The Herb Calycophyllum spruceanum (family:Rubiaceae) known to have several medicinal properties. Methanolic extract of bark was administered to albino rats to study the anti-inflammatory activity by carrageenan induced models as well as behavioural activity by using actophotometer. The powdered bark of Calycophyllmm spruceanum was extracted using a Soxhlet apparatus with methanol. The obtained extract was assessed for anti-inflammatory activity at 125, 250 and 500mg/kg/b.w. dose using diclofenac sodium as a standard drug and for the behavioural activity of Calycophyllmm spruceanum at a dose of 125, 260 and 500mg/kg/b.w using diazepam as the standard drug. The obtained results were compared with those of the standard. There results of anti-inflammatory activity revealed that the extract exhibits significant reduction of paw volume in comparison with the control group For the behavioural activity, the extract exhibited significant decrease in the mean score which indicated reduction in aggression in comparison with the control group. Considerable depressant anxiolytic effects in association with anti-inflammatory activity as demonstrated in the present study might balance each present other, and thus, the plant would be utilized in different inflammation and excitatory situations.

1. Research
Article
Evaluation
Of Anti-Inflammatory AndBehavourial
Activities
Of
CalycophyllumSprucaenumInAlbino
Rats
Veena
Rani
Inugala, Sadhya B,Vanitha
Prakash
Karnam
Email:
vina913@gmail.com
56
Abstract
The
Herb
Calycophyllum
spruceannm
(family:Rubiaceae)
isknown
tohave
several
medicinal
properties,
Methanolic
extract
of Calycophyllom
spruceanun
bark
was
ad toalbino
rats
tostudy theanti-inflammatoryactivity
bycarrageenan
induced
model
as
well
as
behavioural
activity by usingactoplhotonetre.
Thepowdered
bark
of
Calycoplhyllum
spruCeanum
was
extracted
using aSoxhlet
apparatuswithmethanol.
Theobtained
extract
was
assessed
foranti-inflammatory
activity at 125,250
and
500
mg/kg/b.w.doses
usingdiclofenac
sodium
as
a
standard
drug and
forthebelhavioural
activity of Calycophyllun
spruceanum
atadose
of 125,260
and500mg/
kg/b.w usingdiazepam
as
thestandard
drug, Theobtained
results
were
comparedwiththose
of thestandard.
The
results
of anti-inflammatory
activity revealed
thatthe
extract
exhibited
significantreduction
inpaw
volume
incomparison
withthecontrol
group.
Forthebelhavioural
activity,theextract
exhibited
asignificantdecrease
inthemean
score,
which
indicated
areduction
inaggressionincomparisonwiththecontrol
group.
Considerable
depressant
andanxiolytic effects
inassociation
withanti-inflammatory
activity as
demonstrated
inthepresent
study might balance
each
other,andthus,theplant would
beutilized
indifferent
inflammation
andexcitatory
situations.
Key words:
Actophotometer,CalycophyllumSpruceanun,Carrageenan,Diclofenac
Sodium
Introduction
Inflammation
is atermoriginated fromtheLatin
wordinflammation',
whichindicates
being seton
fire, andit is avital process
in thebody's immune
system,where
it plays aroletoeradicate
and
repair
aninjured tissue
orto n foreign entities
orharmfulagents 1, Multiple drugs havebeen
produced to treatthe diseases
of inflammation
of
chronic
origin. Forexample,glucocorticoidsare
the
steroidal
hormones
thatstimulate
theexpression of
around
130
genes,
which
adds
to thephagocytosis,
anti-inflammation,antioxidative
stress
andblocks
thegenes
of pro-inflammation ,A long duration
utilization
of glucocorticoidsraises
the apoptosis
of growth platehypertrophic
chondrocytes,
which
Veena
Rani
Inugala*, Sadhya B,Vanitha
Prakash
Karnam
Department
ofPharnmacology,
SSJ
CollegeofPharmacy,
Vattinagulapally,
Hyderabad-75,
Telangana
State,India
*
Corresponding
Author
Date
ofSubmission:
14-Aug-2019,
Date
ofRevision:
28-Aug
2019;Date
ofAcceptance:
30-Aug-2019
decreases
the growth of boneslongitudinally
On the other hand, it is found that around
one
percent of long term utilizers of NSAIDs,
likepatients with severe
inflammatory disorders,
progress
togastrointestinal
(GI) complexities
like
the damage of mucosa
whichleadsto bleeding
1S, A few scientistshave even
shown acute renal
damage asanoutcome
of NSAIDsconsumption
CoJ,
Natural
medicinal
components
presentattentive
major pharmacological
attention
dueto various
uninvestigated
plant products,exhibiting abroad
varieties
of effects
suchasanti-inflammatoryand
anti-cancer
effects[7-8],
Calycoplyllum
spruceanum
grows
straight upto a
height of around
30m,andhasbeenutilizedas
a
resource
of best
lumber
of high density.It generates
sufficient
little, white, aromatic
flowers(during
June
and
July), in turnresultingin seedpods
with
elongated
structure
containingthree
tofive
seeds.
It
may
frequently beseen
around
water,due
tofactthat
itcould
survive
generalperiodic
floodinginthearea.
Howto citethisartícle:
Inugala RV,SandhyaB, Karnam
VPEvaluation
Of Anti-Inflammatory
AndBehavourial
Activities
Of
Calycophyllum
Sprucaenun
InAlbino
Rats.
MJPS
20
19;5(2):56-61.
Manipal Journal
of Pharmaceutical
Sciences
| September 2019 Volume5 Issue 2
Created with Docutain

2. Inugala
RV,etal:Evaluation
OfAnti-Inflammatory
And
Behavourial
Activities...
Justtwo investigations havebeencommunicated
ontheplant. In 2009,scientists
showed
thatit
possesses
powerful antifungaleflects
invitroon
11
usual
skinyeastsandfungi , Whereas
in 2003,
researchers
reportedthejdentification
of various
new
plant constituents
knownasseco-iridoids
andfound
thatthreeof themwere
potent against
Trypansoma
cruzi,thetropicalparasite
Co),Based
on
these
investigations,
includingthetwo
groups
of
phytoconstituents
exhibiting effects
of antifungal,
antibacterial,
andinsecticidal,
researchers
are
now
beginningtoprove
itstraditional
applications
for
difterent
infections
of fungi andbacteria
of theskin,
andit isutilized
as
arepellent of insect.
Themain
aim
of thisprojectistoassess
theanti-inflammatory
andbehavioural
aspectsof methanolic
extract
of
Calycophylhum
spruceanum
barkin Wistarratsby
usingsuitable
experìmental
models.
Materials and Methods
Materials:
The barkof Calycoplyllum
spruceanum
utilized
for the currentinvestigation was
collected
from
Sri Venkateshwara
University, Tirupati, India.
The authentication
of the plant was
doneat the
Departnentof Botany,and
thespecimen
of voucher
was
conserved
at the herbarium
library of the
institute.
Adult Wistar rats werecollected from Sainath
Enterprises,Hyderabad.Theratswere
categorized
intosixgroups
randomly consisting of sixratsin
eachgroup.The rats weighing between
180-200
g were
kept asaseparate group (six rats in each
cage). The animalswere
kept for two to seven
days to acclimatize
to theconditions
of theanimal
room
They were
managed
inprescribed
laboratory
conditions
of temperature22t2 °Candcyclesof
12hours
of light anddark.They were
fed witha
standard
diet (Hindustan Lever, Bangalore)and
sufficient
tap water.
Plantcollectionandextraction
The barkof afreshplant was
collected
and the
adhered
dirt was
rermoved.
Theleaves
were
removed
andcleaned
withdístilled
water,blotted
anddried
by theshade
instead
of sun
light. Theshade-dried
material
was
powderedusing acommercial
mixer:
Thispowderwas
further
sieved
togetafine
powder
andwas
utilizedfor solventextraction.
Around100
gof thepowderwas
keptfortheSoxhlet
extraction
using 1000
mlmethanol.
Thisparticularcycle was
repented
again
and
again,
forhours
toafew
days,
till
the
colour
of thesolvent
faded
away
in thesiphon
of the soxhlet.The extractwas
thenconcerntrated
under
reduced
pressure
andstored
in arefrigerator
before
further
use.
Attheend
of theprocess
of the
hot
extraction,
the
extract
was
filtered
byfilter
paper:
The filtrate was
concentrated and the solvent was
retrieved
by a
rotary evaporator. Theextractwas
then
placedindesiccators
toget ridof renaining
moisture,if present,andlastly, was
preserved inair
tight containers
at4°Cinarefrigerator:. Theextract
was
then
subjectedtoapreliminary phytochemical
evaluation
(carbohydrates,
proteinsand
amino
acids,
alkaloids,
fixed
oils
and
fats,
glycosides,
triterpenoids,
phenolicsandtannins) as
perthestandard
methods
Animal model used for the anti-inflammatory
study(Carrageenan
induced
acute
paw
oedema):
[19-14]
Theeffect
of oraladministration
of 125, 250and500
mg/kg of themethanolic
extract
of CS,20ng/kg
diclofenac
sodium
orvehicle
(saline, 10ml/ kg) was
assessed
for anti-inflammatory in therats.Therats
were
induced
withedema
to thehindpaw
by asub
plantarinjectionof 0.1
mlofcarrageenan
(1%w/v).
One
of thegroups
was
kept as
Normal
control
where
no
treatment
was
given. Swelling in thepawinduced
by carrageenan
injection was
recorded
at0, one,
two
andthreehoursby using aPlethysmometre.
The
animals
were
administered
with the plant extract
one
hourprior totheadministration
of carrageenan.
Measurement
was
doneimmediately beforeand
threehoursafterthe injection of Carrageenan.
Percent
inhibition
was
calculated
as
comparedtothe
Vehicle
control
(100%).
Behavourial
studyof theextract
using
Actophotometre Ciei3
The locomotor
behaviour
of the animals
was
recorded
using anactophotometre.
The animals
were
kept inan
actophotometre
andthebasal
score
of theactivity was
documnented
for fiveminutes.
Manipal
Journal
ofPharmaceutícal
Sciences
| September
2019
| Volume
5| Issue
2 57
Created with Docutain

3. Each
animal
was
administered
withthedrug and
thescore
of activity was
recorded
after30 and
60minutes.
A reduction
in theactivity score
was
considered
asanindex of the central nervous
system
(CNS)depression.
Thestudyprotocolfor
anti-inflammatory
andbehavioural
activities
of the
methanolic
extract
of calycophyllum
sprucaenum
is
presented
inTable
1.
Table
1: Study DesignforAnti-inflammatory
andBehavioural
Activities
of Methanolic
Extract
of Calycophyllumsprucaenum
(MCS)
Antiinflammatory
Activty
GROUP 1:
Control
group
was
administered
with a
normalsaline
58
GROUP 2: Sub
plantarinjectionof
0.ImlCarrageenan
(1%
w/v) andno
treatment
GROUP$: Standard
dose
of 0mg/kg of
Diclofenacsodim
GROUP4: Testdoseof
195
mg/kg of MCS
GROUP5: Testdoseof
250
mg/kg of MCS
Results
GROUP6: Testdoseof
500mg/kg of MCS
InugalaRV,etal:Evaluation
OfAnti-Inflammatory
And
Behavourial
Activities...
S.
No.
1
5
5
6
Phytochemicalinvestigation
Tests
The resultsof the presence
of preliminary
phytochemicalconstituents
in the methanolic
extract
of Calycophyllum
sprUCAenum
are
shown
in
Table2. The variousconstituents
present were:
Alkaloids,
Glycosidesand
Triterpenoids.
Table2: Phytochemical constituents
present in methanolic
extract
ofCalycophyllum
sprucaenum
Carbohydrates
Alkaloids
Behavourial
Study
GROUP1:
Control
group
was
administered
with anormalsaline
Fixedoils and fats
GROUP 2:
Glycosides
Standard
dose
Triterpenoids
of mg/kg/b.w
of Diazepam
GROUP: Testdose
of 125
mg/kgof MCS
GROUP 4: Test doseof 250
mg/kg of MCS
GROUP5:Testdose
of 500
mg/kg of MCS
Proteinsandamino
acids
Phenolicsandtannins
Methanolic
Extract
Effectof methanolic
extract
of Calycophyllum
sprucaenum
onanti-inflammatory
activity by
Plethysmometre
In case
of the effectof methanolic
extractof
Calycophyllum
sprucaenum
onanti-infl
ammatory
activity by the Plethysmometre,the extract
exhibited
a
significantdecrease
inthe
paw
volume
in
comparison
withthecontrol
group
(p<0.01),Here,
thehighest reduction
was
seen
for standard
drug,
followed
by thehighest dose
of extract
atthethird
hourTheresults
of theanti-infl
ammatoryactivity
are
shownin Table3.
Table
3:Effect
of Methanolic
extracts
ofCalycophyllum
sprucaenum
on
anti-inflammatory
activity by Plethysmometre
Groups Meandifference
in pawvolume
(ml)
3hr
Norimalcontrol
Disease
control
Diclofenacsodium
20mg/kg
Extract
(ECS)
125
mg/kg
Extract
(ECS)
250mg/kg
2 hr
0.00 0.00 0.00
0.15±0.06 0.21t0.08 0.34t0.07
0.14+0.05 0.12+0.06 0.09t0.0g**
1hr
0.16£0.06
Discussion
0.15±0.07
0.16±0.06
0.19±0.07 0.16£0.06
0.18±O.08 0.14t0.05*
Extract
(ECS)
500mg/kg
Data
represented as
mean
±S.E.Mvalues
of six animals
each.
#p<0.05and**p<0.01(One-wayANOVA
withDunnet's
post
hoctest); controlwas
compared with theextractandstandard
treated
groups
0.14t0.05** 0. 10±0.01*
Effectof methanolic
extractof Calycophyllum
sprucaenum behavioural activity by
actophotometre
In case
of the effect of methanolic extract on
Calycophyllum
sprucaenum
on
behavioural
activity by
actophotometre,
theextract
exhibited
asignificant
decrease
in the mean
score,which indicatesa
reduction
inaggressionas
comparedtothecontrol
group
(p<0.01).Here,thehighest reduction
was
seen
forthestandard
followed
by thehighest dose
of extract.
Theresults
of thebehavioural
activity
are
shown
inTable4.
Anxietyand
hypno-sedation
are
basicallyregulated
in the CNSthrough the complex of GABAA
receptor Ci9).A variety of medications
that are
ManipalJournal
of Pharmaceutical
Sciences| September 2019| Volume
5 | ssue
2
Created with Docutain

4. appliedto different
disorders
of neurological
and
psychological
mayaltertheGABA
systematthe
level
of theproductionof GABA,which
can
cause
hypnosis
oranxiolysis
inanimals
viapotentiating
theGABA-regulated
postsynaptic
inhibition
by an
allosteric
alteration
of GABAreceptors Ci, and
by adirect
raise
in theconductance
of chloride
or
indirectly through potentiatingGABA-stimulated
conductance
of chlorideat the same
timewith a
depressionof voltage stimulated
Ca++currents
suchas
barbituratesC).
Table4: Effectof Methanolic
extracts
of Calycophyllum
Sprucaenumon
Behavioural
activity by actophotometre
Groups Meanscore
in 10min
30 min
338.3±l1.3
239.6+19,8***
Control
Diazepam
mg/kg
Extract
(ECS)
195
mg/kg
Extract
(ECS)
250
mg/kg
Extract
Inugala
R
V,etal:Evaluation
Of
Anti-Inflammatory
And
Behavourial
Activities...
(ECS)
500
mg/kg
Basal
345.27110.1
352.5+22,2
355.78+13.6
349.66+17.3
295.76+15.9
267.56+
14.22*
357.29t13.22 947.67t15.39**
60 min
342.76+12.5
112.74+1l1.27#**
233.65t16.26***
179,29+15.8***
125.42+
11.7%**
Data represented asimean
± S.E.M valuesof six animals
each.
p<0.05, **p<0.0 land***
p<0.001 (One-way
ANOVAwith Dunnet's
post-hoc test); control was
comparedwithextract
andstandard
treated
groups
were
compared.
In thisinvestigation,CNSdepressant
effect
of
methanolic
extract
of Calycophyllum
Sprucaentnm
was
screened
by alocomotor
activity of theanimal
test.The extractconsiderably decreased
the
locomotor
activity as
observed
in theresults
by the
actophotometretest.
Methanolic
extract
of Calycophyllum
Sprcaenum
contains
different
phytochemical
constituents
like
triterpenoidsaponins(A and
B),exhibitingoleanolic
acidasaglycone,alkaloid
achyranthine, water
soluble
base
betaine,andsteroids.
CNSdepressant
and
anxiolytic activity of ECS
was
suggested
tobe
contributed
by these
phytochemicals
observed
inthe
extract.
Various
medicinal
plants havebeenshown
topossess
CNS
depressant
and
anxiolyticeffect
due
totheavailability
of triterpenoids,
tiosaponins,
Ci)
and
flavonoids
[l3,10g
Phytochemical
screening
of methanolic
extract
of
Calycophylum
Sprucaenum
also
showed
the
presence
of triterpenoid
saponins;
even
thoughflavanoids
were
shown
to beabsent.
Triterpenoid saponins
are
already
reported
topossess
agonistic/facilitator
effects
at thereceptorcomplexof GABAA
1],
which
suggests
thehypothesis
thatthey exhibit
action
as
benzodiazepine-similar
molecules.
Thisis
evident
by behavioural
activities
in animal
models
of CNS
depressionandanxiety Cls,
16],
In this investigation, wealsoscreened
the anti
inflammatory efectof themethanolic
extract
of
Calycoplyllum
Sprucaenum
by experimental
models,
i.e., carrageenan-induced
pawedema
model.
The
carrageenan-induced
pawedema
model
is utilized
to evaluatethe anti- inflammatory effect of a
medication
in theinflammation
acute
phase.Edema
caused
by carrageenan
isthought tobebiphasicel.
13,Thefirstphase(onehour) includes
thesecretion
of histamine
andserotonin, whereas
the second
phase(> one
hour) isregulatedby cyclooxygenase
endproducts. T he connection
between
the two
phasesisgiven by kinin
[1], Themethanolicextract
of Caycoplyllum
Sprucaenum
significantlyinhibited
theformationof edema
in boththefirst andsecond
phases. The anti-edematous
effectof methanolic
extract
of Calycoplyllum
Sprucaenum
in thefirst
phasemight bedue
totheconsiderable
depletionof
histamine
signaling
throughthe
stabilizing
effect
of mastcell, [1319]
anddrect inhibitionof histidine
decarboxylase
gene
transcriptions
and
histamine
H1
receptor
[990,
In the currentinvestigation, the anti-edematous
effect
of themethanolic
extract
of Calycophyllhem
Sprucaenum
continued
inthesecond
phasewiththe
maximal
actionseen
at one
hour.This might be
simplifiedby theprobableinhibition
of thesecretion
and/or
effect
of theprostaglandin
andkininby
extract.
Flavanoids
andsaponinsare
shown
to possess
anti
inflammatory
activity through various
mechanisms
(us,
e] andotherpharmacological
activities
like
analgesic,antioxidant,antimicrobial,antiviral,
anticancer,
anti-diabetic,
andanti-platelet
activities
c2s0,
Hence,
inlight of thecurrent
experimental
data,
Manipal Journal
of Pharmaceutical
Sciences September 2019 | Volume
5 | Issue
2 59
Created with Docutain

5. theanti-inflammatory effect
of theECSmight be
contributed
by itsflavanoids
and
saponins.
Conclusion
Fromthe outcome, wemayconclude
that the
methanolic
extractof CalycophyllumSprucaenum
containsanadequate CNS depressant and
anxiolytic effectwhencomparedwith the
standard.
Triterpenoids andsaponinsmight bethe
phytochemical constituents
thatcontribute
forthis
effect.
Thecentral
depressantandanxiolytic effect
along with strong anti-inflammatory activity as
earlier
demonstrated
in theinvestigations might
complement eachother, amd
thus, might beused
in differentpainful andexcitatory situations.
It
canbe concluded that the methanolic extract
of CalycophyllumSprucaenumpossesses
ananti
inflammatory effectdue to the claimns
of ethno
pharmacology.
This
information
mightbeutilized
to formulatenovelagents to cure
allergic and
inflammatoryailments.
References
InugalaRV,etal:Evaluation
OfAnti-Inflammatory
And
Behavourial
Activities...
1. Ma D1,Gajewski M.Some
aspects
of the
inflammatory process
Folia
Neuropathol. 1998;
36(4):199-204.
2. FranchimontN, Canalis-
E. Management
of glucocorticoid induced osteoporosis in
premenopausal Women with autoimmune
disease.
Autoimmunity reviews.2003 1;
2(4):224-8.
Yona
S,Gordon
S.Inflammation:
Glucocorticoids
turnthemonocyteswitch.
Immunol
CellBiol.:
2007;85(2):81-2.
4. Wu S,
DeLuca
EInhibition
of theproteasomal
function in chondrocytes down-regulates
growth platechondrogenesis
and
longitudinal
bone
growth.Endocrinology.
2006;
147(8):876
1
8
60
5. ChanFK,
Cryer B,
Goldstein
JL, Lanas
A,Peura
DA,Scheiman
JM, Simnon
LS,Singh G,Stillman
MJ,
Wilcox
CM,BergerME.
Anovel
composite
endpointto evaluate
thegastrointestinal(GI)
effects
of nonsteroidal
antiinflammatory drugs
through theentire
GI tract.TheJournal
of
rheumatology.
2010;
37(1);
167-74.
6. Griffin
JM,Gilbert
KM, Pumford
NR.
Inhibition
of CYP2El
reverses
CD4+T-cell
alterations
in trichloroethylene-treated MRL+/+ mice.
ToxicologicalSciences.
2000;
54(2):384-9.
7. Chang J. Medicinal
herbs:drugs ordietary
supplements?Biochemical
pharmacology.2000;
59(3):211-9.
8. Raghav SK, Gupta B, Shrivastava
A, DasHR.
Inhibition
of lipopolysaccharide-inducible
nitric
oxide
synthaseandIL-1ßthrough suppression
of NF-KB
activation
by 3-(1'-1'dimethyl
allyl)-6-hydroxy-
7-methoxy-coumarin
isolated
from
Ruta
graveolens
L.European
journal of
pharmacology.
2007;
29;560(1);
69-80.
9. Portillo
A, Roser
Villa, Blanca
Freixa,Tomás
Adzet.
"AntifungalactivityofParaguayan
plants
used
intraditional
medicine."
J.Ethnopharmacol
2001;
76(1):93-98.
10.Cardona
Zuleta, L. M., et al. "Seco-iridoids
from Calycophyllumspruceanum
Rubiaceae."
Plytochemistry.
9003;642):549-53.
11.
Kokate,
CK, Purohit,A.P,and
Gokhale,S.B.
2004.
Practical
Pharmacognosy,2ndedition.
Vallabh
Prakashan, NewDelhi, 466-47o.
12.M.DiRosa,J.P.
Giroud,D.A.
VWilloughby
Studies
onthe mediators
of the acuteinflammatory
response
induced
in rats in differentsitesby
carrageenan
andturpentineJ Pathol,1971;104
(1971); 15-29.
13.
D.Salvemini,
Z.Q.
Wang,
PS.
Wyatt,et
al.
Nitric
oxide:
akey mediator
intheearly andlatephase
of carrageenan-induced
ratpawinflammation
Br J Pharmacol, 1996; 118:829-838
14.S. Shashidhara, A.V. Bhandarkar, M. Deepak
Comparative evaluation
of successive
extracts
of leaf and stem bark of Albizzia lebbeck
for
mast
cellstabilization
activity Fitoterapia, 2008;
79: 301-302
15.Galani
VJ, PatelBG.Effectof hydroalcoholic
extract of saphaeranthus indicusagainst
experimentallyin duced
anxiety,depressionand
convulsaions
in rodents.Int J Ayurveda Res.
2010; 1:87-92.
16.
KadamU,
Bhosale
A.
Zopiclone
(Cyclopyrrolone):
ANovel
Hypnosedative;
HypnosedationCaused
by ZopicloneDoes
Not Impair Memory
Learning inAlbinoMice.
CNS
Neurosci
Ther.
2010; 16:180-184.
ManipalJournal
ofPharmaceutical
Sciences
| September
2019
| Volume
5 | Issue
2
Created with Docutain