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Diuretic Activity of Centella asiatica (L.) Urban

Authors:
Hamdard
Medicus
Vol. 60, No.3, 2017
Diuretic Activity of Centella asiatica (L.) Urban
Mahmood Qureshi', Mansoor Ahmadi, Mahjabeerr', Noor Jahan ', Iftekhar Ahmad Baig',
Nudrat Fatima", Hina Sharif'?", Zahid Khan! and Syeda Kiran"
.
,
'Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi,
2Federal Urdu University of Science and Technology, Karachi,
3Dow College of Pharmacy, Karachi,
'Metropolitan University, Karachi,
Pakistan.
*Email: hina.shahbaz80@gmail.com
Abstract
Diuretics are urinary output increasing
water pills used as a co-therapy to treat edema,
hypertension, lungs, liver and kidney dysfunctions
and diabetes. Centella asiatica (L.) Urban is
(Apiaceae) reported to possess all the above
mentioned conditions. Therefore it was evaluated
for its diuretic activity by using metabolic cages
for measuring urinary output after drug
administration. The methanol extract of leaves
and petioles of
C.
asiatica (L.) Urban plant
was administered to Swiss albino mice, weighing
between 22-28 g, in doses of 300 and 500 mg/
kg body weight. Acute oral toxicity test was
also conducted in animals to find out the safety
of drug at high doses. The test compound was
found to be safe up to 4g/kg body weight
~therefore it can be used freely as herbal
medicine. At both doses mild significant diuretic
activity was observed and occurrence was dose
and time dependent.
Keywords
Diuretic, Centella asiatica (L.) Urban,
Toxicity, Mice.
1. INTRODUCTION
Diuretics is a class of medicine also known
as water pills given to increase urinary output
as a result decreasing hypertensive and edemic
conditions. It is specially prescribed in
hypertension, congestive heart failure, liver
cirrhosis, kidney dysfunction and diabetes.
Commonly there are three types of diuretics:
First type is thiazides including chlorothiazide,
HCI-thiazide, metolazone and indapamide-second
type is loop diuretics including torsemide,
furosemide, ethacrynic acid and bumetanide
while third type is potassium-sparing diuretics
including amiloride, spironolactone, triamterene
and eplerenone. Side effects of all these types
are disturbed potassium and sodium levels in
blood, headache, dizziness, thirst, diarrhea,
allergic reaction and sometimes kidney failure
(Marry, 2016).
This v.alubale medicinal herb is a part of
traditional memory enhancing, venous
hypertension, antioxidant, anti-inflammatory,
antiulcer and wound healing medicinal products
(Sushma et al., 2011).
It
contains important
phytoconstituents such as asiatic acid,
asiaticoside, cadiyenol, castelliferol, catechin,
centellasaponins, centelloside, corosolic acid,
madecassic acid, madecassoside, quercetin, rutin
and ursolic acid (Mahmood, 2015). Keeping in
view its use as an anti-inflammatory (Li et aI.,
2009; George et al., 2009; Huang et al., 2011),
venous hypertension (De Sanctis et al., 2001;
15
Hamdard Medicus
Vol. .60, No.3, 2017
lncandela
et
al.,
200 I), diabetes (Cesarone
etal.,
2001; Alqahtani
et al.,
2013), cardiac
(Gnana-pragasarn
et
al.,
2004), liver (Sharma
and Sharma, 2005; Zhang
et al.,
20 II), lungs
(Zhang
et al.,
20 II) and kidney diseases (Pang
et
aI., 20 I0) traditionally also proven by certain
scientific researches, it was decided to search
C.
asiatica
methanol extract for its diuretic
activity.
2. MATERIAL AND METHODS
C.
asiatica
(L.) Urban plant was
purchased from local market of Karachi,
Pakistan and was identified by Dr. Mansoor
Ahmad in the Department
of
Pharmacognosy,
University of Karachi assigned number
20081
MQ/ca-1 to be deposited in departmental
Herbarium. The leaves with petiole.iwere
separated and methanol extract was prepared
usingreported method (Fatima
et al.,
2008).
Toxicity and diuretic activity on healthy, adult
Swiss albino mice of both sexes weighing
between 22-28 g procured from Animal House
of Dow University of Health Sciences.
2.1.
Toxicity Studies
Acute oral toxicity test was conducted on
Swiss albino mice, weight limits 22-28g,
according to the standard method (Schlede
et al.,
1994). The animals were divided into
three groups of six animals each (3 male and
3 female). All animals were kept in quarantine
a week prior to study. The animals were fed
with standard rodent diet and water at
labiturn.
The animals of group I were given orally nonnal
saline (0.5 ml), group 2, 3 and 4 received a
single dose of C.
asiatica
plant extract in normal
saline as I, 2 and 4
g/kg
body weight,
respectively. The animals were observed for
unusual movements immediately and after
30 minutes time interval for six hours after dose
administration.
2.2. Diuretic Studies
The animals were divided into four groups
comprising of three animals. Group I and
Group 2 were test groups provided with
,300 mg/kg and 500 mg/kg body weight dose of
C.
asiatica
extract, Group 3 and Group 4 served
as negative and positive control groups provided
with normal saline (0.5 ml) and furosemide
(10 mg/kg),
respectively. The animals:were
kept separately after grouping in quarantine
for IS days with normal rodent diet and excess
of water. Group I was given normal saline
(0.5 ml) orally by means of graduated feeding
cannula per animal and served as negative
control group for comparison of results. The
plant extract was mixed with normal saline and
given to animals in doses of 300 and 500 rng/kg
body weight. These two groups were marked
as Group 2 and 3, while animals of Group 4
was given furosemide (50 mg/kg body weight)
and served as positive control group. The
animals after the administration of samples were
kept in specially designed metabolic cages
supplied by Techniplast, Italy Model Nalgene
.1700 IS. The water was present in excess and
volUl'!1eof urine passed was recorded after 2,
4 and 6 hours for each animal individually. The
reading of test groups were compared with
negative and positive control groups to find the
study outcome. The whole procedure was
adapted after the method of Caceres
et al.
(1992).
3. RESULTS AND DISCUSSION
3.1. Toxicity Studies
All animals were active and alert showing
normal symptoms while group 3 animals showed
hyperactivity for some time and later
normalized. No mortality was recorded. It
means the test sample is safe upto a dose of
4 g/kg body weight, therefore, can be used as
a drug.
16
Hamdard Medicus Vol. 60, No.3, 2017
3.1. Diuretic Studies
Diuretic assessment was carried out in
albino mice according to the procedure
described in experimental section. The volume
of urine output of individual animal in a
group was measured and collective readings
were recorded as mean±standard deviation
for each group. The readings of both test
groups were compared to the control group for
analysis.
Table
I
shows the volume of urine in
mice after drug administration. The test drug
did not show any prominent diuresis at fi~st two
hours of administration as compared to control,
while standard drug furosemide exhibited
significant diuretic activity. At 4th and 6th hour
the test drug showed marked increase in urinary
output in dose dependent manner. The final
volume of urine for normal saline receiving
animals was 1.9±0.07, for 300 and 500 mg/kg
dose of C. asiatica was 2.8±0.32 and 4.2±1.03,
respectively, and for standard. marketed diuretic
medicine furosemide it was 5.27± 1.86. Table 2
shows the difference in urinary volume obtained
by subtracting theprevious reading from
current reading. The best result at 2nd and 4th
hour was obtained by Furosemide while at 6th
hour by
C.
asiatica extract at 500 mg dose.
Fig.
I
shows comparative result of test drug at
both doses and standard drug at recommended
dose".
4. CONCLUSION
C.
asiatica methanol extract
of
leaves
and petioles was safe up to 4 g/kg body weight
therefore it can be used freely as herbal
medicine. It possesses mild significant diuretic
activity in dose and time dependent manner.
Table l : Diuretic Activity of C. asiatica at Different Time Points-
Group Sample Dose 0.5 hr 2 hr 4 hr 6 hr
(mg/kg)
..
1.
C.
asiatica extract 300 0.22±0.03 0.62±0.21 1.9±0.02 2.8±0.32
2.
C.
asiatica extract 500 0.31±0.02 0.5±0.11 2.2±0.04 4.2±1.03
3. Normal saline 0.5 ml 0.32±0.04 0.8±0.03 1.0±0.05 1.9±0.07
4. Furosemide 10 . 1.02±0.06 3.04±0.09 4.98±0.06 5.27±1.86
Urinary output (ml)
Table 2: Diuretic Activity: Increase ill Urinary Volume with Respect to Time in ml
Group Sample Dose 0.5 hr 2 hr 4 hr 6 hr
(mg/kg)
1.
C.
asiatica extract 300 0 0.40 1.28 0.90
2.
C.
asiatica extract 500 00.19 1.7 2.00
3. Normal saline 0.5 ml 00.48 0.20 0.90
4. Furosemide 10 02.02 1.94 0.29
17
Hamdard Medicus Vol. 60, No.3, 2017
Percentage Increase in Diuresis wih Respect to- Time
450
4.00
350
300
iso
iQO
150
100
SO
o
O.5hrs
2hrs 6hrs
• Lasiatica
aOOma
ac.asiatidl.50OmI( •
Furosem:ide
Fig. 1: Percentage of increase in diuresis with reespect to control
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525.
Hamdard Medicus
Vol. 60, No.3, 2017
Pharmacognostic and Phytochemical Standardization of Leaves Extract of
Lagerstroemia speciosa L. Pers.
Iftikhar Ahmad Baig', Mansoor Ahmadi, Mahjabeen/, Noor Jahan ', Mahmood Qureshi',
Nudrat Fatima', Hina Sharif" and Syeda Kiran "
'Research Institute of Pharmaceutical Sciences. Faculty of Pharmacy, University of Karachi,
lDepartment of Pharmacology, Faculty of Pharmacy, Federal Urdu University, Karachi,
3Department of Pharmacology, Dow College of Pharmacy Karachi, Karachi, -;
'Department of Pharmacognosy, Faculty of Pharmacy, Hamdard University,
Shahrah-e-Madinat al-Hikrnah; Karachi-74700,
'Department of Pharmacognosy, Faculty of Pharmacy, Jinnah University for Women, Karachi.
'Department of Pharmacy. Metropolitan University Karachi, Karachi,
Pakistan.
+Email: suns_kiran@hotmail.com
Abstract
Lagerstroemia speciosa
L. Pers. belongs
to family
Lytharaceae.
It exhibits a wide.range
of therapeutic effects as an efficacious natural
remedy for diabetes, besides being used for the
treatment of diuretic, febrifuge, stimulant and
purgative.
It
is standardized on the basis of
phytochern ical and pharmacognostic characters.
Pharmacognostic evaluations of powdered
leaves of this plant has green and yellowish
brown color. It had bitter taste with
characteristic odor. Microscopic evaluation of
transverse section of
L. speciosa
L. tissue
manifested upper epidermis, palisade
parenchyma, group of vascular of vascular
bundles, parenchyma, spongy parenchyma, lower
epidermis, xylem vessels, phloem tissues, rosette
aggregate
calcium oxalate crystals in
parenchyma cell, and collenchymas.
Phytochem ica
I
analys is for standard ization
demonstrated the presence of alkaloids, sterols,
tannins, terpenoids.isaponins, phenols, flavonoids,
carbohydrates and proteins. Fluorescence study
showed in visible light and ultra violet (UV)
light at 254 nm and 366 nrn. In TLC test solvent
system of chloroform-methanol-water (80:20:2)
showed 6 major phytochemical agents and
solvent system of Ethyl acetate-Methanol-Water
(100:16.5:13.5) revealed the presence of
5 different chemical components.
Keywords
Lagerstromeia speciosa
L. Pers.,
Lytharaceae,
Standardization, Phytochemical,
Pharma-cognostic,
0
iabetes.
1. INTRODUCTION
Lagerstroemia speciosa
L. Pers. is a
member of family Lytharaceae commonly
known as Banaba or Jarul.Jt grows widely in
tropical countries including India,Philippines,
Malaysia, southern China, Korea, Australia and
Oceania (de Padua
et al..
1997). It is a tall tree
that reach to '20 to 25 m in height, and flowers
while it is still a shrub. In the last decade of
20
th
centuary,
L. speciosa
L. became center of
attention for the researchers globally. Several
in vivo
and
in vitro
experiments confirmed its
strong hypoglycemic action (Guy Klein et aI.,
2007).It contain corosolic acid, a pentacylic
30
Hamdard Medicus Vol. 60, No.3, 2017
triterpene acid which is glucose lowering
and insulin mimetic agent (Ashnagar et al.,
2013). It is popular as an anti-diabetic, diuretic,
febrifuge, stimulant and purgative (Tanquilut
et al., 2009). In the Philippine, the tea made from
the leaves of L. speciosa L. has been used as
a beverage for the treatment and prevention of
diabetes mellitus (Quisumbing, 1978).
Phytochemical. literature survey of
Lagerstroemia speciosa
L.
I
Compounds Plant Part/Group References
Betulinic acid Extracts from the Augusta el al., 2013
Maslinic acid aerial parts
Ursolic acid
Corosolic acid Toshihiroer al., 2012
Ethyl gallate Extracts from the leaves Shouhong et al., 2010
Gallic acid
Pentacylic triterpenes
Oleanolic acid Ethyl Acetate extract from Wenli et al., 2009
Arjunolic acid the leaves
-
Asiatic acid
23-hydroxyurosilic acid r-
.-
Flosin B
Stachyurin
Casuarinin ..
Casuariin
Epipunicacortein A
2,3-(S)-hexahydroxydiphenoyl-a/
I3-d-glucose
l-aonanol Petroleum ether extracts of Rahman et al., 2009
the leaves
Compound LI Leaves Rahman et aI., 2009
Compound L2
:
3-0-tnethyl-ellagic acid 42-sulfate Leaves Bai et al., 2008
~sitosterol
Ellagic acid Leaves Bai el al., 2008 .
31
Hamdard Medicus Vol. 60, No.3, 2017
Compounds Plant Part/Group References
Methyl ellagic acid derivatives
3-0-methylellagic acid Leaves Bai et al., 2008
3,32-di-O-methylellagic acid
;
3,4,32-tri-O-methylellagic acid
3,4,8,9,10 pentahydroxydibenzo
[b,dJpyran-6-one -;
Corosolic acid Leaves and extracts Vijaykumar et al., 2006
31-norlargerenol acetate Leaves Ragasa et al., 2005
24-methylenecycloartanol acetate
31-methylenecYc;loartanol acetate
Largerenol acetate
Tinotufolins C
Tinotufolins 0
Lutein-
...
Phytol .
Sitosterol
Sitosterol acetate
Cycloeucalenol
Valoneic acid dilactone Leaves Unno et al., 2004
Ellagic acid
-
Triterpene pentacyclic acids Extracts from the leaves Yoshihito Okada et al.,
3 beta, 23-"dihydroxy-l-ox-olean- 2003
12-en-2S--oic acid 2003
Valoneaic acid dilactone Leaves Hosoyama et al., 2003
Lagerstroemin Leaves Hattori et al., 2003
,.Takeo Hayashi et al.,
Ellagitannins Aqueous acetone extract of
Lagerstroem in, the leaves 2001
Flosin B
Reginin A
Lageracetal Leaves Masaji Takahashi et al.,
1973
32
Hamdard
Medicus
Vol. 60, No.3, 2017
2. MATERIALS AND METHOD
2.1.
Sample Collection
The plant material leaves of Lagerstroemia
speciosa L. was purchased from local market
at Karachi, Pakistan in November-December
2012 and identified by Prof. Dr.. Mjinsoor
Ahmad, Department of Pharmacognosy, Faculty
of Pharmacy University of Karachi. Receipt
specimen (L8-
111212012)
was deposited in the
herbarium of University of Karachi.
Fig. 1: Leaves of
Lagerstroemia speciosa
L. Pers,
2.2.
Chemicals and Reagents
The chemical reagents and solvents
procured in the research study of L. speciosa L.
involves, methanol, ethanol, ethyl acetate,
chloroform, ether, sulphuric acid, hydrochloric
acid, acetic acid, ferric chloride, acetic
anhydride, n-butanol, sodium hydroxide,
potassium hydroxide, carbon tetrachloride, nitric
acid, lead acetate were purchased from (Merck,
Germany) .. Aniline from (BDH, England),
Tween 20 (Riedel-de Haen, Germany) and
formaline (Fluka, Switzerland).
2.3.
Sample Preparation
The phytochemical standardization of
L. speciosa L. was accomplished on extract of
leaves and seeds of the drug.
2.3.1.
Powder Drug Preparation
The dried leaves of L. speciosa L. were
cleaned and cut into small pieces then ground
it in the grinding machine, powdered material
was than sifted with the help of musline cloth
to obtained fine particle size of the drug materiaL
2.3.2.
Extraction
The dry leaves of
L. speciosaL:
were
coarsely ground into powdered form then Instilled
in methanol and put it for 15 days at ordinary
temperature for further percolation process.
Then filtered off the methanol extract then added
methanol again in the retained extract under
same condition to obtained second MEOH
extract. This process was repeated thrice to
get the maximum yield of phytochemical
components. Finally the combined methanol
extract subjected to rotary evaporator to' get
residue after evaporation under reduced pressure
and controlled temperature. This dried methanol
extract of the leaves .of
1.
.speciosa
L.
was
used for the research experiments. Ethanol,
aqueous and ethyl acetate extract were. also
used for identification of chemical constituents.
_.
2.4. Instrumentation
The following instruments have been used
for the Pharmacognostic studies and
phytochemical standardization of leaves extract
M
the plant.
Rotary evaporator(Eyela, Japan) was used
for extraction and drying under reduced pressure.
Shaker (SS-80 Japan) was used for shaking the
mixtures of solvents and plants extracts.
Electronic Microscope (LABOV AL-4,
Germany) was used for microscopic examination
of powder dnig.U.V Lamp (original Hanau
33
Hamdard Medicus Vol. 60, No.3, 2017
254 nm, fluotest) was used for defection of
compound in TLC monitoring. Physical Balances
(Libror A;EG-120 Shimadzu, Japan and Libror
EB-3200 .
0
Shimadzu, Japan) were used for
weighing purpose.TLC Plates (20
x
20
ern,
silica
gel 60 pre-coated plates, fluorescence at 254 nm
Merck, Germany) and pre-coated TLC Plates
(5
x
10 ern, silica gel 60 T254, 0.2 mm thick)
were used for the detection of phytoconstituents.
3. Pharmacognostic and Phytochemical
Standardization of Drug
Standardized diagnostic characteristics are
one of most important feature of identification
of a drug. Some characteristic features are given
below:
3.1. Organoleptic Evaluation
Organoleptic studies were performed on
leaves and its powder including macroscopic
and-microscopic evaluation.
Macroscopic evaluation
Leaves were broad and ovate, green to
yellowish brown in colour, 12-25
ern
wide and
8-12 cm long with characteristic odor and bitter
.taste.
. The leaves powder was fine in nature with
has slightly bitter taste with characteristic odor
and green and yellowish brown color in
appearance.
Microscopic Evaluation
Microscopic studies of the transverse
section of L. speciosa L. showed midrib, upper
epidermis
(I)
palisade parenchyma (2); group
of vascular bundles(3); parenchyma (4); spongy
parenchyma (5); lower epidermis (6); xylem
vessels (7); phloem tissue (8);rosette aggregate
calcium oxalate crystalsin a parenchyma cell
(9),
and collenchymas (10) as shown in
Fig.
2.
Fig. 2: Transverse section of leaf of
L.
speciosa L.
Surface views of L. speciosa L. lamina under microscope
showed presence of upper epidermis (A) showing striated
cuticle (cu), and Lower. epidermis with anomocytic
stomata (B), as shown in Fig. 3.
-
.~
..
Fig. 3: Microscopic characteristics of
surface views of
L.
speciosa L. lamina.
Microscopic studies of powdered leaves of
L.
speciosa L.
manifestedfollowing observations. (1) Part of the lamina
in s~ctional view, showing the upper epidermis, palisade
parenchyma and part of the spongy parenchyma;
(2) upper epidermis in surface view, showing the striated
cuticle; (3) group of lignified fiber; (4) spiral vessels;
(5) rosette aggregate crystals of calcium oxalate;
(6) reticulate vessels; (7) lower epidermis in surface view
showing anomocytic stomata; (8) fibrovascular tissue
and parenchyma cell as shown in Fig. 4.
34
Hamdard Medicus Vol. 60, No.3, 2017
"
Fig. 4: Microscopic studies of powdered leaves of
L.
speciosa
L.
3.2. Identification of Chemical Constituents
The chemical tests for identification of
phytochemical components have been executed
on each extract i.e. aqeous, ethyl acetate and
methanolic extracts of leaves of L. speciosa L.
Chemical test revealed the presence of alkaloids,
sterols, tannins, terpenoids, saponins, phenols,
flavonoids, carbohydrates and proteins as shown
in Table 1.
3.3. Fluorescence Analysis
Fluorescence study of powdered leaves
of Lagerstroemia speciosa L. .was, performed
by treating the powder with different chemicals
solvents and detected the emergence of different
colors under observation at normal light and
UV light at the wavelength of 254 nm and
366 nm as shown in Table 2.
3.4. Thin Layer Chromatography
In thin layer. Chromatography (TLC)_
of methanol extract which was performed by
Table
1:
Identification of Chemical Constituents by DifferentReagents
.
,
"l
C)
"l
O'l
-
"l
'0
"l
~
'0
~.
"l
'0
~
"l
C
'0
l-
e
c
'0
0
'c
0
.-
0
'0
'Q:j
C C CC
~
Crude extract
-;
l-
V
00
..c
.-
C)
c
cu
0
~
.-
~
c. c.
..c
,.
0
I-
~
I-
::;: r.r.:;
E--
~~
.Q
~
C)
r.r.:;
fi:
I-
,
E--
~
:.
U
Aqueous +
-
+ + +
-
+
-
-
Ethanol. ++
:
+
A
- -
- - -
-
Ethyl acetate xx x x x x x x x
Methanol + + ++ +
A
- -
-
-
Positive
(+);
Negative (-); Abundant
(++);
not analyzed
(x) -
35
Hamdard Medicus Vol. 60, No.3, 2017
Table 2: Fluorescence Analysis of Powder Drug
Observations under
-;
S.No. Treatment of powders Visible light UV light ~ light
(254 nm) (366 nm)
I.
Dry powder . Green Dark green Brownish
green
2. Powder +Brownish Brownish Orange
-:
NaOH in MEOH (1.0 N) yellow green
,
3. Powder +Green Blackish Dark
FeCI3(5%) brown brown
4. . Powder +Light brown Reddish Purple
H2S04(50%)
,
.
brown
5. Powder +Light brown Dark brown Reddish
HCI (1.0 N) brown
--
..;
6~ , Powder +Reddish Brownish Light green
HN03(50%) brown green
7. Powder +Brownish Green Dark green
NaOH in water (1.0 N) yellow
.>
reconstituted in the relevant solvents, in two
different solvent systems including Chloroform-
Methanol-Water (80:20:2) and Ethyl acetate-
Methanol-Water (100:16.5:13.5). TLC showed
the presence of 35 phytoconstituents in both
the solvent systems observed under UV light
on both wavelengths (i.e. 254 nm and 366 nm).
4. RESULTS AND DISCUSSION
In the current research work, the
pharmacognostic evaluation and phytochemical
standardization of leaves powder as well as
extract of Lagerstroemia speciose L. were
performed. Although plant is well known and
its leaves have significant antidiabetic potential
along with other use so it is very important to
establish its standardization for its proper
utilization as media. In different countries of
South Asia L. speciosa is used to treat diabetes
mellitus and several other kinds of diseases (de
padua et
st.,
1997; Quisumbing, 1978;
Matsuyama, 2006). It is well-known anti-
diabetic, diuretic, febrifuge, stimulant and
purgative agent used in, stomach ailments,
diarrhea, bowel movements, aphthous stomatitis
and kidney inflammation, dysuria and other
36
Hamdard Medicus Vo!' 60, No.3, 2017
urinary functions seeds considered to have
narcotic properties; also employed against
aphthae (de padua et al., 1997; Fernando et al.,
2004). The leaves of L. speciosa L. contain
significant amounts of tannins and large amounts
of corosolic .acid, which has previously been
shown to possess antidiabetic properties: A
decoction of 20 g of old leaves of dried fruit in
100cc of water was found to have the equ ivalent
effect to that of 6 to 7.7 units of insulin (Hayashi
et al., 2002; Murakami et al., 1993).
It is
known
that insulin resistance resulting from visceral
fat
accumulation
is involved in various metabolic
disorders. There are several reports on chemical
properties of
L.
speciosa L. given below in
support of 'our results.Coroso lic Acid,
lagerstroemin and gallotannins are major
antidiabetic principles which may stimulate the
glucose transport activity (Toshihiro et al., 2012;
. Ashnagar et al., 20~ 3; Stohs et aI., 2012).
L. speciosa L. inhibit DNA binding nuclear
factor - kappa8 thus prevent diabetes induced
cardiomycyte hypertrophy (Ichikawa et al.,
2010). Ellagitannin lagerstroemia stimulate insulin
·Iike glucose uptake and inhibit adipocyte
differentiation
(Bai
et al., 2008).
It
is also useful
against hyperuricemia by inhibition of xanthine
oxid~se (Unno et aI., 2004). Recent research
also reported the antioxidant (Anil et al., 2010),
hepatoprotective (Lad et al., 2011), antibacterial
(Laruan et al., 2013) and anti-obesity activities
of the plant.
Upon screening of methanolic and
ethylacetate extract of L.speciosa through
various chemical reactions for different groups
of compounds, positive results were obtained
for the presence of alkaloids, sterols tannins
.
'
,
terpenoids, phenols, flavonoids and
carbohydrates in all extracts. Saponins were
present in aqueous and methanolic or the
presence. However, there were no significant
sign of the proteins in all extracts.
Fluorescence analysis of powdered of leaf
of L. speciosa L. was carried out. The
powdered drug was treated with different
reagents or solvents and their characteristics
colors were observed. The 'fluorescence
behavior in different solvents in visible
and UV (254 nm) and (366 nm) were studied
and recorded. Fluorescence behavior of plant
was normal.
Thin layer chromatography of leaf extract
of L. speciosa L. was performed iIi two solvent
systems, chloroform-methanol-water (80:20:2)
and ethyl acetate-methanol-water (100: 16.5:
13.5). The TLC plates were observed under
UV light at 254 nm and 366 nm. The extracts
displayed characteristic chromotographic bands.
The distance of these spots/bands were
measured. The Rr value for each spot were
calculated and recorded. This TLC information
provides help in marker approach regarding
quality, presence of number of compounds of
different chemical types. Identification of leaves
of
L.
speciosa L. pharmacopial standard
through powder microscopy and histological
examinations was also extablished.
5.
CONCLUSION
The purpose ..of research was to provide
the standards and information about identity,
authenticity and quality of L. speciosa
L.
for
its medicinal uses or future investigations.
Literature survey has revealed that
L.speciosa L. has substantial potential towards
the treatment of diabetes and various other
diseases. The diversified actions of this plant
t _
are due to the presence of different types of
chemical compounds.
6. REFERENCES
I. Anil, P., Manish, S., Garvendra, S.R., Vijay, B.
and Tarachand, K. (2010). In vitro antioxidant
studies of Lagerstroemia speciosa leaves.
Pharmacognosy Journal. 2( 10):357-360.
38
Hamdard Medicus Vol. 60, No.3, 2017
2. Ashnagar, A.. Ghanad, A.R. and Motakefpour, M.,
(2013). Isolation and identi fication of major
chemical components found in the leaves of
lagerstroemia indica plants grown in the city of
Tehran, Iran. International Journal ofChem. Tech.
Research.
5(
I):478-481.
3. Augusta Caligiani, Giulia Malavasi,' Gerardo Palla,
Angela Marseglia, Massimiliano Tognolini, Renato
Bruni. (2013). A simple GC-MS method for the
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and ursolic acid contents in commercial botanicals
used as food supplement ingredients. Food
Chemistry.
136(2):735-741.
4. Bai, N.. He, K., Roller, M., Zheng, B., Chen.
X,
Shao, Z. et al. (2008). Active compounds from
Lagerstroemia speciosa insulinlike glucose' uptake
stimulatory/inhibitory and adipocyte differentiation-
inhibitory activities in 3T3-L
I
cells. Journal of
Agricultural and Food Chemistry. 56: 11668-1 1674.
5.
de Padua, L.S .. Lugod, G.e. and Pancho. J.Y.
(1997).
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6. Fernando, E.S., Sun, B.Y .. Sun, M.H., Kong, HY.
and Koh, K.S. (2004). Flowering plants and ferns
of Mt. Makiling. ASEAN-Korean Environmental
Unit, Seoul, Korea.
7. Guy Klein, Jaekyung Kim, Klaus Himmeldirk,
Yanyan Cao and Xiaozhuo Chen. (2007).
Antidiabetes and anti-obesity activity of
Lagerstroemia speciosa. Evidence-Based
Complementary and Alternative Medicine. 4(4):401-
407.
8. Hattori, K., Sukenobu, N .. Sasaki, T., Takasuga, S.,
Hayashi, T., Kasai, R .. Yamasaki, K. and
~ Hazeki, O. (2003). Activation of insulin receptors
by Lagerstroemin. The Japanese Journal of
Pharmacology. 93( I):69-73.
9. Hayashi, T., Maruyama, H., Kasai, R., Hattori, K.,
Takasuga, S., Hazeki,
0.,
Yamasaki, K. and
Tanaka, T. (2002). Ellagitannins from Lagerstroemia
soeciosa as activators of glucose transport in fat
cells. Planta Med. 68(2): 173-175.
10. Ichikawa, H., Yagi, H., Tanaka, T., Cyong, J.e.,
Masaki, T. (20 I 0). Lagerstroemia speciosa
extr.act inhibit TNF-induced activation of nuclear
factor-x B in rat cardiomyocyte H9c2 cells": Journal
of Ethndpharmacology. 128( I ):254-256.
II. Katta Vijaykumar, , Papolu B. Murthy, Sukala
Kannababu, B. Syamasundar and Gottumukkala Y.
Subbaraju. (2006). Quantitative determination of
corosolic acid in Lagerstroemia speciosa leaves,
extracts and dosage forms. International Journal of
Applied Science and Engineering. 4(2): I 03-114.
12. Lad, P.N., Patel, N.C., Shah, Y.N. and Mesariya,
P.S. (2011). Evaluation of hepatoprotective and
antioxidant activity of Lagerstroemia speciose Pers.
Int. 1. Pharm. Res. Development. 3(6):110-117.
13. Laruan, L.M.V .A., Teodora D. Balangcod, Kryssa
Balangcod and Vilma Vallejo, (20 13).
Phytochemical and antibacterial study of
Lager str oemi a speciosa (L.) Pers, and its
ethnomedicinal importance ..to indigenous
communities of Benguet Province, Philippines.
Indian Journal of Traditional Knowledge.
12(3):
379-383.
14. Matsuyama, F., Fukushima, M., Ueda, N., Egawa,
K., Takemoto, 1., Kajimoto, Y., Yonaha, N.,
Miura, T., Kaneko, T., Nishi, Y., Mitsui, R., Fujita,
Y., Yamada, Y. and Seino, Y. (2006). Effects of
corosolic acid on post-challenge plasma glucose
levels. Diabetes Research and Clinical Practice.
73:174-177'-
15. _ Mizanur Rahman, S.M., Shayla Pervin, Mohammad
. Abdul Quader and Amzad Hossain, M. (2009).
Phytochemical studies of the petroleum ether extract
of the leaves of Lagerstroemia speciosa Linn. Indo.
1. Chem.
9(3):470-4703.
16. Muraka~i, c, Myoga, K., Kasai, R., Ohtani, K.,
Kurokawa, T., Ishibashi, S., Oayrit, F., Padolina,
W.G. and Yamasaki, K. (1993). Screening of plant
constituents for effect on glucose transport activity
in Ehrlich ascites tumour cells. Chem. Pharm. Bull ..
(Tokyo). 41:2129-2131.
17. Okada, Y.. Omae, A. and Okuyama, T. (2003).
A new triterpenoid isolated from Lagerstroemia
speciosa
(L.).
Pers. Chemical and Pharmaceutical
Bulletin.
51(4):452-454.
18. Quisumbing, E. (1978). Medicinal Plants of the
Philippines. Katha Publishing, Quezon City,
Philippines, pp. 640-642.
19. Ragasa, Y., Hiu Tian Ngo and John A. Rideout.
(2005). Terpenoids and sterols from Lagerstroemia
speciosa. Journal of Asian Natural Products
Research.
7(1):7-12.
20. Shouhong Gao, Qin Zhan, Jingxian Li, Qi Yang,
Xia. Li, Wansheng Chen and Lianna Sun. (2010).
LC-MS/MS method for the simultaneous
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Hamdard Medicus
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determination of ethyl gallate and its major
metabolite in rat plasma. Journal of Biomedical
Chromatography. 24(5):472-478. 24.
21. Stohs, S.J., Miller, H. and Kaats, G.R. (2012).
A 'review of the efficacy and safety of Banaba
(Lagerstroemia speciose L.) and Corosolic acid.
Phytotherapy Research. 26:317-324.
22. Takahashi, M., Osawa, K., Sato, T., Ueda, J. and 25.
Fujita, Y. (1973). The chemical structure of the
new component "lageracetal" from the leaves of
Lagerstroemia speciosa
(L.).
Yakugaku Zasshi.
93(7):861-863. 26.
23. Tanquilut, N.C., Tanquilut, M.R.C., Estacio,
M.A.C., Torres, E.B., Rosario, J.C. and Reyes,
B.A.S. (2009). Hypoglycemic effects of
Lagerstroemia speciosa (L.). On alloxan-induced
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diabetic mice. Pers. Journal of Medicinal Plants
Research. 3( 12): 1066-1071.
Toshihiro, M., Satoshi, T. and Torao, I. (2012).
Management of diabetes and its complications with
Banaba (Lagerstroemia speciosa L.) and corosolic
acid. Evidence-Based Complementary and
Alternative Medicine. (5): Article ID 871495.
Unno, T., Sugimoto, A. and Kakuda, T. (2004).
Xanthine oxidase inhibitors from the leaves of
Lagerstroemia speciosa
(L.).
Pers. Journal of
Ethnopharmacology. 93(2-3):391-395.
Wenli Hou, Yanfang Li, Qiang.Zhang, Xin Wei,
Aihua Peng, Lijuan Chen and Yriquan Wei. (2009).
Triterpene acids isolated from Lagerstroemia
speciosa leaves as a-glucosidase inhibitors. Journal
of Physiotherapy Research. 23(5):614-618.
Hamdard
Medicus
Vol. 60, No.4, 2017
Anxiolytic Activity of Bark Extract of
Salada reticulata
Wight
Iftikhar Ahmad Baig', Mansoor Ahmadi, Mahjabeen, Noor Jahan
3,
Hina Sharif" \
Mahmood Quresh
i',
Nudrat Fatima', Zahid Khan- and Syeda Kiran"
I
Research Institute of Pharmaceutical Sciences. faculty of Pharmacy. University of Karachi.
2Faculty of Pharmacy. Federal Urdu University. Karachi.
"Department of Pharmacology. Dow College of Pharmacy Karachi. Karachi.
'Department
of Pharmacognosy. Faculty of Pharmacy. Hamdard University.
Shahrah-c-Madinat al-Hikrnah, Karachi-74700.
~Department of Pharmacognosy. Faculty of Pharmacy. Jinnah University for Women. Karachi.
"Department of Pharmacy. Metropolitan University Karachi. Karachi.
Pakistan.
*
Email: hinashahb3zS0.tZ.gmaiI.com
Abstract
Salacia ret iculata
Wight, member of
family
Celastraccae,
is a traditional antidiabetic
drug. Salacinol. kotalanol,
ponkorinol,
salaprinol,
and their corresponding de-o-sulfonated
compounds. mangiferin, kotalagenin 16-acetate
and various proanthocyanidin oligomers are the
major
chern ical
com ponents present in its bark.
The neuro-pharmacological stud ies on.
S. reticulata
Wight included open field, cage
crossing, rearing. traction time, stress induced
swirnrn ing. I
ight and dark exploration tests
and compared
with
diazepam. Significant
anxiolytic
acti v
ity of bark extract of
S. reticulata
Wight at 300 mg dose affecting central nervous
system was noticeable. The CNS effects in
these experiment may possibly be regulated
through
gamma-am ino butyric acid (GABA)
receptor.
Keywords
Sul ac ia
reticul
at a
Wight,
Cel astrace ae ,
diazepam.
Anxiolytic
activity, gamma-am
mo
butyric acid
(GABA)
receptor.
L
INTRODUCTION
Solatia rcticulata
Wight
(Celastraceaei
is a woody climbing shrub with greenishbrown
bark, indigenous to India and Sri Lanka.
It
is
also cultivated in Pakistan and other countries.
Roots, stems, and leaves extracts of
S. reticulata
have been used in Asia from
centuries for the cure of hyperglycem ia and
other ailments including asthma, amenorrhea,
gonorrhea, hemorrhoids, inflammation, rheumatic
and skin diseases (Arunakumara
et al..
2010).
S.
reticula/a
has also been used as a dietary
supplement in Japan as preventive care for
hyperglycem
ia
and
for weight loss (Erika
Kahinao
et al.,
2006). The phytochemicals and
their structure are presented in rig.
I
and in
Tables
I
and 2. Constituents having hypoglycemic
activity includes kotalanol,
ponkorinol. salacinol,
sa
lapr inol, and their corresponding de-O-
sulfonated compounds. Other major chemical
constituents
wh
ich have been isolated from
different parts of plant includes kotalagenin 16-
acetate, mangiferin, and various proantho-
cyanidin oligomers (Akase
et al..
20
II).
Various
in-vivo
and
in vitro
research on
Salacia
extracts revealed that it affect the
enzymes that influence carbohydrate and lipid
metabolism including a-glucosidase, aldose
reductase and pancreatic lipase (Koga
et aI.,
19
Hanidard Medicus
Vol.
60,
No.4, 2017
OH OH
Ho~~~R
HO OH R
=
CH
3
CI, CF
3
NO]
7
A) 3'-O-Bcnzylated Salacinol
Rl
=
OH, R2:: H
R1 = H, R2
=
OH
8. R1
=
OH,
R2
=
H
9. R,
=
H. RZ
=
OH
C) De-O-sulfonated Ponkoranol
r1:~
f'
r
Y
-OH
~s)
OH OH
HO ~~
HO
011
r,Boponliixanol i71
~i'td)lt.
!')'(I-Qli
5'-f:f]H :
3'll-OH,
S'll-Or-!
3'-~pH '3'!i·OH. S'u-Oti
E) Neoponkoranol
OH OH OH
A ~ A
/OH
(~T
'I '-/
A-y-s" OH OH
HO ' , -
;, x
~'~
HO OH
G) Ponkoranol
OH OH OH
::~OH
~s)
OH OH
HO . ",---\
Hd
OH
Neokotalanol
B) Neokotalanol
j~:i
~I ,]_.
HO""(8~ Sri
LI
:
\
HO
011
naosatapnnot ~81 ~'.\ OH
D) Neosalaprinol
OH
-~OH
:
.:
HO~ OH
HO OH
Neosalacinol
F) Neosalacinol
OH
OH
~~,,/"-.
: OH
S~ ~
OH
HOH
OS03
HO OH
H) Ponkoranol
Hnmdard Medicus
Vol. 60. No.4, 2017
~
,,'-;.s~
OS03-
HO' ;~
He) OH
I) SalaprinoI
J)
Salacinol de-O-sulfonate
K) Kotalanol
dc-O-sulfonate L) Quinonemethide
~Oi
V
=
f '\
OH (JH
y1) 15-a.-hydroxyfricdcclan-3-onc N) Lcrnbachol A
~y~
"
~
0) Lernbachol C
P) Lernbachol D
21
Hamdard Medicus
Vol. 60, No.4, 2017
OH
OH
Q)
Pristemirin R) 13-mcmbercd sulfoxide
HO
OH
OH
OH
OH 0
S) l-deoxy-t-thio-darabinofuranose T) Magniferin
HO OH OH
Kobllanol
(C12H24012S2)
Salacinol
(C9H1S09S2)
U)
Kotalanol
V) Salacinol
Fig. 1:
Structures
of
different chemical constituents
from
Salacia
reticulaui
Wight
A)
Tanabe
et
(/1.. 2012; B, F,
G)
Muraoka
et al.,
20
II; C} Eskandari
et al.,
20
I I: D, E) Xie
('I
al ..
20
II:
H, I,
J,
K) Muraoka
et
(1/.,
'2010(1); M, N,
0,
P,
Q)
Rarnamoorthy
et al..
20
I
0; R) Ozaki
e! al.,
2008; S) Yoshikawa
et al.,
2002;
T) Yoshikawa
et al.,
200
I:
V,
V) Yoshikawa
et al., 1998.
Hamdard Medicus
Vol. 60, No.4, 2017
Table
1:
Chemical Constituents of S.
reticulata
Wight
S.No. Chemical constituents References
I.
Celasterol Dhanabalasingham
et al.,
1996
2. De-O-sulfonated Ponkoranol Eskandari
et
01.,
20·1
i
3.
Isoiguesterinol Dhanabaiasingham
et
01..
1996
4.
Kotalanol
Muraoka
et al.,
2010
and Yoshikawa
et al., 2002
5. Kotalanol de-O-sulfonates
6. Lehmbachol A
7.
Lehmbachol C Ramamoorthy
et al..
20
i
0
8.
Lehmbachol
0
9.
Neokotalanol Xie
et al.,
2013
10. N eopon korano 1 Xie
et al., 2011
II.
Neosalacinol Muraoka
et al.,
20
I I
12. Neosalaprinol Xie
et aI.,
201
I
13.
Ponkoranol
Muraoka
et
£11..
2010
14. Pristi
111
erin Ramamoorthy
et al.,
2010
15.
Quinonemethide
16. Salacinol Muraoka
et al.,
2010
and Yoshikawa
et al., 2002
-
17.
Salacinol de-O-sulfonate Muraoka
et aJ.,
2010
18.
Salaprinol
19.
Tingenone Dhanabalasingham
et aI.,
1996
20. f3-amyrin
-.
21. 13-membered sulfoxide Ozaki
et al.. 2008
---------
22. 15-u.-hydroxyfriedeelan-3-one Rarnarnoorthy
et
01.,
2010
23.
l-dcoxv-I-th io-darabinofuranose
Yoshikawa
et al.. 2002
--_._-
----
24.
22 r~-h:;.
droxytingenone
----
_
..
-
DhanabalaSingham
et al.,
1996
25. 30-hydrox),pristimerin
-----
---.-
.
26. 3'-O-b(:Jlz\"latcd salacinol Tanabe
et al..
2012
23
Hamdard
Medicus
Yol. 60, No.4, 2017
Table 2: Pharmacological Activities of Salacia reticulata 'Wight
S.No. Activity/ Model of Study Pharmacological Reference
Parts used activities
1. Anti-inflammatory activit),
Leaves hot-water Arthritic mice Anti-rheumatic Sekiguchi
et al.,
extract Mice
(in-l'itro)
activity and
2012
Anti-
inflammatory
Bark extract
In-vivo
and Anti- inflammatory Sekiguchi
et al.,
in vitro
trials
2010
were done using
mice as models
2.
Antimicrobial
Chloroform and
Staphylococcus
Antimicrobial
Choudhary
et al.,
methanolic extract
aureus, Bacillus
200S
subtilis,
Pseudomonas
aeurginosa
and
Escherichia coli
were used in
antibacterial activity
testing
3.
Anti-obesity effect
Whole Plant Differentiation of Inhibition of fat Shimada
et
al.,
extract 3T3-L
1
adipocytes accumulation in
2011
(in-vitro)
adipose
tissues,
Plant extract Obese TSOD mice anti-obesity Akase
et aI.,
2011
powder (Kothalahim (metabolic syndrome
Japan Co. Ltd., model) and non-
Tokyo) obese TSNO mice
(Control of TSOD)
Leaves extract
In-vivo
and Fat accumulation Shimada
e/ al.,
in-vitro
preventive effect and
2010
anti-obesity effect
Aqueous extract' Male Wistar fatty Anti-obesity Kishino
et al.,
aerial parts rats Hypo cholestreamic
2009
Hypoglycemic
Aqueous extracts Male mice for a Anti-obesity
lrn
et al.. 2008
of root and stems period of 9 weeks
Water soluble
In vitro
study by Anti-obesity Yoshikawa
et al.,
extract analyzing the peri-
2002
uterine fat storage of
female Zucker fatty
rats and male rats
Hamdard Medicus
Vol. 60, No.4, 2017
S.No. Activity/ Model of Study Pharmacological Reference
Parts used activities
4.
Anti-Oxidant
Aqueous and
In vitro
study using Anti-oxidant and Yoshikawa
et al.,
methanolic extract of oxidative stress-
Hepatoproteciive
2002
roots and stems induced liver injury
In
mice
5.
Hypoglycemic activity
Combination with Mice
(in-vivo)
Hepatic Enzyme Yokotani
et a/.,
valerian and black Inhibition 2013
cohosh
Leaves and root Double blind placebo- Pred iabetes Shivaprasad
et al.,
bark extracts controlled study management and 2013
hypolipidemic
Diastereomers Rats
(in-vivo)
Hypoglycemic Tanabe
et a/.,
isolated from leaves 2012
Aqueous extract
In-vivo
investigation Hypoglycemic and Kishino
et a/..
using male wistar fatty hypolipidemic 2009
rats for 6 weeks
Aqueous extract of
In vitro
investigation Hypoglycemic
Im
et al., 2009
stem part of the liver cells of
KK-Ay diabetic mice
Thiocyclitol isolated
In-vivo
investigation Hypoglycemic Oe
et al., 2008
from the water using Wistar rats
-
extract of the plant
Extract of the roots IS-day and 30-day Hypoglycem ic Chandrashekar
treatment of the
et al., 2008
extract
Aerial parts Clinical trial Antidiabetic Jayawardena
6.
Immune modulation activity
Leaves extract. Rat small intestine. Increase in cell- Oda
et al.,
2011
mediated immunity
7.
Lipid lowering activity
Ethanolic extract of Pancreatic lipase Lipid-lowering Koga
et al.,
2013
aerial parts activity
in-vivo
activity
25
Hamdard Medicus Vol. 60.
1\0.
4, 2017
S:No. Activity/ Model of Study Pharmacological Reference
Parts used activities
Leaves, root and Clinical trial Anti-hyperlipidemic Shivaprasad et al.,
bark extracts 2013
-
...
--
Proanthocyanidin Mice (in-vivo) Anti-hyperlipidemic Koga ct a1., 2012
oligomers isolated
from leaves
8.
Safety
--
-------
Plant extract Mice for a period Safe, non-toxic Im et aI., 2008
of 3 weeks
Water soluble extract Guinea pigs Safe, non-antigenic Shimoda et a1.,
and non-phototoxic 2001
2013). Anti-inflammatory (Sekiguchi et aI., 2010
and 2011), immunomodulatory (Oda et a1., 2011)
antioxidant (Yoshikawa et al., 2002) and
antimicrobial activities (Choudhary et a1., 2005)
have also been reported.
2. MATERIAL AND METHODS
2.1. Sample Collection
The research specimen Salacia reticulata
Wight bark was procured from local market at
Karachi, Pakistan in November-December 2012.
Fig. 2a: Dried bark of Salacia reticulata Wight
Prof. Dr. Mansoor Ahmad, Department of
Pharmacognosy, Faculty of Pharmacy,
University of Karachi Pakistan identified the
plant. Voucher specimen (SR-1112120l2) was
deposited in the herbarium of University of
Karachi.
2.2. Animals
Mice (25-30 g) were purchased from
animal house of Research Institute of
Pharmaceutical Sciences (RIPS).
Fig. 2b: Dried bark of Salacia reticulata Wight
Hanidard Medicus
Vol. 60. No.4, 2017
2.3. Drug
Valium (Diazepam. Roche Pakistan Ltd.)
was used as standard drug.
2.4. Sample Preparation
The neuropharmacological experiments of
S. reticulata
were performed on bark extract
of drug.
Dry plant (5 kg) material was comminuted
into small
frauments
and macerated in 2.5 liters
'-
of methanol for 15 days at room temperature.
The extract
was
filtered and residue was
re-
soaked in methanol. This procedure was
repeated thrice. The methanol extract was
evaporated under reduced pressure in a rotary
evaporator to acquire residue. which was used
for various experiments.
2.5. Instrumentation
The
following
instruments were used for
the neuropharmacological research of
S. reticulata.
Rotary evaporator
(Eyela.
Japan)
was used for evaporation of plant extract and
shaker (SS-80 Japan) for shaking the extract
prior to evaporation. Physical balances (Libror
AEG-120
Shimadzu.
Japan and Libror E8-3200
D Shirnadzu.
Japan).
3. ASSESSEMENT OF
NEUROPHARMACOLOGICAL
ACTIVITY
CNS
activiry was
studied by Open field
test. Head dip test. Cage crossing test, Rearing
test, Traction time. Forced
swimm
ing test and
Light and dark exploration test. All the eNS
related tests \\ ere performed in a calm and
peaceful em ironment.
In each test. animals were divided into 4
groups:
Group-A
for control.
Group-B
and
Group-C were treated with 300 mg/kg and
500 mg/kg doses of crude extract, respectively,
while
Group-D was
treated
with
diazepam
(2 mg/kg) served as standard. Each group
contain 5 animals. The crude drug and diazepam
were diluted in distilled water and administered
orally. The control animals were also treated
orally with' the same volume of saline as the
crude extract. In all the tests observations were
made after 30 to 40 minutes of treatment.
3.1.
Open Field Test
The open field apparatus designed in the
laboratory consisted of 76x76 em square area
with opaque walls 42 em high. The floor was
divided into 25 equal squares. Testing was
performed by method as described earlier
(Kennett
et al..
1985 and Turner 1965). Animals
were transferred from their home cages and
placed in the center square of the open field
(one at a time). The open field test was noted
in the control and treated animals in numbers
for 30 minutes.
3.2. Head Dip Test
It
is an exploratory test which was
performed according to the method described
previously
(Sanchez-Mateo et al..
2002; Kasture
et al.,
2002 and Oebparsad
et al.,
2003). The
head dip box was also designed in laboratory.
The head dip test
was
noted in the control and
treated animals in numbers for 30 minutes.
3.3. Cage Crossing Test
The test
performed
on mice in a specifically
designed apparatus .\vith rectangular shape. The
experiment was performed according to the
method described by Florence
et al.,
2000. The
cage crossings were noted in the control and
treated animals in numbers for 30 minutes.
3.4. Rearing Test
Rearing is also an exploratory behavior
test which was executed
follow
ing methods
described earlier
(Sanchez-Mateo
et al.,
2002;
Kasture
et al..
2002 and Sakina
et al.,
1990).
27
Hamdard Medicus
Vol. 60, No.4, 2017
The rearing was noted in the control and treated
animals in numbers for 30 minutes.
3.5. Traction Time
Mice (25 to 30
g) were
used. Traction
time was evaluated following the method by
Sanchez-Mateo
et al.,
2002; Kasture
et al.,
2002 and Oebprasad
et aI.,
2003. The traction
time was noted in the control and treated animals
in seconds.
3.6. Forced Swimming Test
It
was performed according to Sanchez
et aI.,
2002 and Turner 1965. The swimming
time was noted in the control and treated animals
in minutes. Mobility and immobility time of
control and treated animals were noted for
6
III
inutes.
3.7. Light and Dark Exploration Test
The apparatus consisted of a Plexiglas box
with two components 20x20 ern each.
Experiment was
performed
by method described
by Crawley and Goodwin, 1980. The light and
dark exploration time was noted in the control
and treated animals for 10 minutes.
4. RESULTS AND DISCUSSION
4.1. Open Field Test
Resu
I
ts showed sign ificant acti v ity
(141.4±5.37) at 300 mg dose in comparison to
diazepam (96.83±40.09), standard drug and
control (203.66±4.15).
4.2. Head Dip Test
Head dip test showed the highly significant
activity (21.67±3.242) at 300 mg dose and
significant value (32.33±3.084) at 100 mg dose
in comparison to diazepam (19.83±1.307) and
control (59.17±0.982).
4.3. Cage Crossing
Te~il
This activity results were slightly significant
(15.958±3.084) at 100 mg close and (16.191±
3.242) at JOO mg dose in comparison to diazepam
(17.333± 1.307), standard drug and control i.e.
(40. I66±0982 ).
4.4.
Rearing Test
This activity displayed highly significant
value (6.833±1.471) at 100 mg dose and at
300 mg dose (7.3J±1.751) in comparison to
diazepam standard drug (12.1666± 1.722) and
control (32.33J±3.723) as shown in Fig. 6.
4.5.
Traction Time
This activity exhibited highly significant
results (24.66±2.423 sec.) at 300 mg dose and
significant value (J6.10±1.466
sec.)
at 100
rng
dose in comparison to diazepam (21.50±
0.245 sec.) and control (7.17:::0.375 sec.) as
indicated in Fig. 7.
4.6. Forced Swimming Test
This activity displayed highly significant
results (3.23±0.0IS min.) at 300 mg dose in
comparison to diazepam (4.46±0.002 min.),
standard drug and
control
i.e. (2.39±0.002 min.).
4.7. Light and Dark Exploration Test
In light exploration activity, test no
significant results were obtained Fig. 9a. The
dark exploration activity displayed highly
significant results (7.01±0.021 min.) at 300
mg
dose and significant results (6.59±O.028 min.)
at 100 mg dose,
in
comparison to diazepam
(7,49±0.007 min.), standard drug and control
(6.29±0.00J min.) as shown in Fig 9b.
In the present study, nenropharmacological,
effects of
S. relic
II/a/([
was
investigated
in mice
to determine its anxiolyiic and antidepressant
potential.
S.
reticulate
Wight is a member
of
. family
Celastraceae
is a
woody climbing
shrub
which have
long been
LIsee! ill
India and
Sri
Lanka to treat diabetes and other health problems
Hamdard
Medicus
Vol. 60, No.4, 2017
2~O
,..-.,
C
o
.2DD
L
E
::l
Z
'-"
o
UO
:J
~
'-' IDD
+-'
c:
o
E
o
>
c
50
~
(1'----=
'.
Control
lOO
Treatment (mg/kg) Diazepam
300
Fig. 3: Assessment of extract of S. reticulata on neuropharmacological activity by open field method
Control 10.5 ml Saline), Treated (S. reticulata extract) and standard drug (Diazepam 2
mg/kg)
70
60
v:
\...
:J
.c
'D
,...
c
::l
Z
:J
40
:J
,...
,...
~
)0
-
,...
:J
,...
:J
lO
>
~
10
I)
Control lOO 300
Treatment
(mg/kg)
Diazepam
Fig .
..f
.-\sscsSll1cnt of extract of S. reticulata on neuropharmacological activity
by
head dip test
Control (().) 1111Saline 1. Treated
(S.
reticulate extract) and standard drug (Diazepam 2
rug/kg)
29
Hamdard Medicus Vol. 60.
;'\io.
4, 2017
"S
,.-...
-40
:J::
:..
0
.D
l!
E
::l
Z
3D
'--"'
0
0
:ZS
E
4-
20
0
.•...
c
IS
0
E
(\)
>
ID
0
;E
.5
0
Control 100 300
Treatment (rng/kg)
Diazepam
Fig. 5: Assessment of extract of S. reticulata on neuropharmacological activity by Cage crossing test
Control (0.5 ml Saline), Treated (S. reticulata extract) and standard drug (Diazepam 2 mg kg)
30
~ 2D
E
4-
o
is
.•....
c
o
r-
r-
C)
;>
10
c
""
,c..
D'----
Control 100 300
Treatment (mg/kg)
Diazepam
Fig. 6: Assessment of extract of S. reticulate on neuropharmacological activity
by
rearing test
Control (0.5 ml Saline), Treated (S. reticulate extract) and standard drug (Diazepam 2 mg. kg)
Hamdard Medicus
Vol. 60, No.4, 2017
J:)
---.
u:
2.'1
-::;
c
~
..J
:.;
o
.n
o:
~
:.;
E
15
c
~
10
..J
-
~
5
t=
0
Control 100 300
Treatment (mg/kg)
Diazepam
Fig. 7: :-\~~('~~ment of extract of S. reticulata on neuropharmacological activity by traction time test
Control
10.)
rnl
Saline). Treated (S. reticulata extract) and standard drug (Diazepam 2 rug/kg)
J5
3
,-
,.-..
_.)
c
~
,
:.;
-
E
c-,
1.5
~
..c
~
~
OJ
()
Control 100 Diazepam300
Treatment (mg/kg)
Fi~.
Sa:
Assessment of extract of S. reticulata on neuropharmacological activity by
forced swimming test (mobility time)
Contr«]
In.)
III
I Saline
j.
Treated
(S.
reticulate extract) and standard drug (Diazepam 2 mg/kg)
31
Harndard Medicus
Vol. 60. No.4, 2017
5
45
~4
c
~
3.5
<;»
Q)
3
E
.....
>. 25
•....
.
-
.-
2
.0
0
E
1.5
E
......
05
0
Control
100 300
Treatment (mg/kg)
Diazepam
Fig. 8b: Assessment of extract of S. reticulata on neuropharmacological activity
by
forced swimming test (immobility time)
Control
(0.5
rn1 Saline). Treated (S. reticulata extract) and standard drug (Diazepam :2 rug/kg)
3.5
3
c
.-
~ 2.5
'-'
.....
..c
b1)
~ 2
c
C
1.5
Q)
0..
on
Q)
E
E=
0.5
o
Control
100 300
Treatment (mglkg)
Diazepam
Fig. 9a: Effect of crude methanoJic extract of S. reticulata on mice in light exploration test
Control
(0.5
ml Saline), Treated (S. reticulata extract) and standard drug (Diazepam :2 rug/kg)
Hamdard Medicus Vol. 60, No.4, 2017
7.6
7.4
,.-..
:::
7.2
~ 7.0
~
'--
6.8
~
--::J
c
6.6
.•..
:::
o
6.4
Co
tr:
o
6.2
E
r-
6.0
5.8
5.6
Control
roo
JOO
Treatment (mg/kg) Diazepam
Fig. 9b: Effect of crude methanolic extract of S. reticulata on mice in dark exploration test
Control
(0.5
ml Saline). Treated (S. reticulata extract) and standard drug (Diazepam 2 rug/kg)
like asthma, amenorrhea, gonorrhea,
hemorrhoids. inflammation, rheumatic and skin
diseases. In Japan S. reticulata is a famous
dietary supplementary for the management of
diabetes and obesity.
It
consists of kotalagenin
16-acetate. kotalanol, mangiferin, ponkorinol,
salacinol and salaprinol which contributes to its
medicinal action.
The open field activity showed significant
activity (141.5:::5.37) at 300mg dose in
comparison to diazepam (96.83±40.09) and
control (203.66=4.165).The same activity in
other parameter. head dip test exhibited highly
significant activity (21.66±3.242) at 300 mg dose
and significant
value
(32.33±3.084) at 100 mg
in comparison to diazepam (19.83± 1.307),
standard drug and control (59.166±0.982); in
cage crossing activity results are highly
significant (lS.958±3.084) at 100 mg dose and
SEM (16.191±3 .242) at 300 mg dose in
comparison to control (40.166±0982) and
standard drug (l7.333±1.307), respectively.
Rearing test displayed highly significant value
(6.833±1.471) at 100 mg dose and (7.333±1.75I )
at 300 mg dose in comparison to standard
(l2.1666±1.722) and control (32.333±3.723);
traction time activity exhibited highly significant
results at 300 mg dose (24.66±2.423 sec.) and
100 mg dose (16.10 1.466 sec.) in comparison
to standard (21.50±0.245 sec.) and control
(7.166± 0.375 sec.); force swimming test
displayed highly significant results (3.23±0.0
18
min.) at 300 mg dose in comparison to standard
(4.46±0.002 min.) and control (2.39±0.002 min.);
in light exploration activity test no significant
results were found. The dark exploration activity
displayed highly significant at 300 mg dose
(7.0l±0.021 min.) and significant results at 100
33
Hamdard Medicus
Vol. 60, No.4, 2017
mg dose
(6.S9±0.028
min.) in comparison to
standard drug (7.49±0.007 min.) and control
(6.29±O.003 min.). These results indicate that
anti-depressant action of the extract of S.
reticulate
affects
CNS.
This is because in some
cases extract results are very significant as
compared to diazepam. Moreover, its effects
on contraction and relaxation of muscles are
also quite prominent. The
CNS
action in these
experiments may be regulated through Gamma
am ino butyric acid (GABA) receptor.
5. CONCLUSION
The present research ind icated the
presence of significant anxiolytic potential of
S.
reticulata
bark extract. This plant may be
used as an alternative natural treatment of
existing drugs available to cure anxiety and
depression. However, it should be used with
the precaution by hypoglycemic patients because
of its strong sugar lowering property.
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Kobata.. K. and Wada,
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(2012). Anti-proliferative
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