measured at 0 min after
vortexing. Thereafter, samples were placed at
37 °C in water-bath and absorbance was again
measured after 4 min. Ascorbic acid as a standard
(100-1000 µM) was processed in the same way12.
Safety evaluation and dose selection—Safe dose of
aqueous extract of T. aestivum was evaluated
according to OECD guidelines number 40713.
Animals—Young male rats (30) of Wistar strain
weighing 150-250 g were procured from National
Institute of Nutrition, Hyderabad. The experimental
animals were housed in a temperature controlled
room (24±1 oC) with 12:12 h L:D illumination
cycle. All animals were allowed free access
to distilled water and fed on a commercial diet.
The experimental protocol has been approved
by the Institutional Animal Ethical Committee
(NCOP/IAEC/approved/18/2010).
Induction of osteoporosis and experimental
design—Animals were divided into following five
groups of 6 rats each. Gr. I were negative control
animals treated with saline. Osteoporosis was
induced in male rats by injecting methyl prednisolone
sodium succinate, a glucocorticoid (GC) in a dose
of 10 mg/kg thrice a week for four weeks by
subcutaneous route14; Gr. 2 animals received GC
alone; Gr. 3 were GC treated animals which received
risedronate sodium (20 µg/kg, sc) as a standard,
five times a week for the next 30 days; Gr. 4 were
GC treated animals which received the aqueous
extract of T. aestivum (200 mg/kg) daily by the
oral route for the next 30 days; Gr. 5 were GC treated
animals which received aqueous extract of
T. aestivum (400 mg/kg) daily by oral route for the
next 30 days.
Biochemical analysis—After 60 days, rats were
anaesthetized with enflurane and blood was
withdrawn from the retro orbital plexus and collected
into dry test tubes. It was centrifuged at 3000 rpm for
10 min for the separation of serum. The separated
serum was used for biochemical analysis.
Bone mineral content markers—Calcium was
estimated by OCPC method15 in which it complexes
with o-cresolphthalein complexone and phosphorous
was estimated by molybdate UV method16.
Bone resorption
markers—Creatinine
was
estimated by Jaffe’s kinetic method17. Serum specific
alkaline phosphatase was estimated by PNPP
kinetic method18 in the serum using standard kits
with a semi auto analyzer (Erba Chem 5 Plus V2).
BANJI et al.: TRITICUM AESTIVUM & GLUCOCORTICOID INDUCED OSTEOPOROSIS IN RATS
Hydroxyproline was estimated in urine using
modified Switzer and Summer19. The levels of
serum chemistries of bone mineral content and
bone resorption markers were measured using a
semiautomatic analyzer (ERBA Chem-5 Plus V2,
Transasia Bio-Medicals Ltd, Mumbai).
Physical and biomechanical parameters—Animals
were sacrificed on 60th day after blood withdrawal.
Femur and tibia were isolated, defleshed and
immediately weighed. The length of bones was
determined using dial calipers. Assessment of their
biomechanical strength was done by using Universal
testing machine (UTE 40 HGFL; Fuel Instruments
and Engineers Ltd).
Three point bending test of femur and tibia—The
bones were placed horizontally on two supporting
bars separated at a distance of 15 mm and was loaded
by a rounded press. Bones were subjected to test with
loading at a speed of 1 mm/min each mm
corresponding to 40 Newton (N) perpendicular to the
bone longitudinal axis. Variables estimated were yield
load which is the force causing the first detectable
bone damage and ultimate load which is the force at
which fracture occurs20.
Compression test of femur and tibia—The bones
were placed on the flat metallic stage and compressed
until it fractured. The reading was recorded in
Newtons21.
Statistical analysis—In vitro data are expressed as
mean±SD. In vivo data are expressed as mean±SE.
Analysis of data was done by One-way ANOVA
followed by Dunnett multiple comparison tests using
Graph Pad InStat version 3.10 for Windows 2009
(Graph Pad Software). The statistical significance was
set as 0.05 (P<0.05).
155
Results
Phytochemical screening—The presence of
carbohydrates, amino acids, proteins, tannins, and
flavonoids were detected in AETA. Three amino
acids were clearly detected in the AETA with
Rf values of 0.5, 0.76 and 0.47 which coincided
with the Rf values of standard amino acids leucine,
tyrosine and asparagine. The presence of ascorbic
acid was detected in the aqueous extract of T. aestivum
with an Rf value 0.75 which was similar to standard
ascorbic acid.
In vitro evaluation of wheat grass powder—The
content of chlorophyll a in wheat grass was found
to be 0.66±0.03 mg/g dry weight and the content
of chlorophyll b was 0.15±0.02 mg/g dry weight.
The total carotenoid content was 1.02±0.07 mg/g.
FRAP assay—Antioxidant activity of 1 mg/mL
of AETA was equivalent to 300 µ mol/L of ascorbic
acid.
Safety evaluation—AETA was found to be safe up
to a dose of 2000 mg/kg body weight.
Effect of AETA on biochemical parameters—GC
induced biochemical changes by reducing serum
calcium and phosphorus levels to 8.3±1.0 and
4.03±0.78 mg/dL respectively which was reversed by
risedronate. AETA (400 mg/kg) caused a significant
rise in serum calcium and phosphorus compared
with GC treated group (P<0.05, Table. 1). GC
produced an elevation in levels of alkaline
phosphatase and creatinine. Treatment with
risedronate and AETA (400 mg/kg) evoked a
reduction in the levels of alkaline phosphatase and
creatinine compared with GC treated group (P<0.05).
GC treated group exhibited a significant rise in
Table 1—Effect of aqueous extract of T. aestivum on biochemical parameters in osteoporosis induced by GC in rats
[Values are mean ± SE from 6 rats each]
Biomarker
Gr 1
Control
Gr 2
GC
Gr 3
Res
Calaium
10.6±0.9
8.3±1.0*
10.4±0.07#
(mg/dL)
Phosphorous
5.0±0.65
4.03±0.78*
4.8±0.87#
(mg/dL)
Alkaline Phosphatase
129±2.0
262.5±2.4*
223.0±2.1*#
(U/L)
Creatinine
0.42±0.01
0.64±0.04*
0.50±0.02#
(mg/dL)
Hydroxyproline
15.8±1.2
17.0±0.68
16.3±0.57
(mmol/L)
P<0.05 as compared with the control, # GC treated group.
Res-Residronate, AETA-Aqueous extract of T. aestivum, GC-Glucocorticoid
Gr 4
AETA
(200 mg/kg)
9.2±0.79#
Gr 5
AETA
(400 mg/kg)
9.7±1.05#
4.45±0.6*
4.7±0.34#
255.6±2.06*
243.6±1.8*#
0.58±0.006*
0.47±0.007#
16.4±0.54
14.03±1.06
#
156
INDIAN J EXP BIOL, FEBRUARY 2014
urinary hydroxyproline levels compared to negative
control (P<0.05). Treatment with risedronate and
AETA produced a decline in the levels of urinary
hydroxyproline compared to GC treated group
(P<0.05, Table.1).
Effect of AETA on physical and biomechanical
parameters—A significant decrease in femur and
tibia weight and length was observed in methyl
prednisolone treated rats compared to negative control
group (P<0.05). Bone weight and length significantly
increased on treatment with residronate, and AETA
respectively compared with GC treated group
(P<0.05, Fig. 1a and b).
Three point bending test of femur and tibia—The
ultimate load tolerated by femur and tibia in rats
treated with GC declined to in the three point bending
test and compression test compared with the control
Fig. 1—Effect of the aqueous extract of T. aestivum on (a) bone
weight and (b) bone length in GC induced osteoporosis in rats.
[Values are mean±SE from 6 rats each. GC=glucocorticoid,
Ris=risedronate, AETA= aqueous extract of T. aestivum.
P values: *<0.05 compared with control, #<0.05 compared with
GC treated group].
(P<0.05). Load tolerated by risedronate treated rats
increased in both the three point and compression test.
Maximum load required to fracture the femur and
tibia increased significantly following treatment with
AETA (400 mg/kg) compared with GC treated group
(P<0.05, Fig. 2a and b).
Discussion
The present study evaluated the effect of the
aqueous extract of T. aestivum on osteoporosis
induced by glucocorticoids as they alter skeletal
integrity by affecting bone metabolism, reduce the life
span of osteoblasts and inhibit osteoblastogenesis22.
Glucocorticoids create an imbalance in the rhythm
between bone formation and bone resorption, thereby
increasing the risk of fractures23. Glucocorticoids
hinder the synthesis of collagen and affect
differentiation of osteoblasts causing rapid bone
Fig. 2—Effect of the aqueous extract of T. aestivum on
biomechanical strength using (a) three point test and
(b) compression test in GC induced osteoporosis in rats
[Values are mean±SE from 6 rats each. GC=glucocorticoid,
Ris-risedronate, AETA=aqueous extract of T. aestivum.
P values: * <0.05 compared with control, #<0.05 compared with
GC treated group].
BANJI et al.: TRITICUM AESTIVUM & GLUCOCORTICOID INDUCED OSTEOPOROSIS IN RATS
loss24,25. Owing to this, glucocorticoids are considered
ideal candidates for experimental induction of
osteoporosis in rodents.
Calcium salts in bone are embedded in collagen
fibrils of which 13% is hydroxyproline26. During bone
loss, breakdown of collagen fibrils occurs, resulting
in the loss of hydroxyproline in urine and is
considered to be an index of bone resorption27. AETA
was found to substantially reduce the excretion of
hydroxyproline thereby maintaining bone structure.
Treatment with AETA produced a significant decline
in the levels of serum alkaline phosphatase in
the osteoporosis induced animals. Serum alkaline
phosphatase is a biochemical marker of bone
turnover, good indicator of internal bone activity and
is used to monitor metabolic bone disease28.
Calcium is abundant in bone and is essential
to maintain bone mineral density. A significant
reduction in the level of calcium was observed
on treatment with GC. This may be because GC
enhances urinary excretion of calcium and reduces
its intestinal absorption. The levels of phosphate
in serum were also significantly altered in the GC
treated group. Decline in the levels of phosphate
could be due to enhanced renal excretion and
alteration in their transport across the brush border
membrane. Treatment with AETA produced
an appreciable increase in the levels of calcium and
phosphorous. AETA contains calcium which may
contribute to the maintenance of calcium homeostasis
and potassium which buffers metabolic acid loads
as activation of osteoclasts occurs due to lowering
of pH29. Minor minerals like zinc and iron may be
responsible for activation of osteoblast enzymes and
collagen hydroxylation30.
Bone mineral density is dependent on the supply
of number of dietary factors like minerals
and vitamins31. Formation of collagen which is a
prerequisite for normal bone development relies on
several nutrients32. Studies reveal that 15-day old
seedlings of T. aestivum are power houses of minerals
like calcium, potassium, phosphorous, magnesium,
iron, zinc and copper9. Abundant presence of ascorbic
acid or vitamin C has been reported in the seedlings
of T. aestivum9; therefore the aqueous extract of
15-day old seedlings of T. aestivum was utilized in
this study. Vitamin C is essential for the formation of
type I collagen and healthy bones. Collagen needs to
undergo hydroxylation so as to retain its structural
integrity33. Ascorbic acid serves as an essential
157
coenzyme required to convert proline to
hydroxyproline which plays a pivotal role in
maintaining collagen structure. In addition, decreased
bone density and increased bone loss has also
been associated with low protein diet34. Proteins
present in AETA could promote bone health
presumably by both these mechanisms.
Biomechanical tests have enabled us to discern
the effect of AETA on the femur and tibia of
osteoporotic animals. Physical parameters show
increased weight and length of femur and tibia in
AETA treated animals indicating improvement in
the structure of the weight bearing bones.
Biomechanical studies revealed improvement in the
integrity of skeletal structures with AETA thereby
reducing the risk of osteoporosis.
Free radicals enhance bone loss, by damaging the
osteoblast cells35. In vitro tests revealed the
antioxidant potential of AETA which may help in the
restoration of bone formation.
In conclusion, AETA has substantial capability
of reducing the risk of glucocorticoid induced
osteoporosis. Therefore supplementation of diet
with T. aestivum as a nutrient will be economically
justifiable as compared to the high cost of current
therapies.
Acknowledgement
Support by Nalanda Education Society, Nalgonda508001, India is acknowledged.
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