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The anti-inflammatory potential of Spinacia oleracea leaf extract

Authors:
  • DSKM College of Pharmacy, Bhopal, India

Abstract

Background: Spinacia oleracea is widely used as food and in the treatment of urinary calculi, as a laxative, in difficult breathing, inflammation of the liver, and jaundice. However, no studies have been done on its anti-inflammatory activity. Materials and Methods: To evaluate the anti-inflammatory activity of the methanolic aqueous fraction (MAF and water extract (WE) of Spinacia oleracea leaf in an acute inflammation model. was administered in rat hind paw, in chronic inflammation model, cotton pellet–induced granuloma was performed. Results: The water extract of Spinacia oleracea and its methanolic aqueous fraction at 600 mg/kg dose showed significant (p < 0.001) inhibition of inflammation in both acute and chronic anti-inflammatory models. The potency of the extracts was compared with the standard diclofenac sodium (5 mg/kg). The methanolic aqueous fraction and water extract showed a dose-dependent increase and decrease in catalase content. The highest protein content was found in the water extract.
Journal of Natural Pharmaceuticals, Volume 2, Issue 2, April-June, 2011
80
Address for
correspondence:
Asst. Prof. Alok Kumar
Shukla,
Radharaman College of
Pharmacy, Radharaman
Group of Institutions,
Bhadbada Road,
Ratibad, Bhopal –
462002, Madhya
Pradesh, India.
E-mail:alokshukla51@
rediffmail.com
Radharaman College of
Pharmacy, Radharaman
Group of Institutions, Ratibad,
Bhopal, Madhya Pradesh,
India
Anti-in ammatory potential of Spinacia oleracea leaf extract
Anil Nagar, Alok Kumar Shukla, Papiya Bigoniya
ABSTRACT
Background: Spinacia oleracea is widely used as food and in the treatment of urinary calculi, as a laxative,
in dif cult breathing, in ammation of the liver, and jaundice. However, no studies have been done on its anti-
in ammatory activity. Materials and Methods: To evaluate the anti-in ammatory activity of the methanolic
aqueous fraction (MAF and water extract (WE) of Spinacia oleracea leaf in an acute in ammation model.
was administered in rat hind paw, in chronic in ammation model, cotton pellet–induced granuloma was
performed. Results: The water extract of Spinacia oleracea and its methanolic aqueous fraction at 600 mg/
kg dose showed signi cant (P < 0.001) inhibition of in ammation in both acute and chronic anti-in ammatory
models. The potency of the extracts was compared with the standard diclofenac sodium (5 mg/kg). The
methanolic aqueous fraction and water extract showed a dose-dependent increase and decrease in catalase
content. The highest protein content was found in the water extract.
Key words: Carrageenan, cotton-pellet granuloma, in ammatory, Spinacia oleracea
INTRODUCTION
In ammation is de ned as the local response
of living mammalian vascularized connective
tissue to the injury caused by various
exogenous and endogenous stimuli. It is a
body defense reaction in order to eliminate
or limit the spread of injurious agents as well
as to remove the consequent necrosed cells
and tissues.[1] In ammation helps to clear
the infections and along with the repair it
makes wound healing possible, both have
considerable potential to cause harm. For
example, in ammatory reactions underlie
life-threatening anaphylactic responses to
insect bites or drugs, as well as, chronic
diseases such as rheumatoid arthritis and
atherosclerosis. An initial inflammatory
stimulus triggers the release of chemical
mediators from plasma or cells, which
then regulate the subsequent vascular and
cellular responses.[2] Plants had been used
for medicinal purposes long before recorded
history. Ancient Chinese and Egyptian
papyrus writings describe medicinal uses
of plants. African and Native American
indigenous cultures used herbs as healing
rituals, while others developed traditional
medical systems (such as Ayurveda and
Traditional Chinese Medicine), wherein
herbal therapies were used. Researchers
found that people in different parts of the
world have a tendency to use the same or
similar plants for the same purpose. There
are so many plants which possess reported
anti-in ammatory activity like Curcuma
longa Linn., Glycyrrhiza glabra Linn.,
Cyperus rotundus Linn., Tylophora indica
Linn., Berberis aristata DC., Mesua ferra
Linn., and Psoralea corylifolia Linn.[3]
Spinacia oleracea Linn. is commonly
known as “Spinach” belonging to family
Chenopodiaceae and is native to central and
southwestern Asia. Spinach is a favorite
vegetable among Indians in the winter
season and is a dietary powerhouse, full of
vitamins and minerals such as vitamin A, C,
D, K, and folic acid. The leaves have been
used in the treatment of febrile conditions,
inflammation of lungs, and bowel. The
seeds are laxative and cooling. The leaves
of spinach are demulcent or soothing agents,
diuretic, and laxative.[4-6] They have been
used in the treatment of dif cult breathing,
inflammation of the liver, and jaundice.
It cools and moistens and promotes the
urine ow. The edible leaves are rich in
minerals, particularly in iron and calcium,
and are recommended for anemic persons.
Original Article
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DOI:
DOI: 10.4103/2229-5119.83961
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The present study was undertaken to evaluate the
anti-in ammatory activity of Spinacia oleracea in an
experimental rat model considering its antioxidant
property.
MATERIALS AND METHODS
Plant Materials
Fresh, young, healthy leaves of Spinacia oleracea were
collected from the local market of Bhopal (Madhya
Pradesh) during the winter season (November, 2009).
The leaves were authenticated by Dr. Madhuri Modak,
Department of Botany, M.V.M., Bhopal, and a voucher
specimen (No. Ch, 2010) was kept for future reference.
Preparation of Extract
Water Extract
Weighed powdered leaves (200 g) were extracted with
double distilled water at room temperature for 24 hours.
The extractive solution was ltered with Whatman lter
paper and vaccum dried at 40 – 50oC. The obtained crude
water extract (brown colored sticky mass) was stored
in the refrigerator. The percentage yield of the water
extract was 23.59%.
Methanolic Aqueous Fraction
Weighed powdered leaves (200 g) were defatted with
petroleum ether (40 – 60oC) for 72 hours in a Soxhlet
apparatus. The marc was dried to remove traces of
petroleum ether. The defatted plant material was again
subjected to hot continuous extraction with methanol
in the Soxhlet apparatus for 36 hours. The methanolic
extract was then evaporated under reduced pressure in a
rotary evaporator and concentrated to dryness and stored
in a cool dark place. The dried extract was dissolved in
distilled water and passed through a Whatman lter
paper. Following ltration, the ltrate was dried on water
bath below 50oC and weighed again. The percentage yield
of MAF was 23.06%.
Animals
Male albino rats weighing between 150 and 200 g were
used in the experiments. The animals were placed
randomly and allocated to different treatment groups.
The animals were housed in polypropylene cages with
paddy husk bedding at a temperature of 22 ± 2oC and
relative humidity of 65 ± 5%. A 12: 12 hour, light: dark
cycle was followed. All the animals were allowed free
access to water and feed, the standard commercial
pelleted chaw (Hindustan liver). All the experimental
procedures and protocols used in this study were reviewed
by the Institutional Animal Ethics Committee (IAEC) of
the Radharaman College of Pharmacy (Approval number
IAEC/RCP/2010/12) and were in accordance with the
guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA),
Chennai, India.
Acute toxicity studies (LD50)
Acute Oral Toxicity (AOT) of MAF and WE were
determined using Swiss albino mice. The animals were
fasted for three hours prior to the experiment and were
administered a single dose of extracts suspended in 5%
gum acacia (the dose ranged from 500 – 2000 mg/kg at
various dose levels), and observed for mortality up to
48 hours (short-term toxicity). Based on the short-term
toxicity, the dose of the next animal was determined as
per the Organization for Economic Co-operation and
Development (OECD) guideline 423.[7] All the animals
were also observed for long-term toxicity (14 days).
Phytochemical Analysis
The extracts of Spinacia oleracea were subjected to the
phytochemical tests for the presence of carbohydrates,
avonoids, tannins, phenolic compounds, saponins, and
steroids.[8]
Chemicals and Reagents
Chemicals used in the present study were Autozyme
total protein reagent (Accurex Biomedical Pvt. Ltd.,
Mumbai), Diclofenac sodium (Biochem Pharma, Mumbai),
Carrageenan (S.D. Fine Chemicals Limited, Mumbai),
Nitro-blue tetrazolium (NBT), and Hydroxylamine
hydrochloride, (S.D. Fine Chemicals Limited, Mumbai).
All other chemicals and reagents used were of analytical
grade.
Experimental design
The rats were divided into the control group (Group – I),
standard drug-treated group (Group – II), MAF-treated
group (Groups – III, IV, V), and the WE-treated group
(Groups – VI, VII, VIII) comprising of six in rats each.
Group – I rats received only the vehicle (5% gum acacia,
1 ml/100 gm). Rats of Group – II were treated orally with
5 mg/kg of diclofenac sodium as a standard drug. Rats
of Group – III to Group – VIII were treated orally with
(200, 400, 600 mg/kg) MAF, and WE, respectively, by
orogastric cannula.
Carrageenan–induced rat paw edema
The MAF, WE, diclofenac, and vehicle were administered
to the animals of the respective groups, one hour prior to
the injection of 0.1 mL of 1% freshly prepared carrageenan
in normal saline in the sub-planter region of the right
hind paw. The paw volume was measured initially
and then in the rst, second, and third hours after the
Carrageenan injection, by using the Plethysmometer
(Jyoti Scienti c, India). The anti-in ammatory effect of
MAF and WE were calculated by the following equation:
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Journal of Natural Pharmaceuticals, Volume 2, Issue 2, April-June, 2011
82
% = 100
ct
c
VV
Edema Inhibition V
Where, Vt represents the paw volume in drug-treated
animals and Vc represents the paw volume of the control
groups animals.[9]
Cotton pellet–induced granuloma
The rats were anesthetized with Ketamine (50 mg/kg,
ip) and sterile cotton pellets weighing 20 ± 1 mg were
implanted subcutaneously into the groin region of each
rat. Vehicle, MAF, WE, and diclofenac were administered
orally to the animals of the respective groups for seven
consecutive days, starting from the day of cotton pellet
implantation. On the eighth day, the animals were
anesthetized and the implanted cotton pellets were
carefully dissected and made free from the extraneous
tissues. The wet pellets were dried in an oven at 60°C
for 24 hours and weighed. Increment in the dry weight of
the implanted pellets was taken as a measure of formed
granuloma weight. The anti-proliferative effect of MAF
and WE was compared with the control.
% = 100
ct
c
WW
Inhibition W
Where, Wc represents the pellet weight of the control
group animals and Wt represents the pellet weight of
the drug-treated group animals.[10]
Biochemical estimation
The collected, dried, and weighed, granulation mass
was placed in sealed tubes containing 10 ml of 6 N HCL.
The sealed tubes were heated at 110°C for 24 hours to
hydrolyse these tissues. The hydrolysate was cooled and
excess acid was neutralized by titration with 10 N NaOH,
using methyl red as an indicator. The volume of neutral
hydrolysate was made up to 20 ml with distilled water.
This was used to estimate catalase, superoxide dismutase
(SOD) and protein content.
Estimation of the catalase
The catalase was determined following the method of
Aebi[11] as H2O2 decomposition. The reaction mixture
contains hydrolysate (20 μl) and a phosphate buffer (0.1
M, pH 7.2, 2 μl). The reaction was initiated by adding 1
ml of 30 mM H2O2 and the decrease in absorbance was
determined at 340 nm after every 15 seconds for 60
seconds. The results were expressed as H2O2 decomposed
/ minute / mg of protein, using 71 as the molar extinction
coef cient of H2O2.
Estimation of superoxide dismutase
Superoxide dismutase (SOD) was estimated as per
the nitro-blue tetrazolium (NBT) reaction method of
Giannopolitis and Ries.[12] The reaction mixture contained
hydrolysate (100 μl), sodium carbonate (0.05 M, 1 ml),
NBT (0.01%, 0.4 ml), and EDTA (1 mM, 0.2 ml). Zero
minute absorbance was noted at 560 nm and the reaction
was initiated by the addition of 0.4 ml of hydroxylamine
hydrochloride (2.4 mM). The reaction mixture was then
incubated at 25°C for ve minutes and the reduction of
NBT was noted. One enzymatic unit of SOD was the
amount of protein in the form of the enzyme present in
100 μl of the sample required to inhibit the reduction of
24 mM NBT by 50% and was expressed as unit / mg of
protein.
Estimation of total protein
Total protein was measured as per the Biuret method of
Strikland.[13] The reaction mixture contained hydrolysate
(10 μl) and autozyme total protein reagent (1 ml). The
reaction mixtures were mixed well and incubated for
ve minutes at 37°C. The absorbance of the standard
and test sample against the blank was measured at 546
nm. The intensity of the color was directly proportional
to the protein concentration in the specimen.
Statistical Analysis
The results were expressed as Mean ± S.E.M. The
statistical comparison was performed using one way
analysis of variance (ANOVA) to assess the statistical
significance, followed by Turkey Karman multiple
comparison test. A p value of less than 0.05 was considered
statistically signi cant.
RESULTS
Phytochemical screening
The qualitative phytochemical analysis of Spinacia
oleracea WE and MAF showed a presence of carbohydrates,
avonoids, tannins and phenolic compounds, saponins
and steroids. The content of avonoids and phenolic
compounds was higher in WE.
Acute toxicity studies (LD50)
The extract-treated animals were observed for mortality
up to 48 hours (short-term toxicity) and for 14 days (long-
term toxicity). The study indicated that the MAF and WE
of Spinacia oleracea was non-lethal and safe up to 2000
mg/kg, p.o. dose.
Effect of methanolic aqueous fraction and water
extract on carrageenan-induced rat paw edema
The methanolic aqueous fraction at 600 mg/kg dose and
WE in all the tested doses showed a highly signi cant
(P < 0.01 to P < 0.001) decrease in rat paw edema induced
by carrageenan. MAF at 600 mg/kg dose showed 58.82%
suppression, whereas, WE at the same dose showed
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Journal of Natural Pharmaceuticals, Volume 2, Issue 2, April-June, 2011 83
73.52% suppression of paw edema volume, which was
extremely signi cant and comparable to the standard
drug diclofenac sodium [Table 1].
Effect of MAF and WE on cotton pellet granuloma
The results indicate that MAF and WE of Spinacia
oleracea at the dose levels of 600 mg/kg produced, 44.80
and 54.09% decrease in the weight of the granuloma
respectively, compared to the control. The standard
drug, diclofenac sodium (5 mg/kg) treated group showed
67.75% inhibition in granuloma weight, compared to the
MAF of Spinacia oleracea. WE showed a more potent
effect on both carrageenan-induced paw edema as well
as cotton pellet granuloma growth with 73.52 and 54.09%
reduction respectively, compared to 58.82 and 44.80%
inhibition by the former [Table 2].
Effect of MAF and WE on catalase, superoxide
dismutase activity, and total protein content
The methanolic aqueous fraction and WE showed dose-
dependent increase and decrease in catalase content,
respectively. The WE dose showed signi cant (P < 0.05)
decrease in catalase content compared to control only at
600 mg/kg. The standard drug diclofenac sodium–treated
group showed an extremely signi cant (P < 0.001) decrease
in the catalase level with 295 ± 14.53 μ mole / minute /
mg of granulation tissue protein, compared to very high
catalase activity (704 ± 81.40 μ mole / minute / mg of
protein) in the vehicle control group. Both the extract-
treated group and the diclofenac sodium administered
group showed an extremely significant (P < 0.001)
increase in the superoxide dismutase (SOD) level in
implanted cotton pellet compared to the control group.
MAF showed a signi cant dose-dependent decrease in the
protein content of the implanted cotton pellet, whereas,
WE showed an extremely signi cant dose-dependent
increase in protein content. The highest protein content
was found in WE 600 mg/kg dose (3.29 ± 0.054 mg / 100
mg of granulation tissue). The results are tabulated in
Table 3.
DISCUSSION
The present study was focused on the anti-in ammatory
activity of the water extract and methanolic aqueous
fraction of the S. oleracea leaf. Different extracts of
S. oleracea leaves were subjected to phytochemical
screening, out of which the methanol extract, methanolic
aqueous fraction, and water extract were found to contain
more number of anti-in ammatory phytochemicals, such
Table 1: E ect of MAF and WE of Spinacia oleracea leaf on carrageenan-
induced rat paw edema
Treatment Dose
(mg/kg)
Edema volume a er
three hours
% Decrease in
edema volume
Control (vehicle) - 0.85 ± 0.07 -
Standard Drug
(Diclofenac sodium)
5 0.15 ± 0.02 *** 82.35
Methanolic Aqueous
Frac on (MAF)
200 0.53 ± 0.05 *37.64
400 0.47 ± 0.03 *44.70
600 0.35 ± 0.04 *** 58.82
Water Extract
(WE)
200 0.44 ± 0.14 ** 48.00
400 0.32 ± 0.06 *** 61.76
600 0.22 ± 0.08 *** 73.52
The values are expressed as Mean ± S.E.M, n = 6 in each group, ***P < 0.001,
**P < 0.01, *P < 0.05, when compared to the control group.
Table 2: E ect of MAF and WE of Spinacia oleracea leaf on co on pellet–
induced granuloma
Treatment Dose
(mg/kg)
Dry weight of
co on pellet (mg)
% Inhibi on
Control (vehicle) - 91.5 ± 1.44 -
Standard Drug
(Diclofenac sodium)
5 29.5 ± 0.28 *** 67.75
Methanolic Aqueous
Frac on (MAF)
200 72 ± 0.40 *** 21.31
400 60.75 ± 0.69 *** 33.60
600 50.5 ± 1.02 *** 44.80
Water Extract
(WE)
200 63 ± 0.81 *** 31.14
400 53.75 ± 0.84 *** 41.25
600 42 ± 0.40 *** 54.09
The values are expressed as Mean ± S.E.M, n = 6 in each group, ***P < 0.001,
**P < 0.01, *P < 0.05, when compared with the control group
Table 3: E ect of MAF and WE of Spinacia oleracea leaf on the catalase ac vity, superoxide dismutase, and total protein level of implanted co on
pellets in rats
Treatment Dose (mg/kg) Catalase ac vity
(μ moles H2O2 consumed
/ minute / mg protein)
Superoxide dismutase
ac vity
( units / mg of protein)
Total protein
(mg of protein / 100 mg of
granula on ssue)
Control (vehicle) - 704 ± 81.40 1.47 ± 0.017 0.18 ±0.003
Standard Drug
(Diclofenac sodium)
5 295 ± 14.53 *** 1.75 ± 0.002 *** 2.09 ± 0.014 ***
Methanolic Aqueous Frac on (MAF) 200 523.6 ± 9.97 ** 1.73 ± 0.024 *** 2.03 ± 0.358 ***
400 704 ± 11.43 ns 1.72 ± 0.024 *** 1.57 ± 0.166 ***
600 739 ± 4.04 ns 1.71 ± 0.029 *** 1.06 ± 0.154 *
Water Extract
(WE)
200 760 ± 15.12 ns 1.82 ± 0.004 *** 0.89 ± 0.080 ns
400 637.6 ± 11.89 ns 1.72 ± 0.021 *** 1.81 ± 0.234 ***
600 542 ± 12.12 *1.62 ± 0.057 *** 3.29 ± 0.054 ***
The values are expressed as Mean ± S.E.M, n = 6 in each group, ***P < 0.001, **P < 0.01, *P < 0.05, ns – non-signi cant when compared with control group.
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as, carbohydrates, tannins and phenolic compounds,
saponins, avonoids, and steroids. Hence, an in vivo
study was carried out with methanolic aqueous fraction
and water extract of S. oleracea in animal models, to
investigate the anti-in ammatory activity.
In ammation comprises of three phases namely acute,
sub-acute and chronic. In acute inflammation, due
to increased permeability of blood vessels, uid and
granulocytic cells accumulate at the site of injury. This
reaction often triggers a systemic response such as, fever,
leucocytosis, protein catabolism, and altered hepatic
synthesis of acute phase proteins, such as C-reactive
protein.[1]
The development of edema in the paw of the rat after the
injection of carrageenan is due to the release of histamine,
serotonin, and prostaglandin-like substances. According
to Vinegar et al.,[14] the carrageenan-induced paw edema
is a biphasic event. The rst phase occurs within an
hour and is partly due to the trauma of injection and
also attributed to the release of histamine, serotonin,
and kinins. The second phase of edema is due to the
release of prostaglandins (PGs), protease, and lysosome.
The second phase is sensitive to most clinically effective
anti-in ammatory drugs.[15] Prostaglandins play a major
role in the development of the second phase of reaction,
which is measured after three hours of in ammation
induction.[16,17]
The signi cant ameliorative activities of the extracts
(MAF and WE) and the standard drug (Diclofenac
sodium) were observed in the present study. The WE,
at a dose of 600 mg/kg, suppressed the second phase
of carrageenan-induced in ammation in an extremely
signi cant manner, with 73.52% suppression of edema
volume compared to 58.82% of MAF and 82.35% of
diclofenac sodium. This clearly shows the presence of the
dose-dependent, anti-in ammatory activity in the MAF
and WE of the S. oleracea leaf.
Subcutaneous implantation of a foreign material like
cotton pellet or sponge elicits a chronic in ammatory
reaction, which becomes evident in the form of granuloma.
It can be quantified by recording the dry weight of
granulation tissue.
Granuloma represents both the exudative and
proliferative phase of in ammation. Proliferation of
the broblast and formation of new blood vessels is
one of the main features observed during formation
and maturation of granulation tissue. Granuloma thus
represents intermingling of healing and in ammation
to form a mass composed of in ammatory cells, area of
granulation and brous tissue.[18] The healing process
is modulated by a finely tuned interaction between
inhibitory and growth factors. The cotton pellet
granuloma method has been widely employed to assess
the transudative, exudative and proliferative components
of chronic in ammation. The in ammatory granuloma
is typical, the repair phase of in ammation starts with
proliferation of the broblast as well as multiplication of
small blood vessels. These proliferating cells penetrate
the exudates producing a highly vascularized, reddened
mass, known as granulation tissue. The uid absorbed
by the pellet greatly in uences the wet weight of the
granuloma and the dry weight correlates well with the
amount of glaucomatous tissue formed.[19]
It is well known that free radicals are endogenously or
exogenously associated with the pathogenesis of various
diseases such as parkinsons disease, alzeimer type
dementia, atherosclerosis, diabetes, cancer, arthritis,
and the aging process.[20] Free radicals and other reactive
oxygen species (ROS) are derived either from normal
essential metabolic processes in the human body or
from external sources such as exposure to X-rays,
ozone, cigarette smoking, air pollutants, and industrial
chemicals.[21] In ammation is a complex process and
ROS plays an important role in the pathogenesis of
in ammatory diseases.[22] Thus, antioxidants that can
scavenge ROS are expected to improve these disorders.
Edible plants contain a wide variety of chemicals such
as avonoids and other phenolic compounds that have
potential antioxidant activity through a number of
different mechanisms. Catalase is a hemoprotein
enzyme, which is present in most cells and catalyses
the decomposition of hydrogen peroxide to water and
oxygen. This is one of the endogenous antioxidant
mechanisms that protect cells from the oxidative damage
caused by hydrogen peroxide and the reactive hydroxyl
radical. This enzyme catalyses the decomposition of
H2O2 and OH.[23] Superoxide dismutase (SOD) is a
metallo protein and is the rst enzyme involved in the
antioxidant’s defense by lowering the steady level of
O2-. Anti-in ammatory drugs suppress the growth of
the granulation tissue. In the present study, the effect
of MAF and WE on the implanted cotton pellet–induced
granulation tissue formation of rat was assessed.
Additionally important biochemical parameters like
catalase, SOD, and total protein were also assessed, as
they play an important role in the pathophysiology of
the chronic in ammatory process. The MAF and WE of
S. oleracea leaves in the diclofenac sodium-treated group
showed a marked and statistically signi cant decrease
in the dry cotton pellet weight in comparison to the
control group. Thus, the result indicates that WE have
a higher transudative and anti-proliferative activity
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than the MAF of S. oleracea leaf. In the present study
treatment, MAF and WE showed a complex co-relation
between the growth of granulation tissue and the anti-
oxidant enzyme parameter. MAF showed decreased
weight of granulation tissue along with a decrease in
protein content. It also showed high SOD and moderate
catalase content increase. WE showed a moderately high
increase in protein content, but a decrease in the total
weight of the implanted cotton pellet and at the same
time a moderate decrease in catalase and increase in
SOD content.
In ammation is the body`s natural reaction to invasion
by an infection agent, toxin or physical, chemical or
traumatic damage. One of the purposes of in ammation
is to protect the site of an injury. Chronic systemic
in ammation is not contained to a particular tissue, but
involves the lining of blood vessels and many internal
organs and systems.
The generation of ROS is a crucial process in the
pathogenesis of wound tissue damage. Over-production
of ROS results in oxidative stress thereby, causing
cytotoxicity and delayed wound healing therefore,
elimination of ROS could be an important strategy in
healing of chronic wounds. Estimation of antioxidants
like SOD and catalase in the granulation tissue is also
relevant, because these antioxidants hasten the process
of wound healing by destroying the free radicals.[24]
These enzymes are known to quench the superoxide
radical and thus prevent the damage of cells caused
by free radicals. Granulation, collagen maturation and
scar formation are some of the many phases of wound
healing, which run concurrently, but are independent
of each other.
Water extract showed high anti-in ammatory activity
along with a decrease in weight of granulation tissue
and a decrease in catalase, but an increase in the protein
content and SOD level. Water extract by virtue of its anti-
in ammatory activity decreased cellular proliferation,
but increased collagen synthesis at the tissue implanted
wound site, as evidenced by an increase in the total protein
content of the granulation tissue. Biological activities
of the skin are due to the interaction of glycosamino
glycans, a major component of the extracellular matrix,
with various binding proteins. The water extract may
have protected the granulation tissue from oxidative free
radical damage and thereby stimulated wound healing.
The methanolic aqueous fraction of methanol extract
showed an anti-in ammatory pro le by decreasing the
total weight and protein content of the granulation tissue,
while increasing the content of the antioxidant enzyme
catalase and SOD. Estimation of an anti-oxidant like
SOD and catalase in the granulation tissue is relevant,
because these enzymes enhance the anti-in ammatory
response of the drugs by destroying the free radicals.
The bioactivity of the avonoids is tightly correlated
with their chemical structure and action mechanisms,
mostly inhibitory on enzymatic systems involved in
cellular activation.[25] Bergmen et al.,[26] reported the
presence of an antioxidant component in S. oleracea
as, both avonoids and β-coumaric acid, in the leaves.
The potent antioxidant activity of S. oleracea has also
been reported by Sisodia et al.[27] and Bhatia et al.,[28] as
a protector of radiation-induced oxidative damage, due
to the presence of rich carotinoid contents (β-carotene,
lutein, zeaxanthine). Several polyphenolic compounds
act as potent cyclooxygenase inhibitors, combating with
oxidative stress in the body and maintaining a balance
between oxidants and antioxidants, to improve human
health. Antioxidants reduce the risk of chronic diseases
and prevent disease progression by either enhancing
the body`s natural defenses or by supplementing it
with dietary ingredients. Antioxidants of natural
origin, such as polyphenols (tannins, avonoids), act by
donating electrons to the intermediate radicals formed
in oxidative stress or tissue damage, which help in
inhibition of peroxidation.[29] Plants rich in avonoids,
bio-flavonoids, and other phenolic compounds are
known for their anti-in ammatory and antioxidant
activities.[30,31] Flavonoids are generally good scavengers
of free radicals possessing potent antioxidant activity.
The action shows that the extracts have the capability
of reducing the synthesis of mucopolysaccharide and
collagen, and the number of broblasts, which are
natural proliferative events of granulation in tissue
formation. Wound healing or repair is a natural process
of regenerating dermal and epidermal tissue and may
be categorized into three phases, namely, in ammation,
proliferation, and remodeling phases.
Better collagenation seen under the in uence of the
extract may be because of the presence of avonoids,
Phenolic, and saponins, which are responsible for the free
radical scavenging activity, and are believed to be some
of the most important components for wound healing and
anti-oxidant activity. Rats treated with water extract
and methanolic aqueous fraction at a dose of 600 mg/kg
showed a signi cant increase in the activity of SOD in
the granulation tissue when compared with the control.
This enzyme is known to quench the superoxide radical,
and thus, prevent the damage of cells caused by free
radicals.[32]
The water extract of S. oleracea possesses good anti-
in ammatory and antioxidant activity along with a wound
Nagar, et al.: Anti-in ammatory activity of Spinacia oleracea
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Journal of Natural Pharmaceuticals, Volume 2, Issue 2, April-June, 2011
86
healing property. The results of this study substantiate
the ameliorative effect of S. oleracea aqueous extract
on asthamatic in ammation and its potent antioxidant
pro le.[33]
S. oleracea scavenges free oxygen radicals and increases
the SOD and catalase levels in granulation tissue,
in turn increasing the antioxidant defense system.
Schirrmacher et al., reported the antioxidant and
anti-lipid peroxidative effect of S. oleracea in healthy
individuals, reinforcing the in-vivo antioxidant activity
that was found.[34] Based on the results of the current
study and other reported scienti c evidences it can be
concluded that the water extract of S. oleracea leaves
have a potential anti-in ammatory and wound healing
ef cacy. S. oleracea could be a fairly economic therapeutic
agent for wound management, as a pro-healer and an
anti-in ammatory agent. The current study explores
the acute and chronic anti-in ammatory potential of
S. oleracea with its in-vivo antioxidant potential, but
it cannot establish the exact phytopharmacological
correlation. The ef cacy of the plant against allergen-
induced and immunologically related inflammation
should also be assessed, to substantiate its usefulness
in in ammatory diseases.
The outcome of this study has encouraged us to carry
out a detailed phytopharmacological exploration of
S. oleracea, to nd the entire active principal responsible
along with the mode of action, which is in progress in
our laboratory.
REFERENCES
1. Mohan H. Text Book of Pathology. 4th ed. New Delhi: Jaypee Brothers,
Medical Publishers (P) Ltd; 2002. p. 114-60, 432-6.
2. Robbins C. Pathologic Basis of Disease. 7th ed. Philadelphia: Saunders;
2004. p. 47-118, 1304-11.
3. Shah BN, Nayak BS, Seth AK, Jalalpure SS, Patel KN, Patel MA. Search
for medicinal plants as a source of anti-inflammatory and anti-arthritic
agents-A review. Pharmacogn Mag 2006; 2:77-86.
4. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd ed. Dehra Dun:
International Book Distributors; 1987. p. 2078-9.
5. Nandkarni KM. Indian Materia Medica. Vol 1. New Dehli: Popular
Prakashan; 1976. p. 1164-6.
6. Doijode SD. Seed storage of horticultural crops. Binghamton: Food
Product Press; 2001. p. 262-6.
7. OECD/OCDE, OECD Guidelines for the testing of chemicals,
revised draft guidelines 423: Acute Oral toxicity- Acute toxic class
method, revised document. CPCSEA. Ministry of social justice and
empowerment, Govt. of India, 2000.
8. Kokate CK, Purohit AP, Gokhle SB. Pharmacognosy. 37th ed. Pune: Nirali
Prakashan; 2007. p. 607-11.
9. Winter CA, Risley EA, Nuss GW. Carrageenan-induced edema in hind
paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp
Biol Med 1962;111:544-7.
10. Darcy PF, Haward EM, Muggleton RW, Townsend SB. The Anti-
inflammatory action of Griseofulvin in experimental animals. J Pharm
Pharmacol 1960;12:659-65.
11. Aebi H. Catalase, In methods of enzymatic analysis. Vol2. New York:
Academic press; 1983. p. 673-84.
12. Giannopolitis N, Ries SK. Superoxide dismutase. Occurrence in higher
plants. Plant Physiol 1977;59:309.
13. Strikkland RD, Freeman M, Gurule ET. Copper binding by protein in
alkaline solution. Anal Chem 1961; 961:545-52.
14. Vinegar R, Schreiber W, Hugo R. Biphasic development of carrageenan
edema in rats. J Pharmacol Exp Ther 1969;166:96-103.
15. Crunkhorn P, Meacock SC. Mediators of inflammation induced in the
rat paw by Carrageenan. Br J Pharmacol 1971;42:392-402.
16. Dirosa M, Giround JP, W illoughby DA. Studies of the acute inflammatory
response induced in rats in different sites by carrageenan and
terpentine. J Pathol1971;104:15-29.
17. Dutta S, Das S. A study of the anti-inflammatory effect of the leaves
of Psidium guajava Linn. on experimental animal models. Pharmacogn
Res 2010;2:313-7.
18. Hurley JV. Muirs Textbook of Pathology. London: Anderson JR;
1985. p. 4.
19. Swingle KF, Shideman FE. Phases of the inflammatory response to
subcutaneous implantation of cotton pellet and their modification
by certain Anti-inflammatory agents. J Pharmacol ExpTher 1972;185:
226-34.
20. Kumar R, Phani Kumar G, Chaurasia OP. In Vitro antioxidant activity
of methanolic extract of Rhodiola imbricata Edgew. Pharmacogn J
2010;2:157-61.
21. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants
and functional foods: Impact on human health. Pharmacogn Rev
2010;4:118-26.
22. Conner EM, Grisham MB. Inflammation, free radicals and antioxidants.
Nutrition 1996;12:274-7.
23. Arivazhagen P, Thilagavathy T, Selvam PC. Antioxidant lipoate and tissue
antioxidants in aged rats. J Nutr Biochem 2000;11:122-7.
24. Dissemond J, Goss M, Wagner SN. The role of oxidative stress in the
pathogenesis and therapy of chronic wounds. Hautarzt 2002;53:718-23.
25. Ilepo MT, Basile A, Miranda R, Nappo C. Immunopharmacological
properties of flavonoids. Fitoterapia 2000;71:1015-93.
26. Bergman M, Varshavsky L, Gottlibe HE, Grossman S. The antioxidant
activity of aqueous spinach extract: Chemical identification of active
fractions. Phytochemistry 2001;8:143-52.
27. Sisodiya R, Yadav K, Sharma KV, Bhatia AL. Spinacia oleracea modulates
radiation-induced biochemical changes in mice testis. Indian J Pharm
Sci 2008;70:320-6.
28. Bhatia AL, Jain M. Spinacia oleracea L. protects against gamma
radiations: A study on glutathione and lipid peroxidation in mouse
liver. Phytomedicine 2004;11:607-15.
29. Phan TT, Wang L, See P, Greyer RJ, Chan SY, Lee ST. Phenolic compounds
of chromoleenaorata protect cultured skin cells from oxidative
damage: Implication for cutaneous wound healing. Biol Pharm Bull
2001;24:1373-9.
30. Umamaheswari M, Chatterjee TK. Effect of the fractions of Coccin
iagrandis on Ethanol-Induced cerebral oxidative stress in rats.
Pharmacogn Res 2009;1:25-34.
31. Ilavarasan R, Mallika M, Venkataraman S. Anti-inflammatory and
Antioxidant activities of Cassia fistula Linn. Afr J Tradit Complement
Altern Med 2005;2:70-85.
32. Liu F, Ooi VE, Chang ST. Free radical scavenging activities of mushroom
polysaccharide extracts. Life Sci 1997;60:763-8.
33. Heo JC, Park CH, Lee HJ, Kim SO, Kim TH, Lee SH. Amelioration of
asthmatic inflammation by an aqueous extract of Spinacia oleracea Linn.
Int J Mol Med 2010;25:409-14.
Nagar, et al.: Anti-in ammatory activity of Spinacia oleracea
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Journal of Natural Pharmaceuticals, Volume 2, Issue 2, April-June, 2011 87
Cite this article as: Nagar A, Shukla AK, Bigoniya P. Anti-in ammatory
potential of Spinacia oleracea leaf extract. J Nat Pharm 2011;2:80-7.
Source of Support: Nil. Con ict of Interest: None declared.
34. Schirrmacher G, Skurk T, Hauner H, Grassmann J. Effect of Spinacia
oleracea L. and Perilla frutescens L. on antioxidants and lipid peroxidation
in an intervention study in healthy individuals. Plant Foods Hum Nutr
2010;65:71-6.
Nagar, et al.: Anti-in ammatory activity of Spinacia oleracea
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... It has also been speculated that there are other effects of the phytochemicals present in spinach and strawberries in addition to antioxidant protection. The anti-inflammatory potential of spinach leaf extract was confirmed by Nager [25]. These observations emphasized the fact that phytochemicals present in spinach leaf extract possess antioxidant, antiallergic, antiinflammatory, antiviral, antiproliferative, and anticarcinogenic properties [26]. ...
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Objective: Spinacia oleracea L. extract has been reported to possess potent antioxidant activity in several animal models of neurodegenerative disease but its anti-inflammatory role has not been established much. Thus, the present study had been undertaken to evaluate the possible neuroprotective mechanism of S.oleracea against Lipopolysaccharide (LPS) induced PD. Methods: LPS, a bacterial endotoxin was injected intraperitonially (5mg/kg b.wt) in balb c mice and various neurobehavioral tests, biochemical, RT-PCR and neurochemical studies were performed at day 21. Results: LPS injection in mice produced PD like symptoms as evident from impaired motor functions total locomotor activity, catalepsy and rotarod, increased oxidative burden (MDA, GSH, SOD), enhanced mRNA expression of proinflammatory cytokines and decreased dopamine turnover in striatum. However, spinach leaf extract (50mg/kg, i.p) administration intaperitonially for 21 days significantly attenuated the LPS induced behavioural, biochemical, neurochemical and cellular alterations. Enhanced mRNA expression of proinflammatory cytokines (TNF-α and IL-1β) have also been observed following LPS injection which was significantly attenuated by the S.L.E (50mg/kg, i.p) administration for 21 days. Conclusion: Collectively, these observations suggest a possible anti-inflammatory effect of S.L.E against LPS induced neurodegenerative conditions.
... It has also been speculated that there are other effects of the phytochemicals present in spinach and strawberries in addition to antioxidant protection. The anti-inflammatory potential of spinach leaf extract was confirmed by Nager [25]. These observations emphasized the fact that phytochemicals present in spinach leaf extract possess antioxidant, antiallergic, antiinflammatory, antiviral, antiproliferative, and anticarcinogenic properties [26]. ...
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Context: Use of herbal medicine throughout the world is increasing. Plants still remaining the primary source of supply of many important drugs used in modem medicine. Spinacia Oleracea i,e spinach leaves contain more number of anti-inflammatory phytochemcicals such as carbohydratie, tanins and phenolic compounds, saponins, flavinoids and steroid. Better collagenation seen under the influence of the flavinoids, phenolic compound and saponins which is responsible for the free radical scavenging activity and are believed to be some of the most important component for wound healing and antioxident activity. Spinacia Oleracea scavenges free oxygen redicals and increases the catalase level in granulation tissue. Plants still remaining the primary source of supply of many important drugs used in modem medicine. Considering its medicinal value and availability in our country this study was undertaken to evaluate the antiinflammatory effect of the Spinacia Oleracea leaf extract in rat models. Material and Methods: The experiments were carried out on 30 (thirty) Swiss male albino rats. They were collected from the ICDDRB, Dhaka. The rats were of male weighing between 150- 200gm which were divided randomly into 5 groups each having 6 rats. Groups were labeled as group-I, group-II, group-III, group-IV and group-V. The anti inflammatory effect of Spinacia Oleracea leaf extract in experiment rat were evaluated and compared with the anti inflammatory effects of aspirin and hydrocortisone. The study was prospective experimental type and was conducted in the department of Pharmacology, Dhaka Medical College, Dhaka, from July 2011 to June 2012. Result : Administration of mathanolic extract and water extract of Spinacia Oleracea leaf at a dose of 200mg/kg body weight orally produced a significant (P<0.05) anti-inflammatory effect, and the percentage of inhibition of oedema formation was 28.75% and 40.79% respectively. There were highly significant (P<0.05) percentage of inhibition of oedema formation was observed in aspirin (40.52%) and in hydrocortisone (47.71%). Conclusion: Spinacia Oleracea leaf extract, possess significant anti-inflammatory activity in rats. J Dhaka Medical College, Vol. 27, No.1, April, 2018, Page 88-93
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