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Vol 12, Issue 3, 2019
Online - 2455-3891
Print - 0974-2441
EVALUATION OF ANTIPSORIATIC ACTIVITY OF AQUEOUS EXTRACT OF BRASSICA
OLERACEA VAR. CAPITATA AND ETHANOLIC EXTRACT OF MENTHA SPICATA LEAVES ON
Received: 01 December 2018, Revised and Accepted: 21 January 2019
ABSTRACT
The objective of this study is to evaluate the antipsoriatic activity of aqueous extract of Brassica oleracea var. capitata leaves (BOAE) and
ethanolic extract of Mentha spicata leaves (MSEE) on Imiquimod-induced Psoriasis-like dermatitis.
Imiquimod induction method for psoriasis-like dermatitis was used for the evaluation of antipsoriatic activity. Extracts were tested at
a dose of 100 mg/kg body weight (b.wt.) and 400 mg/kg b.wt. in Swiss albino mice. These test doses were compared with the Retino-A (0.025%)
treated group which serves as a standard group. Parameters studied in the test include changes in body weight, spleen weight and spleen index,
neutrophils, lymphocytes, eosinophils, monocytes, hemoglobin, red blood cells, platelets, packed cell volume, mean corpuscular volume, mean
corpuscular hemoglobin (MCH), MCH concentration, epidermal thickness, erythema, desquamation, skin thickness, superoxide dismutase, catalase,
lipid peroxidation of skin and percentage orthokeratotic values, drug activity, and relative epidermal thickness.
MSEE at dose of 400 mg/kg b.wt has shown more effect when compared with MSEE at low dose, BOAE topical and oral.
From the study, it was concluded that the above extracts have promising anti-psoriatic activity.
Brassica oleracea var. capitata, Mentha spicata, Psoriasis, Imiquimod, Retino-A (0.025%).
INTRODUCTION
Psoriasis is a condition in which the skin cells build up characterized
by red skin, scales, and dry patches resulting in severe itching. It is a
chronic, painful disease for which there is yet no cure. Psoriasis is a
disorder of the skin, which occurs when the immune system sends out
faulty signals, resulting in the speeding-up of the skin cell cycle [1,2].
It is a noncommunicable disease which can occur at any age. It affects
patients physically as well as mentally. Psoriasis involves the skin and
nails and is also associated with a number of comorbidities. The reported
prevalence of psoriasis in countries ranges between 0.09% and 11.4%,
making it a serious global problem. The etiology of psoriasis still remains
unclear [3]. Although there is a reported suggestion that psoriasis could
be an autoimmune disease, no autoantigen that could be responsible
has been defined yet. In psoriasis epidermal hyperproliferation,
abnormal keratinocyte differentiation can be seen. However, the cause
of the loss of control of keratinocyte turnover is idiopathic. Early and
active psoriatic lesions are characterized by intraepidermal penetration
of activated polymorphonuclear leukocytes, which causes uncontrolled
release of reactive oxygen species (ROS) which leads to pre-oxidative
damage of skin membranes, which worsens the condition by increasing
the severity of the lesions. ROS activates phospholipase A2 and thus
increase the release of the mediators of arachidonic acid. Prostaglandin
produced by the cyclooxygenase pathway also contributes to psoriasis
by dilating capillaries present in the dermis of the skin, stimulates
keratinocyte cell growth and increase in leuckocyte infiltration [4].
Availability, affordability, side effects on prolonged usage of allopathic
drugs have become a greatest concern for psoriasis. Hence, this
study was aimed at assessing the antipsoriatic activity using herbal
extracts of Brassica oleracea var. capitata and Mentha spicata leaves.
These two plants are well-known plants. B. oleracea var. capitata
is commonly known as cabbage and M. spicata plant is commonly
known as spearmint. Both these plants are cultivated in India and
has a wide usage. As B. oleracea va r.capitata and M. spicata was used
conventionally, but there is no reported data so an attempt was made
to evaluate the antipsoriatic activity of B. oleracea var.capitata and M.
spicata [5,6].
MATERIALS AND METHODS
The fresh leaves of B. oleracea var. capitata were collected from the local
market. The leaves of the M. spicata plant were collected from the
agriculture area near Tirupati in Chittoor District, AP, India. Both the
plants were authenticated by Dr. K Madhava chetty, Assistant Professor,
Department of Botany, S.V University, Tirupati. Then the plants were
washed with distilled water to remove dirt and soil. Then, the collected
M. spicata plant leaves were dried under shade at room temperature for
10–15 days. The dried plant leaves were powdered by using a grinding
mill to obtain a coarse powder and then passed through 40 mesh sieve.
(Mentha spicata)
The powdered material was subjected to extraction using ethanol
by the solvent extraction method. Initially, 100 g of crude powder
was taken and packed in a packing paper. This pack was placed in a
Soxhlet extractor and extracted with ethanol, the extraction was carried
out until the extract becomes colorless. The extract was then filtered
with what man filter papers (No.1) and the filtrate was evaporated to
dryness in a rotary evaporator at 40° [4]. The obtained crude extract
was stored in a refrigerator at 4°C until the time of use.
The percentage yield of the extract was calculated by using the following
formula
%yield=weightof theextract
weight of theplantmaterial ×100
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2019.v12i3.30693
Research Article
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Husna and Reddy
(Brassica oleracea .capitata)
The fresh leaves of B. oleracea var.capitata were collected and washed
thoroughly under running water. Then, the leaves were blended with
a blender. After that, the blended plant material was pressed to get
the pure extract of B. oleracea var.capitata. This extract was treated
with Kaolin. Then, the extract was concentrated on the hot plate. The
pure organic part of the sample was thus prepared [7] and stored
at 4–5°C. This final extract was redissolved in sterile water before
experimentation for the evaluation of the antipsoriatic activity.
PRELIMINARY PHYTOCHEMICAL SCREENING
The standard screening test of the extract was performed for various
plant constituents. The crude extract was screened for the presence
or absence of secondary metabolites using standard procedures [8,9]
[Table 1].
ACUTE ORAL TOXICITY
An acute toxicity study was carried out as per OECD-423 guidelines.
ANTIPSORIATIC ACTIVITY
All experimental procedures and protocols used in this study were
reviewed by the Institutional Animal Ethical Committee (IAEC) and
Committee for the purpose of control and supervision of Experiments
Brassica oleracea capitataMentha spicata
Normal BOAE MSEE
Oral
Neutrophils (%) 13.5±4.1 7.26±4.1 11.3±4.68b8.1±4.1c9.2±3.4c8.6±5.2b10.4±3.9c
Lymphocyte (%) 62.2±4.14 91.6±4.8 67.0±5.7c85.6±3.7c75.2±9.5b81.8±7.5c71.0±11.9b
Eosinophils (%) 2.7±1.78 7.3±1.2 2.2±1.3c4.6±1.3c4.0±1.5b6.2±1.10c2.2±1.3c
Monocyte (%) 2.12±1.60 7.0±1.5 1.9±1.43b4.2±1.48c2.2±1.3c4.0±1.58b1.84±1.35c
Hemoglobin (g/dl) 11.31±0.5 7.89±0.62 10.79±0.53c9.38±0.53b10.16±0.53b9.66±0.49c10.36±0.4c
RBC (milli/cumm) 4.4±0.7 2.11±0.61 3.97±0.47c2.78±0.40c3.7±0.5b3.28±0.68a3.61±0.3c
Platelets (lakhs/cumm) 2.65±0.4 1.65±0.3 2.51±0.65a1.94±0.4c2.21±0.4c2.00±0.5b2.31±0.40c
PCV 15.26±1.3 31.8±1.6 17.0±1.2c23.4±1.83b19.2±1.56c21.8±1.6b17.9±1.6c
MCV 43.35±1.03 54.8±1.5 44.9±1.04c52.5±1.5c49.3±1.45b51.0±1.4b46.2±1.0c
MCH 14.86±0.89 23.3±1.6 17.4±1.5c22.1±1.3b19.3±0.7c21.3±0.8c17.9±0.9b
MCHC 26.6±1.0 32.1±2.1c27.0±0.8c30.3±1.8a29.0±1.3b32.2±1.2c
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed
hemoglobin concentration, RBC: Red blood cells, SEM: Standard error of mean, MSEE: Ethanolic extract of Mentha spicata leaves, BOAE: Aqueous extract of Brassica
oleracea var. capitata leaves
Brassica oleracea capitata Mentha spicata
Normal BOAE MSEE
Oral
Orthokeratosis (%) 62.3±1.2 12.18±2.02 60.04±2.3c19.63±2.9b40.6±2.2c32.8±2.8b55.4±1.9c
Drug activity (%) - - 54.01 5.18 24.31 18.08 32.1
Relative epidermal thickness (%) 71.4±4.6 100.0±9.02 67.5±5.18c52.3±9.65a56.9±4.0c54.3±2.7c65.87±3.7b
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Brassica oleracea capitataMentha spicata
Normal Control BOAE MSEE
Oral
Body weight 24.25±1.1 17.4±0.98 23.0±0.85 19.4±1.06 20.95±1.04 20.2±1.5 21.8±1.3
Spleen weight 1.2±0.49 5.1±1.46 2.51±0.93 4.47±1.03 3.18±0.650 2.84±1.0 2.9±1.4
Spleen index 5.28±0.925 16.65±0.89 5.94±1.32 12.00±0.56 9.5±0.94 10.6±1.3 6.2±1.0
All values were expressed as mean±SEM, (n=6).Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Name of the test Results
MSEE BOAE
Alkaloids +ve +ve
Carbohydrates +ve +ve
Proteins +ve +ve
Phenols +ve +ve
Flavanoids +ve +ve
Amino acids +ve +ve
Glycosides +ve +ve
Saponins +ve -ve
Terpenes -ve +ve
Tannins +ve +ve
+ve: Presence of compounds, -ve: Absence of compounds. MSEE: Ethanolic
extract of Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
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on Animals (CPCSEA) rules were in accordance with the guidelines of
IAEC (Regd.No.1521/PO/a/11/CPCSEA). The anti-psoriatic activity
of plant extracts was reviewed through different tests [Tables 2-9].
Male Swiss mice (weighing 20–30 g) at the age of 8 to 11 weeks were
used for the study. Animals were allowed for free access to water and
standard chow diet up to the end of the 23-day experimental period.
Brassica oleracea capitataMentha spicata
Normal BOAE MSEE
Oral
SOD (units/mg) 20.24±0.932 8.71±0.843 18.18±1.01c12.68±1.8a13.48±1.2b14.98±0.943c16.24±0.98b
CAT (units/mg) 6.85±0.6 2.01±0.932 4.63±0.683c3.69±0.915b4.02±0.74c3.87±0.98a4.29±0.87b
LPO (n.mol/mg) 2.09±0.587 15.31±0.73 4.83±0.673c4.4±0.88a3.84±0.72b4.2±0.62b3.86±0.54c
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Brassica oleracea capitataMentha spicata
BOAE MSEE
Oral Oral
On 7th day 4.3±0.34 4.0±0.45b4.8±0.57a4.2±0.78a4.9±0.56b3.9±0.34b
On 12th day 4.5±0.46 2.10±0.32c4.3±0.34b3.8±0.44b4.0±0.60a3.5±0.56b
On 21st day 4.8±0.54 1.10±0.22c3.5±0.53b2.75±0.46a3.1±0.35b1.54±0.23c
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Brassica oleracea capitata Mentha spicata
Normal BOAE MSEE
Oral
On 7th day 0.12±0.01 1.51±0.11 1.43±0.11c1.97±0.3a2.01±0.1c1.89±0.3a2.4±0.3b
On 12th day 0.14±0.02 1.87±0.12 1.01±0.1c1.8±0.1c1.4±0.29a1.63±0.1c1.2±0.2b
On 21st day 0.11±0.03 2.03±0.2 0.39±0.1c1.5±0.134c1.2±0.2b1±0.1c1.01±0.1c
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Brassica oleracea capitataMentha spicata
BOAE MSEE
Oral
On 7th day 4.0±0.4 3.9±0.2c4.2±0.3a4.6±0.3b3.7±0.2c4.3±0.16c
On 12th day 4.5±0.2 2.4±0.1c3.6±0.2b2.4±0.27a3.0±0.19c2.4±0.2b
On 21th day 5.2±0.17 1.8±0.15c2.9±0.3c2.3±0.18b2.9±0.14c1.02±0.12c
All values were expressed as mean±SEM, (n=6). Statistical significance: aP<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
Brassica oleracea capitataMentha spicata
Erythema BOAE MSEE
Oral
On 7th day 4.0±0.4 3.9±0.2b4.2±0.3b4.6±0.3b3.7±0.2c4.3±0.16c
On 12th day 4.5±0.2 2.4±0.1c3.6±0.2c2.4±0.27b3.0±0.19b2.4±0.2b
On 21th day 5.2±0.17 1.8±0.15c2.9±0.3a2.3±0.18b2.9±0.14c1.02±0.12c
All values were expressed as mean±SEM, (n=6). Statistical significance: ap<0.05, bp<0.01, cp<0.001 were compared with disease control (one-way ANOVA followed by
Mentha spicata leaves, BOAE: Aqueous extract of Brassica oleracea var.
capitata leaves
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antioxidant studies that were carried out are lipid peroxidation (LPO)
[18], catalase (CAT) [19], and superoxide dismutase [20].
Blood was collected through retroorbital route and stored in K2
ethylenediaminetetraacetic acid vial for the complete blood count.
Blood was analyzed for the following parameters as hemoglobin (Hb %),
red blood cell (RBC), lymphocytes, neutrophils, eosinophil, monocyte,
platelet count, packed cell volume (PCV), mean corpuscular volume
(MCV), mean corpuscular hemoglobin (MCH), MCH concentration
(MCHC) were estimated with the help of hematology analyzer (Medonic
CA620, Boule, Sweden).
The results are expressed as mean±standard error of the mean.
Comparison between the treatment groups and control were performed
using Graph pad prism06 version by ANOVA method followed by
multiple comparisons of Dunnet’s method.
RESULTS
Morphological findings
Scales and redness on the skin of mice was observed after application
of Imiquimod
Assessment of psoriasis using psoriasis area and severity index
Morphological characters and health status, such as water and
food consumption, behavior signs, body weight, cardiovascular
signs, and respiratory patterns of all mice were normal throughout
the experimental period. Two or three days after starting the IMQ
application, it was observed that the dorsal skin exhibited signs of
erythema, scaling, and thickening (Fig. 2). Thereafter, the intensity
of psoriasis-like symptoms of diseased mice progressively increased
in severity until the end of the treatment (day 16). However, mice
in Group I treated daily with Vaseline did not show any signs of
inflammation on the dorsal skin (Fig. 1). The independent PASI scores
showed the continuous increase in levels of inflammation after IMQ
application from day 1 to day 7, before initiation of treatment with
Retino-A, aqueous extract of B. oleracea var. capitata (BOAE), ethanolic
extract of M. spicata (MSEE). The intensity of PASI scores reached a
peak on the seventh day after IMQ treatment which indicates successful
induction of psoriasis-like dermatitis in the IMQ-treated mice.
However, there was a statistically significant decrease in psoriasis-like
symptoms beginning on day 9, the 2nd day after initiation of treatment
with Retino-A (group III), BOAE (groups IV, V), MSEE (Groups VI, VII).
These symptoms consistently declined until day 16. The individual
The animals were divided into seven groups. Each group consists of five
mice. All the animals were shaved on the back using hair removal cream
(Veet). After 24 h of hair removal, 62.5 mg of Imiquimod (translating
3.125 mg of active compound) was applied on the shaved back using
the applicator brush for 7 consecutive days for all the animals except
for normal group [10].
regular feed
induced with Imiquimod 5% cream at a dose of 62.5 mg (translating
3.125 mg of active compound) on the shaved back topically for 7
consecutive days. Mice were given acetate in drinking water for
same as Group-II animals. On the 8th day, Retino-A 0.025% was
applied topically for the other 16 days [12]
animals. On the 8th day, B. oleracea var. capitata extract was diluted
with distilled water and given orally at a dose of 400 mg/kg b.wt.
for 16 days
animals. On the 8th day, B. oleracea var.capitata extract was applied
topically as a 0.4% concentration for 16 days
animals. On the 8th day, M. spicata extract was diluted with distilled
water and given orally at a dose of 200 mg/kg b.wt. for 16 days
animals. On the 8th day, M. spicata extract was diluted with distilled
water and given orally at a dose of 400 mg/kg b.wt. for 16 days.
On every alternate day, the thickness of skin and ear thickness was
measured using a digital micrometer (SLB Works). The increase in skin
thickness was taken as a measure of skin inflammation [13].
The severity of inflammation on the back skin- an objective scoring
system was developed based on the clinical Psoriasis Area and Severity
Index (PASI). This is an online application. Erythema, induration, and
desquamation were scored independently on a scale from 0 to 4: 0,
none; 1, slight; 2, moderate; 3, marked; 4, very marked.
Body weight of the animals is assessed before the experiment and on
the last day after 2 h of treatment, animals were weighed, and weight
was recorded [14].
At the end after 2 hs of final treatment mice were weighed then
skin of mice was collected
The skin that was isolated was stored in separate containers containing
10% formalin in saline. Longitudinal histology sections of animal skin
were and stained with hematoxylin-eosin [15,16].
The spleen from each mouse was isolated and was weighed.
Splenomegaly was evaluated by calculating the ratio of the weight of
the spleen to the body weight [17].
The spleen index is calculated using formula,
Spleen index=spleen weight/body weight × 100
Skin tissues were collected from the experimental animals, and the tissues
were homogenized to carry out the In vivo antioxidant parameters. The
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PASI scores and the cumulative scores of all groups from days 1 to 16
are depicted in Fig. 1-7. Comparing with the IMQ-treated group, BOAE,
MSEE treated groups showed a significant inhibitory effect on IMQ-
induced psoriasis-like dermatitis. The route-dependent reduction in
the PASI inflammatory symptoms was observed in the BOAE -treated
groups at doses of 400 mg/kg b. wt. and dose-dependent reduction
PASI inflammatory symptoms were observed in the BOAE -treated
group at dose administrations of 100 mg/kg b.wt., 400 mg/kg b.wt. of
mice. The marked reduction of PASI scores of Retino-A treated group
was comparable to the 400 mg/kg b.wt. treated group.
Histological slides
The histopathology slides helped in evaluating different parameters
Mentha spicata
Brassica oleracea capitata
Brassica oleracea capitata oral
Mentha spicata
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DISCUSSION
Swiss albino male mice were used in this experiment due to its
better tolerance level to Imiquimod compared to female [10]. As the
autoantigen causing psoriasis is not known, there is no permanent cure
for this disease. The medical care whichever available in the market is
treating the disease temporarily.
Natural plant ingredients are often used as novel therapeutic agents to
control the inflammation with reducing side effects. As B. oleracea va r.
capitata and M. spicata was used traditionally, but there is no reported
data so an attempt was made to evaluate the antipsoriatic activity of
B. oleracea va r.capitata and M. spicata [5,6].
Morphological findings and calculating PSAI confirms the attack of
psoriasis-like dermatitis [21]. The decrease of the spleen weight/body
weight ratio and cellularity of PALS after treatment with BOAE,MSEE in
the IMQ-induced psoriasis-like dermatitis mice may be suggestive of a
deficit in T-independent humoral immune responses [22]. Low number
of neutrophils in the blood indicates the presence of infection [23,24].
Increase in lymphocytes that the immune system is working heavily,
which is resulting in hyperproliferation of skin cells [25]. Eosinophils
provide inflammatory signals that accelerate the pathogenesis
of psoriasis [26]. High levels of monocytes indicate autoimmune
disorders [27]. In auto-immune diseases, the immune system attacks
and destroys RBC, platelets [28] and platelets may get trapped in the
spleen [29]. PCV, MCV, MCH, and MCHC results indicate deranged
pattern of iron status [30]. The decrease in the degree of orthokeratosis
indicates destruction of granular layer [31]. Increase in erythema,
desquamation, and skin thickness shows the presence of psoriasis
morphologically [21]. If oxidative stress is persisting, or its level very
high, the protein damage became profound and a decreased superoxide
dismutase, CAT activity may occur [32]. LPO is a useful marker of
oxidative stress because it is linked to increased production of ROS [33].
The histological studies reveal that the skin section treated/induced
with Imiquimod showed a degenerative changes in skin tissue shows
thickened epidermis, increased keratinization decrease in percentage of
orthokeratotic regions, elongation of rette ridges, capillary loop dilation
which indicates the severe damage of skin tissue. Animals treated with
Retino-A and BOAE, MSEE shows normal cytoarchitecture of skin tissue
indicates regenerative changes [34].
CONCLUSION
This study showed that the aqueous extract of B. oleracea. var.
capitata and ethanolic extract of M. spicata leaves exhibits a
significant antipsoriatic activity. Route of administration and also
Dose dependently decreased the relative epidermal thickness of
animal skin as well as other histopathology features. Hematological
parameters have also shown that these plant extracts play a prominent
role in the recovery of psoriatic-like symptoms. The study implies that
B. oleracea e var.capitata and M. spicata leaves could be used as natural
therapeutic drugs to prevent complications related to psoriasis and
authentifies the folk claim of the plant in the use of traditional medicine
for the treatment of psoriasis. Additional clinical investigation of
these extracts is needed to evaluate the efficacy and safety of their
application as dietary supplements with health benefits to psoriasis
patients. Further molecular level investigation is needed to prove its
antipsoriatic activity.
I express my profound thanks to my Father Syed Jowhar Madani & my
Mother S. Sharmila for financial and moral support. I extend my thanks
to all my friends who helped me to carry out the experiment. We extend
our sincere gratitude to Dr. V. Jayasankar Reddy, HOD of Department
of Pharmacology and Dr. P. Jayachandra Reddy, Principal, Krishna Teja
Pharmacy College,Tirupati, India for providing instrumental facilities
and guidance.
S. Ayesha Husna reporting anti-psoriatic activity of aqueous extract
of B. oleracea var. capitata and ethanolic extract of M. spicata leaves
Brassica oleracea capitata
Mentha spicata
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Husna and Reddy
on imiquimod induced psoriasis like dermatitis,preparation of the
manuscript,Dr. V. Jayasankar Reddy supervised the work.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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AQ1
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Author Query???
AQ1: Kindly note in your given word file the reference number 7 and 5 seems to be combined as
single reference. We highlight that reference. So, please check and advice what we do.
