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To,
The Director, Academic Affairs
KLE University Nehru Nagar
Belgaum-590010, Karnataka.
Sub: Submission of Thesis for the award of Doctor of Philosophy in the Faculty of
Pharmacy
Respected Sir,
With reference to the above cited subject, I Madan Lal Kaushik Registration No. DOUN
9012, Full time Ph.D Research Scholar, Faculty of Pharmacy, KLE University, Belgaum
submitting the Ph.D Thesis and Synopsis for the award of Doctor of Philosophy in the Faculty of
Pharmacy Thesis entitled:
“Preliminary phytochemical study and antiarthritic potential of Curcuma zedoaria Rosc
Root” for your kind perusal.
Enclosures:
1) Five hard bound copies each of Ph.D Thesis
2) Five soft copies (CD) of Ph.D Thesis and Synopsis
3) Five hard bound copy of Synopsis
Thanking You
Yours sincerely
Forward by Guide
Madan Lal Kaushik
Ph.D Research Scholar
Faculty of Pharmacy,
Forward by Principal KLE University Belgaum--590010
Date:
To,
The Director, Academic Affairs
KLE University Nehru Nagar
Belgaum-590010, Karnataka.
Sub: Submission of Thesis for the award of Doctor of Philosophy in the Faculty of
Pharmacy
Respected Sir,
With reference to the above cited subject, I Madan L. Kaushik Registration No. DOUN
9012, Ph.D Research Scholar, Faculty of Pharmacy, KLE University, Belgaum submitting the
Ph.D Thesis and Synopsis for the award of Doctor of Philosophy in the Faculty of Pharmacy Thesis
entitled:
“Preliminary phytochemical study and antiarthritic potential of Curcuma zedoaria Rosc
Root” for your kind perusal.
Enclosures:
1) Five hard bound copies each of Ph.D Thesis
2) Five soft copies (CD) of Ph.D Thesis and Synopsis
3) Five hard bound copy of Synopsis
Thanking You
Yours sincerely
Forward by Guide
Madan L. Kaushik
Ph.D Research Scholar
Faculty of Pharmacy,
Forward by Principal KLE University Belgaum--590010
Date:
KLE UNIVERSITY (Formerly known as KLE Academy of Higher Education & Research)
[Established under Section 3 of the UGC Act, 1956 vide Government of India Notification No.F.9-19/2000-U.3(A)]
FORTH HALF YEARLY PROGRESS REPORT OF Ph.D. RESEARCH WORK
Sl.
No. Particulars
1 Name and Address of the Candidate
(Qualification, Designation and
place of working.)
MADAN LAL KAUSHIK M. PHARMA Research Scholar
Faculty of Pharmacy,
J. N. Medical College Campus,
KLE University, Belgaum-590010-
2 Registration No.
( Provisional / Permanent)
DOUN09012 (Permanent)
3 Research faculty
Faculty of Pharmacy
4 Title of Research topic
Preliminary Phytochemicals study and anti-
arthritic potential of Curcuma zedoaria Rosc
root
5 Date of Registration
August-2009
6 Progress report number submitted
Forth Report
7 Period covered
December 31st -2010 to 30 Jun 2011
8 Date of Submission of last progress
report
31st -December 2010
9 Name, Designation and Address of
the Guide
Dr. SUNIL S. JALALPURE M. PHARM. Ph.D
Faculty of Pharmacy, KLE University,
J. N. Medical College Campus, Belgaum-
590010
10 Name and Address of the Institution
( Where research is carried out)
Faculty of Pharmacy
KLE University, J. N. Medical College
Campus, Belgaum-590010, Karnataka
ASPECTS OF HALF YEARLY LAST PROGRESS REPORT 31st JUNE-2011
I PROGRESS IN
THE REVIEW OF
THE
LITERATURE
Yes
Annexure-1
II NEW DATA
ACQUIRED OR
THEORETICAL
BACKGROUND /
TECHNIQUES
DEVELOPED
On the basis of literature survey all procedures
methodologies adopted in the research which are
mentioned in Annexure-II
Annexure-II
III PROGRESS /
STANDARDIZATI
ON IN
RESEARCH
METHODOLOGY
Yes progress in the standardization of Petroleum
ether extract of Curcuma zedoaria Rosc root (CZ).
(Isolation purification and Characterizations)
Annexure-III
Analyze the active constituents by GCMS present
in potent petroleum ether extract of CZ
Preparative Thin layer chromatography:
Curcumin, Demethoxycurcumin, and
Bisdmethoxycurcumin
Colum Chromatography
Curcumin, Demethoxycurcumin, and
Bisdmethoxycurcumin
High performance liquid chromatography:
Curcumin, Demethoxycurcumin, and Bisdmethoxy
curcumin
High performance thin layer chromatography:
Curcumin, Demethoxycurcumin and Bisdmethoxy
curcumin .
UV Spectra: Curcumin, Demethoxycurcumin, and
Bisdmethoxycurcumin
FT-IR Spectra: Curcumin, Demethoxycurcumin,
and Bisdmethoxycurcumin
H1- NMR Spectra: Curcumin,
Demethoxycurcumin and Bisdmethoxycurcumin
Liquid chromatography Mass spectroscopy:
Curcumin, Demethoxycurcumin and
Bisdmethoxycurcumin
II) NO. OF
SAMPLES
PROCESSED
Sample (Petroleum ether extract) was proceeded
for Isolation and characterizations
IV DISCUSSION OF
THE WORK
DONE
INCLUDING ANY
NEW FINDINGS
Isolation and Characterizations of active
compounds present in petroleum ether extract of
CZ which are responsible for anti-arthritic activity.
Annexure-IV
V FUTURE PLAN
OF WORK
Publications of research work Annexure-V
VI REFERENCES
(NEW & MORE
RELEVANT)
Yes Mention new and more Reference Annexure-VI
VII HIGHLIGHTS
(IN BRIEF) OF
PREVIOUS
PROGRESS
REPORTS.
In previous progress Pharmacological evaluation of
CZ extracts was performed which includes
following contents.
Annexure-VII
Acute Toxicity Study
Anti-arthritic activity of CZ, FCA-induced arthritic
in rats
Induction of Arthritis (Paw volume)
Behavioral observations
Physiology profile
Biochemistry profile
Nitric oxide synthesis
Vascular permeability
Histopathology of joints
Radiographic of joints
Anti-inflammatory activity of CZ, against
Carrageenan and Histamine
Organ to Body weight ratio (Toxicology)
EXTENDED RESEARCH WORK FOR ASPECTS OF HALF YEARLY PROGRESS
REPORT 31st JUNE-2011
I REVIEW OF
LITERATURE
Review on herbal formulation and its stability
study.
Annexure-VIII
II STANDARDIZATI
ON IN
RESEARCH
METHODOLOGY
Development of single herbal Formulations (A, B, C,
and D), Pharmacological standardization and
accelerated stability study.
Annexure-IX
Preparation of test samples
Selection of animals
Acute Toxicity Study
1. PROGRESS IN THE REVIEW OF THE LITERATURE
On the basis of pharmacological result, petroleum ether, chloroform and ethanol extract was showed
potent antiarthritic activity in rats. Amongst these petroleum ether extract showed highly potent so we
investigate the active constituent present in petroleum ether extract which is responsible for this
activity. As per literature survey, plant contains number of active constituents such as bitter resin,
organic acid, gum, starch sugar2, curcumenol, dihyrocurdione, curcumin, dihydrocurcumin,
Anti-arthritic activity of CZ, single herb
formulations, FCA-induced arthritic in rats
PARAMETERS
Induction of Arthritis
(Paw volume)
Nitric oxide synthesis
Behavioral
observations
Vascular
permeability
Physiology profile Radiographic of
joints
Biochemistry profile Histopathology of
joints
Anti-inflammatory activity of CZ, against
Carrageenan and Histamine
Organ to Body weight ratio (Toxicology)
Statistical analysis
Accelerated stability study of single herb
formulations
I. Formulation-A II. Formulation-B
III. Formulation-C IV. Formulation-D
III Pharmacology screening of Curcuma aromatic
tetrahyrodemothxycurcumin, tetrahydrobismethoxycurcumin, a-curcumene, b-tumerone zerumbone,
zerumbone epoxide, diferuloylmethane, di-p-coumaroylmethane, curcumin, demothxycurcumin and
bisdemothxycurcumin, furanodiene, germacrone, curdione, neocurdione, curcumenol, isocurcumenol,
aerugidiol, zedoarondiol, curcumenone, curzenone and dehydrocurdione
Isolation of curcumin, demothxycurcumin and bisdemothxycurcumin from Petroleum ether extract by
Thin Layer Chromatography
II. NEW DATA ACQUIRED OR THEORETICAL BACKGROUND TECHNIQUES
DEVELOPED
Separation and purification of crude curcuminoids by preparative TLC: and column
Chromatographic Studies.
Preparative TLC of crude extract obtained from pet-ether extract of CZ
Stationary phase: Silica gel G for TLC.
Mobile phase: Chloroform: Benzene: Ethanol (45: 45: 10)
Detecting reagent: 2% Boric acid in Methanol
Preparative TLC plates: The size of TLC plate, which was used for the preparative
TLC, is 20 X 20 cm as proposed by Stahl (1964).
Preparation of TLC plates: Prepared thick slurry of silica gel G using distilled water and
spread on the plates, the thickness of layer was 0.5 - 2 mm, and for avoiding cracking of thick
layers, they are dried for several hours at room temperature before placing them in oven.
Activation of TLC plates: The TLC plates were heated in an oven at 1100
C for 30 min.
Preparation of sample: Pt-Ether extract was dissolved in methanol and used for application.
Preparation of mobile phase: Chloroform: Benzene: Ethanol in the ratio (45 : 45 : 10) was
taken in TLC chamber and after complete saturation it was used for developing the TLC plates.
Application of sample: The sample was applied in the form of band (about 2 cm above from the
bottom of the plate) and allowed to dry at room temperature.
The dried plates were kept in the developing chamber, presaturated with the mobile phase. When
the solvent front covered 3/4th
of the distance, plates were removed and dried at room
temperature.
Identification of bands: For the identification of Curcuminoids, the developed TLC plates were
sprayed with 2% boric acid in methanol spraying reagent and observed for the appearance of
colored bands. The area corresponding to colored bands on the adjacent track was scrapped off.
Collection of residue: The scrapped portion was treated with methanol to dissolution, filtered
and the filtrate was evaporated in a china dish on hot water bath to get the residue (Compound
1).
Colum chromatography of crude curcuminoids obtained from petroleum ether extract
of CZ
Isolation of Curcumin from successive Petroleum ether extract was carried out, as this particular
extract showed four (yellowish red, yellow, light yellow and light yellowish red) spots
respectively. From these, curcumin was isolated by column chromatography.
Column chromatography is one of the most useful methods for the separation and purification of
both solids and liquids. Column chromatography is another solid-liquid technique in which the
two phases are a solid (stationary phase) and a liquid (moving phase). The theory of column
chromatography is analogous to that of thin-layer chromatography. The most common
adsorbents are silica gel and alumina. The sample is dissolved in a small quantity of solvent (the
eluent) and applied to the top of the column. The eluent, instead of rising by capillary action up
as in TLC, flows down through the column filled with the adsorbent. There is an equilibrium
established between the solute adsorbed on the silica gel or alumina and the eluting solvent
flowing down through the column. Column chromatography is generally used as a purification
technique, in which it is possible to isolate desired compounds from a mixture.
Adsorbent : Silica gel activated for column chromatography
Dimensions of the column : L - 45cm, Diameter- outer -3 cm, inner -2.8 cm
Length of adsorbent packed : 31cm
Rate of elution : 5 ml/min. (Till Methanol (10) : Chloroform (90))
Afterwards 5-10 drops/min.
Volume of elute collected : 50 ml (Till 10 :90) Afterwards 5ml
Fraction collected : 165
Type of elution : Gradient elution.
Preparation of sample
1.5 gm of crude curcuminoids, isolated from preparative TLC method was dissolved in
15 ml of methanol and further concentrated.
It was dissolved in 10ml of methanol and mixed with 2.5 gm of silica gel (60-120 mesh
size) and dried in vacuum oven at 45OC. The adsorbed material obtained was transferred
to the column.
Column Packing
100 gm of silica gel was activated in hot air oven at 1100 C for 1 hour.
The glass wool is fixed at the bottom of the column.
The slurry of activated silica was made in pet ether and charged in the column in small portions
by keeping the knob open with gentle taping after each addition, in order to ensure uniform
packing. A small quantity of solvent was allowed to remain at the top of the column in order to
avoid the drying or cracking of the column. Tapping is necessary to avoid the air bubble
formation in the column during packing which otherwise may interfere in the separation. The
packed column was kept undisturbed overnight.
The column was run fast for sometime with pet ether in order to remove any impurities. Prepared
sample was then charged into the column and was allowed to settle. A small cotton pad was
placed above the sample to prevent the mixing of dust particles with the sample. Then it was
eluted with the mobile phase to collect fractions and is concentrated. Each fraction was evaluated
by TLC to know how many different phytoconstituents are there in it.
Fractions showing same number of compounds and Rf values were combined, concentrated and
evaporated to dryness.
TLC of Column Eluates
Adsorbent : Silica gel G
Solvent System : Benzene:Chloroform:Eethanol
Proportion : 45:45:10
Visualizing agent : 2% Boric acid in Methanol [40,41]
.
Characterization of Phytoconstituents using spectroscopy techniques:
All the separated compounds from petroleum ether root extract of Curcuma zedoaria Rosc were
characterized by physical, chemicals and spectroscopy technique such as
UV Spectrum (Ultra violet visible spectroscopy)
FTIR Spectrum
HPLC (High performance liquid chromatography)
HPTLC (High performance thin layer liquid chromatography)
HNMR (proton nuclear magnetic resonance)
LCMS (Liquid chromatography mass spectroscopy)
GCMS (Gas chromatography mass spectroscopy)
Development of Single herbal Formulation214
In the present study, it was thought worthwhile to formulate extracts of in the form of
suspension. The formulation of all bio-active extracts was prepared by applying probability for
that all the bio-active extracts were mixed in equal proportion as per the probability applied and
LD 50 cut off value was calculated and according to the cut off value formulation was done.
1. Formulation contains pet ether, [PEE]+chloroform [CHE] and ethanol extracts [ETE] of root
of Curcuma zedoaria Rosc (CZ)
(Single Herb formulation-A)
2. Formulation contains pet ether [PEE] and chloroform extract [CHE]of root of CZ
(Single Herb formulation-B)
3. Formulation contains pet ether [PEE] and ethanol extract [ETE]of root of CZ
(Single Herb formulation-C)
4. Formulation contains chloroform [CHE] and ethanol extract [ETE] of root of CZ
(Single Herb formulation-D)
Procedure 25
The bioactive extracts will be mixed with light kaolin in a mortar and add compound powder of
tragacanth than orange syrup is added and triturate so as to form a smooth cream. The foreign
particles are removed with the tip of glass rod than benzoic acid solution was incorporated and
amaranth solution previously diluted with chloroform water was added and stir thoroughly so as
to form a uniform mix. Add more of orange syrup up to the required volume.
Methodology for development of single herbal formulations 214
Animals
Female Wistar rats between 2 and 3 months of age weighing 160 ± 40 g were used which
obtained from JNMC, central animal house. All animals were housed in an animal room under
normal condition of 24±1 0C, 12-h light and dark cycle and 55±5% humidity. The study designs
were approved by the Institutional Animal Ethical Committee of K.L.E.’S College of Pharmacy,
Belgaum, India, (Resolution No. 31/7/2010-13).
Acute toxicity study
The acute toxicity studies were carried out according to the guidelines set by CPCSEA, OECD
(425). Starting dose was selected in an increasing order of 175, 550, 1775, 2000 and 5000 mg/kg
b.w. for each extract was evaluated for toxicity studies. Each dose of extracts were subjected to
female Wistar rats and LD50
dose were selected for further pharmacological activity [OECD
guideline 2001].
Basic formula for single herbal formulation
Formulations
Curcuma
zedoaria
root
extracts
Ingred
ients
Qu
an
tity
Quantity required of additives
Light
kaolin
Compound
tragacant
Amaranth
solution
Benzoic
acid
solution
Orange
syrup
Formulation A PEE+CHE
ETE extract
0.6 gm 25% 0.1% 0.05% 0.2% qs to
100ml
Formulation B PEE+CHE
extract
0.4 gm 25% 0.1% 0.05% 0.2% qs to
100ml
Formulation C PEE+ETE
extract
0.4 gm 25% 0.1% 0.05% 0.2% qs to
100ml
Formulation D CHE +ETE
extract
0.4 gm 25% 0.1% 0.05% 0.2% qs to
100ml
Preparation of drug and animal groups
The animals were divided into eight groups of six animals in each. Mineral oil injected in left
ankle joint of rats as normal group received normal saline and FCA injected group as control
group also received normal saline, indomethacin was used as standard drug, rumalaya forte was
used as herbal standard drug (Marketed preparation for arthritic disease,) ethanol and aqueous
extracts of CZ were used as test drugs. Dose calculation was based on w/w of each extract and
doses were selected on the basis of our previous study [10]. Selection of animals groups taken
for experiment were as follows
Normal Group
Normal: Mineral oil+Normal Saline (p.o)
Control Group
Control: FCA+Normal Saline (p.o)
Standard groups:
Standard-I: FCA+10 mg/kg Indomethacin (i.p)
Standard-II: FCA+200 mg/kg, p.o Rumalaya forte
Test groups:
SHF-A: FCA+200 mg/kg, p.o Single herb Formulation-A
SHF-B: FCA+200 mg/kg, p.o Single herb Formulation-B
SHF-C: FCA+200 mg/kg, p.o Single herb Formulation-C
SHF-D: FCA+200 mg/kg,p.o Single herb Formulation-D.
Induction of monoarthritis
Pre-induction Baseline was taken prior to injection of Complete Freund,s Adjuvant (CFA)
measured by left paw volume of each animal at 0 day for the induction of monoarthritis. All rats
were anaesthetized with 40 mg/kg thiopentone sodium intra-peritoneal injections. Once
anaesthetized, 0.1 ml CFA was injected in left ankle joint of rats (Butler et al., 1992). A 26-
gauge needle was introduced into the capsule of the tibiotarsal joint percutaneously by directing
it cephalad, mesiad and superiorly from the midpoint of the inframalleolar fossa until a distinct
loss of resistance was felt approximately 4 mm and complete adjuvant or vehicle injected. With a
true intracapsular injection, a firm resistance to injection was characteristically felt after the
injection of 0.1 ml of CFA fluid (Hong et al., 2008).
Paw edema
The severity of adjuvant arthritis was quantified by measuring the volume of hind paw using
Plethysmometer. Paw volume (ml) was measured on 0 days and thereafter 3, 7, 14, 21, 28,
35, and 42 days of CFA postinoculation. Data were expressed as the increase in paw volume
with respect to day 0 paw volume.
Behavioral observation (open-field test)
For behavioral observations, all the animals were subjected to open-field test before the
induction of arthritis and thereafter 3, 14, 21, 28, 35, and 42 days of postinoculation of FCA
injection. Rat was placed in an open field in the sound-attenuated room. The floor was white
polyvinyl with a black grid dividing open field into 84 squares (10 × 10). Illumination was
provided by a bulb (60 W) placed above the center of the field, while the rest of the room was
darkened.[25,26]
The rat was initially placed in the center of the open field and all the behavior
test were observed for 5 minutes. . After each animal observations test, the open field was
cleaned with wet sponge and tissue paper[27]
and all observations were made between 18.00 and
20.00 hours.
Based on the previous scientific data on behavioral observations of normal rats, the following
behaviors were quantified: (I) Latency time to explore: means that time taken “to start explore
(second)” from insertion time; (II) Ambulatory behavior: means that the rat “crossed grid line”
(horizontal locomotor activity); (III) Rearing, means that the “look for” sometime in air for this it
elevates its head and forepaws, almost standing up, (vertical locomotor activity); (IV) Grooming
behavior: means licking, rubbing the ears, nose, and head or the snout with forepaws and
preening; (V) Urinations: (number of urine passes) considered as anxiety behavior of rat; (VI)
Defecation: (number of boluses pass) also considered as anxiety behavior of rat during open-
field observation.[28]
Measurement of physiology profile
Body weights were observed at 0, 3, 7, 14, 21, 28, 35, and 42 days. All animals were
anaesthetized and blood was collected from retro-orbital plexus of the entire arthritic and non-
arthritic animals in plain EDTA containing tubes respectively. Samples were subjected to
physiological examinations (Jain 1999.,), such as Hemoglobin level, Erythrocyte Sedimentation
Rate (ESR), Red blood cell (RBC), White blood cell, (WBC) (Kale and Kale, 1999). Differential
platelet counts: Neutrophils, Lymphocytes and Monocytes (Jain, 1998).
Measurement of biochemistry profile
Blood were withdrawn from arthritic and non arthritic rats from retro orbital plexus on the last
day of experiment. Serum samples were collected after blood centrifugation at 3000 X g for 10
minutes. These samples were used to determine aspartate amino transferase (AST), alkaline
amino transferase (ALT) blood urea nitrogen (BUN), uric acid, creatinine (Cr), and total protein,
were tested using commercial kit by auto analyser to detect any effects on liver and kidney
functions24
.
Nitric oxide synthesis
Serum was separated from each group of animals. Sodium nitroprusside (5 Mm) in standard
phosphate buffer solution with different serum samples dissolved in standard phosphate buffer
(0.025 M, pH 7.4) solutions were incubated in equal amount at 250C for 5 h. After 5 h, 0.5 ml of
incubation solution was removed and diluted with 0.5 ml of Griess reagent. The absorbance was
read at 546 nm using Shimadzu UV-Visible spectrophotometer (Shriwaikar et al., 2003).
Assessment of vascular permeability
Evan’s blue (50 mg/kg) was administered via the jugular vein into the anaesthetized rat. After 4
h, the anterior and posterior synovial capsules and fat pad were dissected from each ankle joint,
which were small; thus, tissues obtained from four ankles were grouped to form one sample. The
samples were then weighed, and the amount of Evans blue in the sample was estimated using
dye extraction technique (Lam et al., 2004).
Radiography examination
At the end of the experiments, all rats were anesthetized with 40 mg/kg sodium thiopental
intraperitoneal injection. Once anesthetized, the animals were kept on X-ray plates, the
projections of the left ankle joint were taken at day 42. The following parameters were evaluated
blind using the tarsometatarsal region: erosion, a destruction of bony structure resulting in
irregular bone surface; periosteal reaction, a fine ossified line, paralleling normal bone producing
bone thickening; increase in soft tissue which was manifested as an increase in width of the soft
tissue and calcification. The parameters were using score which follows: 0, no sign; 1, mild; 2,
moderate; and 3, severe.[29]
Histopathology examination
Animals were scarified 43 day after the induction of arthritis. Left ankle joint were removed and
post fixed with 10 % formalin for 10 day and then decalcified in 5% formic acid. Left hind paws
were removed from all groups of animals and post fixed with normal saline and then decalcified
in 5% formic acid. Joints were then trimmed, embedded and sectioned at 6 μm. Sections were
then stained with haematoxyline and eosin. Pathology lesions of rats ankle joint were graded on a
blind scale under light microscopic 100 X (Mcdougall et al., 1995) Histological evaluation was
carried out according to the following scale Nil 0, Mild 1, moderate 2 and marked 3 and the
following considered parameters skin conjestion, skin odema, skin inflammatory infiltration,
synovial ulceration, synovial nutrophilic infiltration, synovial lymphocytic infiltration, synovial
macrophages, synovial granulation tissue, synovial granulations tissue, synovial cellular
degeneration, cartilage destruction and bone destruction.
Organ to body weight ratio (toxicology)
All groups were sacrificed after 42 days of last dose for calculating organ to body weight ratio.
isolated and weighed the vital organs viz spleen, thymus, adrenals, stomach, liver, heart, kidney
Lungs and Brain, Evaluation the relationship between organ weight and body weight to
determine which endpoint (organ weight, organ-to-body weight ratio,) is likely to accurately
detect target organ toxicity. This relationship assumes that the ratio of organ weight (Y) to
body weight (X) within each treatment group is some constant μ, i.e., Y/X = μ29
.
% Change in organ body weight ratio = C-C1/C x 100
C denoted by Normal group and C1 denoted by drug
treated+FCA
Anti-inflammatory activity (acute inflammation)
Carrageenan induced paw edema
Before the experiment, food was withdrawn overnight but adequate water was given to the rats.
Dose selected were 200 mg and 400 mg/kg for each formulation. The animals were divided into
eight groups of 6 animals each. All the doses were given orally half an hour before the
administration of carrageenan (Sigma chemical co, St. Louis MO, USA) and histamine into the
plantar side of the left hind paw. The paw was marked with ink at the level of lateral malleolus
and immersed in mercury up to the mark in the plethysmometer.13
The paw volume was
measured after (1 h) injection carrageenan and then every hour till 6 h of each group. The
difference between the initial and subsequent reading gave the actual edema volume. The
average paw swelling is calculated by comparing the normal group with control, Standards and
all treated groups compared with the control, Percent inhibition of inflammation was calculated
by using the formula,
% inhibition = 100 (Vt/Vc x100)14-16
. Where ‘Vc’ represents edema in control.
‘Vt’ is the edema in drug treated group .
Histamine–Induced paw edema:
For the study of Histamine – induced paw edema in the animals were treated exactly the same
method as carrageenan induced model but instead of carrageenan, here 0.1 ml of 1% w/w
histamine in normal saline was used.
Statistical analysis: The values are expressed as means ± S.E.M. The results were analyzed
statistically by one-way ANOVA followed by Dunnett’s multiple comparison tests using Graph
pad prism version. The difference was considered as significant P value less than 0.05, when
ap<0.001,
bp<0.01,
c p<0.05
Accelerated stability studies of formulations216-217
The accelerated stability studies carried out for single-herbal formulations of bio-active
constituents at Temp 40C ± 2 0C at 80 % humidity. The stability studied for the period of three
months. The different parameters such as colour, odour, , viscosity, pH, sedimentation
volumeand redispersibility test will be studied for all the formulations at 1st, 2nd and at 3rd
months. This showed that formulations are stable in nature.
Table 1: Qualitative analysis of extract and isolated compound performed by
chemical test was further supported by thin layer chromatography
Evaluation of the chromatogram
Qualitative
analysis
Under day light Under UV After Spray
366nm
Standard
Curcuminoids
No of Sport
observed
3 3 3
Color of spots Yellowish Yellowish Radish
Rf Values of
each spot
IA-0.47, IB-
0.38, IC- 0.31
IA-0.47, IB 0.38, IC-
0.31
IA-0.47, IB-0.38,
IC- 0.31
Petroleum ether
extract
No of Sport 3 4 3
Color of spots Yellowish Yellowish Radish
Rf Values of
each spot
IA-0.46, IIB-
0.35, IIIC-0.27
IA-0.46, IIB .35, IIIC-
0.27, IVD-0.16
IA-0.46, IIB-
0.35,IIIC-0.27
Isolated
Compound-I
(By Preparative
TLC)
No of Sport 2 3 3
Color of spots Yellowish Yellowish Radish
Rf Values IA-0.46, IIB-
0.35, IIIC-0.27
IA-0.46, IIB-0.35,
IIIC-0.27, IVD-0.16
IA-0.46, IIB-0.35,
IIIC-0.27
Isolated
Compound-II
(By Column)
No of Sport 1 1 1
Color of spots Orange yellow Dark yellow Reddish
Rf Values 1A- 0.46 1A- 0.46 1A- 0.46
Isolated
Compound-III
By Column)
No of Sport 2 2 2
Color of spots Light yellow yellow Reddish
Rf Values 0.35, 0.28 0.35, 0.28 0.35, 0.28
Isolated
Compound-IV By
Column)
No of Sport 1 1
Color of spots Light yellow Yellow
Rf Values 0.34 0.34, 0.34
1
2
3
4
1
2
3
1
2
3
1
2
A B
H
E
D C
F
G
Details of the fractions eluted by the column chromatography
Data for column eluents of methanol soluble fraction of Isolated compound 1.
Fractions Solvent
Combination
( Chloroform :
Methanol )
TLC Studies
Benzene : Chloroform : Ethanol (45:45:10)
Number of
Spots
Colour Rf Values
1-4 100%
Chloroform.
No spot ---- ----
5-8 1% Methanol in
Chloroform.
No Spot ---- ----
9-12 1.5% Methanol in
Chloroform.
No Spot ---- ----
13-22 2% Methanol in
Chloroform.
1 Spot Yellowish
orange
0.46
23-28 3% Methanol in
Chloroform in
No Spot ---- ----
29-40 3.5% Methanol in
Chloroform.
No Spot ---- ----
41-60 4% Methanol in
Chloroform.
No Spot ---- ----
61-82 4.5% Methanol in
Chloroform.
No Spot
---- ----
83-105 5.0% Methanol in
Chloroform.
2 Spot Light yellow 0.35, 0.28
106-114 5.5% Methanol in
Chloroform.
1 Spot Light yellow 0.34
115-125 6.0% Methanol in
Chloroform.
No spot ---- ----
126-131 7% Methanol in
Chloroform.
No spot ---- ----
132-137 8% Methanol in
Chloroform.
No spot ---- ----
138-143 9% Methanol in
Chloroform.
No spot ---- ----
144-150 10% Methanol in
Chloroform.
No spot ---- ----
Characterization of isolated phytoconstituents using U/V spectroscopy
U/V spectroscopy
Qualitative analysis
of samples
No of Phytochemicals
identified
Absorbance Rf value
AU ƛ max
Standard
Curcuminoids
(3) Mixture of three
compound
IA, 62 200 0.39
IB, 107 406 0.46
IC, 141 427 0.59
Petroleum ether extract (5) Mixture of five
compound
IA 122 200 0.37
IB 108 200 0.43
IC 793 428 0.54
ID 148 200 0.75
IE 153 200 0.77
Isolated Compound-I
(4) Mixture of four
compound
IA 203 369 0.54
IB 327 378 0.75
IC 716 428 0.77
ID 244 200 0.34
Isolated Compound-II (1) single compound IA 86 200 0.39
Isolated Compound-III (4) Mixture of four
compound
IA 118 200 0.39
IB 168 200 0.51
IC 73 200 0.49
ID 63 200 0.73
Isolated Compound-IV (2) Mixture of two
compound
IA 46 200 0.48
IB 42 200 0.32
Isolated Compound-V
(2) Mixture of two
compound
IA 48 214 0.38
IB 57 200 0.34
Isolated Compound-VI
(2) Mixture of two
compound
IA 70 200 0.39
IB 50 214 0.49
Characterization of isolated phytoconstituents by HPTLC
Qualitative
analysis of
samples
No of
Phytochemicals
identified
HPTLC Data
Sta
rt
Rf
Max
Rf
En
d
Rf
Sta
rt
Heig
ht
Max
heig
ht
En
d
Heig
ht
Area
%
Standard
Curcuminoids
(3) Mixture
of three
compound
IA, 0.35 0.37 0.39 2.4 73.1 0.7 2.61
IB, 0.40 0.44 0.47 1.6 294 78.2 16.86
IC, 0.47 0.55 0.60 78.2 687.8 3 80.53
Petroleum ether
extract
(5) Mixture
of five
compound
IA 0.29 0.33 0.35 0 54.5 10.8 2.68
IB 0.40 0.45 0.47 1 256.2 125 14.35
IC 0.47 0.52 0.57 125 714.8 32.4 72.04
ID 0.58 0.61 0.62 31.3 72.8 65.9 4.29
IE 0.63 0.66 0.68 66.4 91.6 4.4 6.63
Isolated
Compound-I
(By Preparative
TLC)
(4) Mixture
of four
compound
IA 0.29 0.35 0.37 10.2 50.8 45.7 9.14
IB 0.37 0.43 0.45 45.1 229.3 65.5 27.16
IC 0.47 0.53 0.60 57 379.6 10.1 61.75
ID 0.75 0.78 0.81 5.5 16.8 0.2 1.96
Isolated
Compound-II
(Column)
(1) single
compound
IA 0.44 0.52 0.57 2.4 720.3 1.9 100
Isolated
Compound-III
(Column)
(3) Mixture
of three
compound
IA 0.27 0.34 0.35 6.3 27 17.8 6.27
IB 0.39 0.43 0.45 25 89.4 46.2 13.63
IC 0.63 0.64 0.68 14.7 33.5 3.1 7.94
Isolated
Compound-IV
(Column)
(2) Mixture
of two
compound
IA 0.37 0.39 0.41 6.8 26.7 18.3 72.94
IB 0.67 0.68 0.72 9.6 11 3.2 27.06
Isolated
Compound-V
(0)
0 0 0 0 0 0 0 0
Isolated
Compound-VI
(0)
0 0 0 0 0 0 0 0
A
G F E
B C D
H
HPTLC Photographs showed that A: standard curcuminoids peaks {1 peaks Curcumin, 2
peaks Demethoxycurcumin and 3 peaks Bisdmethoxycurcumin} B: crude extract peaks C:
Isolated compound-I peaks D: Isolated compound-II peak E: Isolated compound-III, F:
Isolated compound–IV peaks G and H showed no peaks it’s were considered as compound-V
and compound-VI. All peaks compared with stand curcuminoids peaks.
HPTLC Track-I-8
represents the
comparative data
Table: I Acute oral toxicity study of Single herb formulations of Curcuma zedoaria Rosc Root in Rats
Formulation-A, B,C, and D. Dose Range: 175, 550,1500, 2000 mg/kg, p.o.
Species: Female Wistar albino rats Duration: 14 days
Guidelines reference: OECD (425)
Extracts (Dose
mg/kg, p.o)
Body Weight animals
Toxicity
Time of
death
Observations
1st
Day 7
th
Days 14
th
Days Onset Stop Skin
Eyes Resp CNS Tre Con Sali Diah Sleep Leth Coma
175
∞ ∞ ∞ x x x x x x x x x x x x x x
550
∞ ∞ ∞ x x x x x x x x x x x x x x
1500
∞ ∞ ∞ x x x x x x x x x x x x x x
2000 ∞ ∞ ∞ x x x x x x x x x x x x x x
5000
∞ ∞ ∞ x x x x x x x x x x x x x X
Each dose of formulation given to rats in an increasing order such as 175, 550,1500, 2000 mg/kg, p.o. and then administered to other
four rats for each increasing order dose
∞= slightly increase the body weight
At 2000 mg/kg no sign of toxicity or death were observed
Formulation-A, B, C, and D, no showed any type of sign and symptom at 2000 mg/kg body weight
Selected doses for Pharmacological activities were 200 mg/kg of each formulation
Annexure-III
III. PROGRESS/ STANDARDIZATION IN RESEARCH METHODOLOGY
Table: 1 Effect of Single herb formulations of Curcuma zedoaria Rosc root on subcutaneous FCA-induced changes in Paw
edema
Paw Edema (ml)
Treatment
Groups
(n=6)
Dose
(mg/kg)
0 day 1 day 6 day 9 day 12day 15
day 18 day 21 day
Normal
5 ml
Control 5 ml
Standard-I 10
Standard-II 200
SHF-A
200
SHF-B
200
SHF-C
200
SHF-D 200
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on intrarticular FCA-induced changes in Paw
edema
Paw Edema (ml)
Treatment
Groups(n=6)
Dose
(mg/kg)
0 day 3 day 7 day 14 day 21day 28
day 35 day 42 day
Normal
5 ml 0.042± 0.020 0.51± 0.073 0.43 ±0.078 0.06 ± 0.033 0.045±0.022 0.05 ±0.025 0.05 ±0.028 0.062 ± 0.026
Control 5 ml 0.028±0.06 2.400 ±0.144
a 2.37 ±0.117
a 2.42 ±0.117
a 2.42 ±0.114
a 2.28 ±0.10
a 2.13±0.076
a 1.97 ±0.067
a
Standard-I 10 0.018 ±0.009 1.567 ±0.084
a 1.67 ±0.072
a 1.65 ± 0.077
b 1.03 ±0.096
a 0.90±0.09
a 0.77 ±0.05
a 0.62 ± 0.047
a
Standard-II 200 0.068 ±0.023 1.467 ±0.089
a 1.62 ±0.070
a 1.50 ± 0.171
a 0.91 ±0.095
a 0.77±0.07
a 0.65±0.043
a 0.57 ±0.056
a
SHF-A
200 0.043±0.01 1.450± 0.076
a 1.65± 0.076
a 1.567± 0.084
a 0.970±0.07
a 0.80±0.03
a 0.73±0.030
a 0.63± 0.040
a
SHF-B
200 0.067± 0.015 1.75±0.152
c 1.87±0.095c 1.817± 0.070
a 1.55± 0.117
a 1.27± 0.06
a 1.21± 0.067
a 1.09± 0.086
a
SHF-C
200 0.056±0.025 1.62±0.06
a 1.80± 0.086
b 1.60±0.110
a 1.20±0.088
a 1.07±0.07
a 0.975±0.07
a 0.79±0.056
a
SHF-D 200 0.065±0.028 1.58±0.0654a
1.77±0.033a
1.47±0.049a
1.28±0.079a
1.06±0.04a
0.695±0.07a
0.62±0.034a
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in Latency time to explore
in rats
Latency time to explore (seconds)
Treatment
Groups
(n=6) (Dose
mg/kg)
0 day 3rd
day 7th
day 14th
day 21th day 28th
day 35 day 42day
Normal
2.50±0.224 4.50±0.42
b 3.33±0.803 2.67±0.333 2.33±0.211 2.00±0.365 2.0±0.258 1.83±0.307
Control
1.83±0.307 6.50±0.764
a 7.16
± 0.31
a 7.17± 0.601
a 7.33
±0.422
a 6.83±0.477
a 5.83
±0.307
a 6.0
± 0.683
a
Standard-I 2.67±0.422 5.17±0.601 5.50±0.764c 3.67±0.494 3.50±0.764 3.17±1.046 2.50±0.428 2.0±0.258
Standard-II
3.00±0.258 5.00±0.577
c 5.33±0.421
c 4.17±0.654
c 3.83±0.601 3.83±0.703 2.83±0.601 2.67±0.494
SHF-A
2.17±0.477 4.50±0.619 5.50±0.719 4.67±0.715 4.00±0.516 3.50±0.764 3.01±0.730 2.33±0.494
SHF-B
2.67±0.422 5.67±0.333 6.67±0.333
b 5.50±0.428
c 4.17±0.601 3.67±0.558 3.50±0.428 3.33±0.494
SHF-C
2.00±0.365 5.00±0.365
a 5.17±0.401
a 4.17±0.307
a 4.00±0.447
b 4.00±0.258
c 2.67±0.211 2.83±0.307
SHF-D 3.17±0.307 5.33±0.211
a 5.67±0.422
a 4.83±0.307
b 4.50±0.224
a 4.17±0.307 4.00±0.258 3.83±0.167
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in Ambulatory behavior in
rats
Ambulatory
Treatment
Groups
(n=6) (Dose
mg/kg)
0 day 3rd
day 7th
day 14th
day 21st day 28
th day 35 day 42day
Normal
46.83±6.10 41.83±5.13 88.17±10.8
c 103.0±5.79
a 68.0±8.41 72.33±9.07 56.50±5.065 48.67±4.51
Control 49.00±5.43 33.00±3.57
a 31.00±2.89
b 23.0±2.236
a 19.50±2.14
a 19.50±2.84
a 18.50±3.54
a 17.00±2.66
a
Standard-I 71.00±4.56 64.17±4.04 66.83±4.27 62.33±2.87 67.83±4.24 63.50±5.09 64.83±5.19 64.83±5.00
Standard-II 53.17±9.99 46.17±7.85 90.33±14.95 70.3±15.67 43.50±8.50 65.83±8.29 59.0±10.82 55.3±10.65
SHF-A
61.17±1.89 51.67±2.74 49.17±2.54 53.50±2.54 53.67±1.09 56.17±1.08 58.50±1.43 59.50±1.73
SHF-B
57.33±4.54 53.17±6.34 46.50±3.91 42.00±4.51 41.50±4.26 37.83±3.83
c 36.83±4.04
c 34.50±3.79
b
SHF-C
58.33±4.08 50.33±4.77
b 41.50±2.53
b 42.67±2.29 52.83±2.75 57.50±3.37 55.50±1.544 57.50±1.65
SHF-D 64.33±3.23 54.50±3.65 47.00±4.95 51.00±4.98 56.00±4.46 58.33±4.41 61.33±4.06 62.33±3.51
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in Rearing behavior in rats
Rearing
Treatment
Groups
(n=6) (Dose
mg/kg)
0 day 3rd
day 7th
day 14th
day 21st day 28
th days 35 day 42 day
Normal
16.17±1.662 7.00±0.58
a 6.50±1.26
a 9.67±1.99 9.500±0.76 10.7±0.803 14.0±1.63 13.17±1.40
Control 12.17±1.922 3.83±0.873
a 2.50±0.428
a 2.00±0.258
a 1.50±0.22
a 1.00±0.516
a 1.17±0.31
a 0.833±0.40
a
Standard-I 15.83±2.088 7.17±1.58
b 13.50±1.38 17.50±1.54 9.33±1.33
c 10.67±1.43 10.33±1.12 9.50±1.607
c
Standard-II 11.50±2.172 6.83±1.25 5.50±1.84 8.17±1.54 9.00±1.69 7.67±2.16 8.67±1.12 8.50±1.41
SHF-A
14.83±1.167 10.17±0.95 6.50±0.43 8.17±0.601 10.17±0.87 11.33±1.33 12.00±1.32 12.83.0910
SHF-B
13.50±1.335 7.67±0.803 5.67±0.760 4.50±0.764 5.50±0.764 5.67±0.882
c 6.17±1.11
c 5.83±1.05
b
SHF-C
15.17±1.54 10.67±0.96
b 7.50±1.088
a 9.00±0.817
c 10.83±1.01 12.8±1.20 13.67±1.20 14.17±1.01
SHF-D 17.50±1.727 11.33±1.56 9.50±1.057
c 12.67±1.12 14.17±1.17 15.00±1.18 14.83±1.20 15.50±1.34
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in Grooming behavior in
rats
Grooming
Treatment
Groups(n=6)
(Dose mg/kg)
0 day 3rd
day 7th
day 14th
day 21st day 28
th Days 35 day 42day
Normal
3.83±0.872 4.33±0.84 4.67±0.882 4.83±0.477
c 1.33±0.211 2.50±0.563 1.50±0.563 1.17±0.307
c
Control
2.50±0.428 3.33±1.02 8.00±1.238 5.83±0.601
c 4.33±0.558 4.17±0.792 3.17±0.543 2.17±0.167
Standard-I
2.00±0.817 2.17±0.307 2.33±0.422 3.67±0.843 2.0±0.730 2.17±0.307 1.83±0.307 1.50±0.342
Standard-II
1.67±0.211 2.83±0.601 2.83±0.872 3.67±0.558
c 2.00±0.365 1.50±0.224 1.667±0.422 1.17±0.401
SHF-A
3.00±0.577 4.50±0.563 5.00±0.365
c 3.167±0.307 2.00±0.365 2.00±0.365 1.667±0.558 1.50±0.224
SHF-B
3.83±0.601 4.17±0.307 5.50±0.224 6.17±0.4773
c 6.83±0.307
a 5.83±0.601
c 5.00±0.577 3.50±0.671
SHF-C
3.50±0.428 5.17±0.478 6.00±0.577
b 4.50±0.428 2.67±0.333 2.33±0.558 2.167±0.477 2.00±0.447
SHF-D 2.83±0.601 4.50±0.562b
5.17±0.601 3.50±0.428 3.17±0.477 3.33±0.494 3.00±0.365 2.00±0.258
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in anxiety behaviour
(Urinations)
Treatment
Groups
(n=6) (Dose
mg/kg)
0 day 3rd
day 7th
day 14th
day 21th day 28th
day 35 day 42day
Normal
0.83±0.307 0.67±0.33 1.33±0.211 1.00±0.365 0.50±0.224 0.33±0.212 0.50±0.224 0.333±0.212
Control 1.17±0.167 0.33±0.211 2.17±0.307
c 1.33±0.422 1.00±0.365 0.67±0.333 0.17±0.167
c 0.167±0.167
c
Standard-I
1.50±0.224 1.00±0.258 0.83±0.167
a 0.67±0.333
a 0.0±0.0
a 0.0±0.0
a 0.0±0.0
a 0.0±0.0
a
Standard-II
1.83±0.307 1.67±0.211 2.00±0.365 0.50±0.342
b 0.17±0.167
a 0.167±0.167
a 0.0±0.0
c 0.0±0.0
a
SHF-A
1.67±0.333 1.33±0.333 1.500±0.224 1.00±0.258 0.667±0.33 0.500±0.224
c 0.33±0.211
b 0.167±0.167
b
SHF-B
2.00±0.258 2.17±0.307 1.83±0.307 1.83±0.167 1.50±0.224 1.167±0.167 1.00±0.258
c 0.833±0.307
c
SHF-C
2.17±0.167 2.50±0.224 1.83±0.401 1.67±0.422 0.83±0.303 0.33±0.212
b 0.17±0.167
a 0.0±0.0
a
SHF-D 1.33±0.494 2.00±0.258 1.67±0.422 1.50±0.500 1.00±0.258 0.500±0.224 0.0±0.0c
0.0±0.0c
Table: 2 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in anxiety behavior
Defecations
Treatment
Groups
(n=6) (Dose
mg/kg)
0 day 3rd
day 7th
day 14th
day 21st day 28
th day 35 day 42day
Normal
4.33±0.760 3.00±0.816
a 2.17±0.543
a 1.00±0.258
a 0.50±0.227 0.0±0.0
a 0.0±0.0
a 0.0±0.0
a
Control 3.83±0.946 2.83±0.703 2.33±0.333 2.50±0.428 2.00±0.365 1.00±0.36b
1.17±0.307b
1.17±0.749c
Standard-I
2.83±0.601 2.83±0.477 3.00±0.577 2.33±0.422 0.50±0.224
b 0.33±0.211
a 0.50±0.347
a 0.33±0.211
b
Standard-II
2.50±0.563 1.33±0.333 1.17±0.307
c 0.50±0.224
a 0.50±0.342
a 0.17±0.167
a 0.0±0.0
a 0.0±0.0
c
SHF-A
2.67±0.333 2.50±0.563 2.17±0.703 1.83±0.601 1.00±0.365 0.50±0.342
b 0.33±0.211
b 0.167±0.167
b
SHF-B
3.17±0.477 2.83±0.307 2.50±0.342 1.33±0.333
b 1.67±0.333
c 1.33±0.333
b 0.83±0.307
a 0.0±0.0
a
SHF-C
2.33±0.211 1.83±0.401 1.67±0.333 1.33±0.211 1.17±0.303 1.00±0.365
c 0.50±0.342
a 0.33±0.211
a
SHF-D
3.00±601 2.17±0.333 1.33±0.307
b 1.17±0.224
b 0.50±0.341
c 0.50±0.167
c 0.17±0.167
c 0.167±0.601
c
Table: 3 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in body weight
Body weight in days
Treatment
Groups
(n=6) (Dose
mg/kg)
Dose
(mg/kg)
0 day 3rd
day 7th
day 14th
day 21st day 28
th day 35 day 42day
Normal
5 ml 157.8±7.31 161.3±4.79 161.3±4.79 161.5±4.48 166.3±4.24 166.3±4.57 167.2±4.32 167.2±4.44
Control 5 ml 172.0±12.5 168.3±12.3 170.2±13.0 160.8±11.9 161.8±10.7 145.5±9.42 143.3±9.39 131.3±9.93
Standard-I 10 167.8±5.09 160.2±4.08 159.7±4.15 161.3±4.39 163.7±4.69 166.3±4.05 168.5±4.62 172.2±5.06
Standard-II 200 164.7±15.9 161.7±15.9 163.3±15.9 164.0±15.9 162.5±16.18 162.2±16.28 161.5±16.77 161.0±17.13
SHF-A
200 161±6.14 158±6.71 157±6.24 158±6.19 162±5.46 166±4.88 167±4.92 167±4.98
SHF-B
200 169±6.09 168±6.47 166±6.46 149±18.3 165±6.46 163±5.96 163±5.88 163±5.58
SHF-C
200 178±3.36 176±3.53 175±3.51 176±3.64 178±3.73 180±3.77 180±3.82 181±3.83
SHF-D
200 170±5.76 169±5.26 169±4.94 171±5.00 172±4.63 174±4.77 175±4.72 177±4.96
Table: 3 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced change in hematology
Hematology profile
Treatment
Groups
(n=6) (Dose
Dose
(mg/kg)
Hemoglobin
g/dl
ESR
mm/h
WBC
×103/mm
3
RBC
×106/mm
3
Differential count mean %
Neutrophils Lymphocyte
s
Monocytes
Normal
5 ml 14.13 ± 0.604 3.817 ± 0.142 10.37 ± 0.436 7.014 ± 0.352 20.00 ± 3.204 76.50 ± 4.311 1.167 ± 0.167
Control
5 ml 11.58 ± 0.292c
12.42 ± 0.86a
11.98±0.632 6.867 ± 0.563 37.17 ± 3.609b
55.00 ± 4.59b
6.833 ± 1.25a
Standard-I
10 13.50 ± 0.183
a 5.750 ±0.519
a 10.65 ± 0.489 6.800 ± 0.139 24.00 ± 2.477
b 74.3 ± 3.62
b 3.500 ±1.20
b
Standard-II
200 13.95 ± 0.272
a 5.883 ±0.364
a 10.53 ± 0.670 7.150 ± 0.397 25.00 ± 2.595
b 70.50 ± 1.91
c 3.167 ± 0.65
SHF-A
200 13.65±0.3713
a 6.717±0.221
a 11.20±0.647 6.983±0.1815 26.50±2.872 72.17±4.771
c 2.167±0.401
a
SHF-B
200 12.05±0.2655 10.25±0.458 11.68±0.566 6.850±0.2487 35.50±2.487
c 61.00±2.352 5.167±0.307
SHF-C
200 12.93±0.211
b 6.383±0.556
a 10.63±0.470 7.000±0.3011 30.33±1.626 68.83±3.458 2.500±0.428
a
SHF-D
200 13.52±0.2798
a 5.817±0.199
a 10.75±0.388 6.583±0.1579 30.00±2.955 71.50±3.181
c 4.500±0.671
Table no: 4 Effect of Curcuma zedoaria Rosc root extracts on Liver and kidney functions of FCA-induced arthritic Rats
Biochemistry profile
Treatment
Groups
(n=6) (Dose
mg/kg)
Dose
(mg/kg)
SGOT
(IU/L)
SGPT
(IU/L)
Total serum
Proteins
g%
Creatinine
mg/dl
Uric acid Blood Urea
nitrogen mg/%
Normal
5 ml 57.37 ± 1.200 62.14 ± 3.601 6.86 ± 0.238 0.742 ± 0.620 5.36 ± 0.040 42.20 ± 2.52
Control
5 ml 56.59 ± 0.972 65.40 ± 4.44 6.75 ± 0.318 0.784 ± 0.690 5.375 ± 0.044 42.21 ± 2.52
Standard-I
10 56.9 3± 3.99 62.73 ± 1.93 6.76 ± 0.369 0.769 ± 0.740 5.596 ± 0.221 47.10 ± 2.73
Standard-II
200 56.93 ± 2.03 64.06 ± 3.232 6.60 ± 0.222 0.730 ± 0.020 5.72 ± 0.223 47.16±2.10
SHF-A
200 57.46±1.508 62.53±3.062 6.66±0.133 0.750±0.025 5.70±0.193 42.16±2.42
SHF-B
200 56.66±2.251 65.41±4.491 6.74±0.1776 0.777±0.022 5.74±0.216 42.14±1.69
SHF-C
200 56.19±2.013 62.23±1.862 6.45±0.20 0.765±0.035 5.69±0.189 42.97±0.909
SHF-D
200 56.51±1.986 62.84±2.812 6.663±0.175 0.760±0.040 5.630±0.2777 42.70±1.501
Table no: 5 Effect of Single herb formulations of Curcuma zedoaria Rosc root on FCA-induced serum nitric oxide synthesis
and vascular permeability
% INHIBITION AT DAY 42
Treatment Groups
(n=6) (Dose mg/kg)
Dose
(mg/kg)
Nitric oxide % Inhibition Concentration of Evans
Blue in (µg/g) in fat pad
% Inhibition
of
Infiltration
Normal 5 ml 4.80±0.4227 -- 43.50±7.78 --
Control 5 ml 9.72±0.657
a -- 172.2±19.8
a --
Standard-I
10 5.25±0.708a
45.98 50.3±8.19a
70.67
Standard-II
200 4.72±0.81
a 51.47 57±6.25
a 66.89
SHF-A
200 4.78±0.4246
a 50.79 62.83±3.53
a 63.51
SHF-B
200 7.30±0.6496
c 24.89 154.0±14.18 10.56
SHF-C
200 5.18±0.6670
a 46.67 90.50±13.63a
47.44
SHF-D
200 5.27±0.4897
a 45.78 73.83±15.94a
57.12
Table no: 4 Effect of Curcuma zedoaria Rosc root extracts on FCA-induced change in organ body weight ratio
Organ body weight ratio at day 43
(mg/gm) Treatment
Groups
(n=6) (Dose
mg/kg)
% Changes±SEM in organ to body weight ratio
Spleen Thymus Adrenal gland Liver
Kidneys
Brain Stomach Heart
Normal -- -- -- -- -- -- -- --
Control 32.9±** -27.6±* 20.8±** 0.23± 0.64± 0.68± 0.31± 0.53±
Standard-I
6.43± -2.24± 3.46± 0.62± 0.83± 0.52± 0.71± 0.62±
Standard-II
2±0. -4.06± 0.93± 0.11± 0.31± 0.59± 0.43± 0.41±
SHF-A
3.41± -5.62± 2.92± 0.38± 0.48± 0.61± 0.55± 0.54±
SHF-B
14.8± -15.2± 11.7± 0.18± 0.67± 0.54± 0.41± 0.59±
SHF-C
7.89± -3.56± 1.86± 0.21± 0.36± 0.49± 0.59± 0.46±
SHF-D
5.67± -4.74± 2.11± 0.31± 0.28± 0.51± 0.39± 0.49±
Table no: 4 5 Effect of Single herb formulations of CZ root extracts on radiology and histopathology of FCA-induced arthritis
in rats
Radiology and Histopathology study
Treatment Groups
(n=6) (Dose mg/kg)
Dose
(mg/kg)
Radiology Score
Histopathology examination
Mean ± SEM %
Normal 5 ml 00.00 -- ---
Control 5 ml 2.833 ±0.167a -- Markedly damaged
Standard-I
10 1.000 ±0.258a 64.70 Mild damaged
Standard-II
200 0.833±0.307a
70.59 Mild damaged
SHF-A
200 1.333±0.4216
a 52.94 Mild damaged
SHF-B
200 2.667±0.211
5.85 Moderate damaged
SHF-C
200 1.167 ±0.307a 58.80 Mild damaged
SHF-D
200 1.500±0.224
a 47.052 Mild damaged
Table no: 4 Effect of Curcuma zedoaria Rosc root extracts on FCA-induced change in Radiology profile
Figure 7: X- ray study showed the efficacy of formulation of CZ on FCA induced arthritic rats on 42 day. Photographs showed
left ankle joint from each group (A) Normal, (B) Control, (C) Standard-I, (D) Standard-II, (E) SHF-A, (F) SHE-B, (G) SHF-C
and (H) SHF-D groups round circles showed inflammation of each joint. Each value are expressed as mean ± SEM. Control
group compared with normal group and drug treated groups compared with control group.
A
H G F
D C B
E
Table: I Accelerated stability study of CZ Single herb formulations
RT-Room temperature: 300± 20C; RH – 65 ± 5 %. / * – Temp - 400± 20C; RH – 75 ± 5 %.
Single herbal Formulation-A
Parameters
0/ INITIAL
1st MONTH
2nd
MONTH
3nd
MONTH
RT 400c RT 40
0c RT 40
0c RT 40
0c
Colour Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow
Odour Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic
Viscosity (cps) 12.6 11.2 15.6 14.7 13.2 12.6 13.1 12.4
pH 4.23 4.28 4.27 4.31 4.05 4.12 4.19 4.21
Sedimentation
Volume 3ml 3ml 3ml 3ml 3ml 3ml 3ml 3ml
Redispersibility
8.88sec 8.12sec 8.76sec 8.57sec 8.43sec 8.32sec 8.56sec 8.37sec
Table: II Accelerated stability study of CZ Single herb formulations
RT-Room temperature: 300± 20C; RH – 65 ± 5 %. / * – Temp - 400± 20C; RH – 75 ± 5 %.
Single herbal Formulation-B
Parameters
0/ INITIAL
1st MONTH
2ND
MONTH
3RD
MONTH
RT 400c RT 40
0c RT 40
0c RT 40
0c
Colour Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow
Odour Characteristic
(Methanol
like)
Characteristic
(Methanol
like)
Characteristic
(Methanol
like)
Characteristic
Methanol
like
Characteristic
Methanol
like
Characteristic
(Methanol
like)
Characteristic
Methanol
like
Characteristic
(Methanol
like)
Viscosity (cps) 12.6 11.2 12.8 11.7 13.2 11.6 12.9 11.4
pH 4.23 4.28 4.42 4.38 4.32 4.29 4.39 4.31
Sedimentation
Volume 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml
Redispersibility
7.75sec 7.62sec 7.76sec 7.57sec 7.85sec 7.62sec 7.46sec 7.37sec
Table: III Accelerated stability study of CZ Single herb formulations
Single herbal Formulation-C
Parameters
0/ INITIAL
1st MONTH
2ND
MONTH
3RD
MONTH
RT 400c RT 40
0c RT 40
0c RT 40
0c
Colour Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow
Odour Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic
Viscosity (cps) 24.0 23.2 22.3 21.2 21.5 21.2 21.9 20.4
pH 4.47 4.31 4.47 4.35 4.32 4.29 4.39 4.31
Sedimentation
Volume 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml
Redispersibility
8.98sec 8.62sec 8.86sec 8.57sec 7.86sec 7.62sec 7.72sec 7..47sec
Table: IV Accelerated stability study of CZ Single herb formulations
Single herbal Formulation-D
Parameters
0/ INITIAL
1st MONTH
2ND
MONTH
3RD
MONTH
RT 400c RT 400c RT 400c RT 400c
Colour Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow
Odour Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic Characteristic
Viscosity (cps) 25.6 24.2 25.4 24.7 27.2 25.6 26.2 25.4
pH 4.53 4.48 4.52 4.41 4.78 4.59 4.49 4.38
Sedimentation
Volume 3ml 3ml 3ml 3ml 3ml 3ml 3ml 3ml
Redispersibility
6.95sec 6.62sec 6.76sec 6.57sec 7.46sec 7.21sec 6.75sec 6.37sec
RT-Room temperature: 300± 20C; RH – 65 ± 5 %. / * – Temp - 400± 20C; RH – 75 ± 5 %.
DISCUSSION OF THE WORK DONE INCLUDING ANY NEW FINDINGS
Isolation, purification and characterization are the new tool for herbal drug to identify the active
moiety in the plant extract. The present study was to investigate the isolation purification and
characterization of active constituent which is responsible for antiarthritic activity. Petroleum
ether extract of Curcuma zedoaria Rosc root showed highly potent active against FCA- Induced
monoarthritis in rats.
Qualitative analysis of extract performed by chemical test was further supported by TLC and
column chromatography
TLC pattern of standard curcuminoids and crude curcuminoids obtained from petroleum ether
extract
Isolated crude curcuminoids obtained from petroleum ether extract of CZ by using preparative
thin layer chromatography with reference to standard curcuminoids. The results showed that
crude curcuminoids showed 3 spot under daylight and 4 sport under UV light at 254 nm with
solvent system chloroform 45: benzene 45: and methanol 10. TLC pattern showed that petroleum
ether extract has curcuminoids when compared with standard curcuminoids Fig.
Column pattern for crude curcuminoids and preparative TLC
Fraction was combined with chloroform and methanol in ratio. Isolated crude curcuminoids
served as compound-I which obtained from petroleum ether extract and further fractionated by
Colum chromatography and analyzed by preparative TLC. Column pattern showed fraction 1-12
were rejected no spot were observed on preparative TLC plate, fraction 13-22 appears single
spot was observed consider as compound-II, fraction 23- 82 were no spot, fraction 83-105 were
observed 2 spot which was consider as compound-III, fraction 106- 114 was observed single spot
consider as compound-IV and fractions 114-150 were observed no spot. All isolated fraction
were analyzed by preparative TLC and compared with standard. On the basis of comparative
study we conclude that compound-I contains curcuminoids , compound-II contains curcumin and
compound-III contains bismethoxycurcuminoids and dismethoxycurcuminoids. Compound-IV
showed single compound which was unknown. All Isolated compound were recrystalised and
subjected to physical property, chemical property, UV, HPLC, HPTLC, FTIR, LCMS and
HNMR for further qualitative analysis.
Physical and chemical property of isolated compounds
Compound-I was obtained in solid form which has dark yellowish red in colure with characteristic
odour and amorphous powder in nature. The solubility was observed in 95% ethanol, acetone,
methanol and sulphuric acid.. The melting point of this compound was determined by Thiele’s
apparatus and found to be 185-187ºC. When these react with 2% boric acid in methanol it showed
radish colour.
Compound-II was obtained from compound which has yellowish colour with charterstic order and
amorphous powder in nature. The solubility was observed in 95% ethanol, acetone, methanol and
sulphuric acid. The melting point this compound was 186 0C. It react with 2% boric acid it
show radish colour.
UV absorption spectroscopy analysis
UV analysis of isolated compounds at 254 nm revealed that petroleum ether extract contains five
compound three peaks and its Rf value were near to standard curcuminoids. Compound-I
showed four compound, compound-II showed single compound which was near to curcumin
peak and Rf value. Compound-III showed four compound, compound-IV, V and VI each were
contains two compound.
High Performance Liquid Chromatography
On the basis of HPLC data petroleum ether extract showed curcuminoids and compound-I
showed curcuminoids compound II showed curcumin peaks when compared with standard
curcuminoids peaks and reaming compounds had not shown any peaks due to less concentration
of the sample and the result are shown in table:
High Performance Thin Layer Chromatography
Isolated compound and petroleum ether extract were subjected to HPTLC for higher resolution.
HPTLC data showed petroleum ether extract showed five peaks, compound-I showed four peaks,
compound-II single peaks, compound-III showed two peaks compound-IV showed two peaks
compound-V and VI no showed any peaks. The above peaks compared with standard
curcuminoids it reveals that compound-I contain curcuminoids and compound-II showed
curcumin and compound-III contains bismethoxycurcumin and dismethoxycurcumin.
FT-Infrared radiation spectroscopy analysis of isolated compounds
Liquid chromatography and Mass Spectroscopy analysis Isolated compound
HNuclear Magnetic Resonance analysis of Isolated compound
DISCUSSION OF THE EXTENDED WORK DONE INCLUDING ANY NEW FINDINGS
In present study Freund’s Complete Adjuvant (FCA) was used to induce monoarthritis in rats
(Butler et al., 2002). To investigate the potential effect of single herb formulation SHF-A , SHF-
B, SHF-C and SHF-D at 200 mg/kg for each formulation of Curcuma zedoaria Rosc root in
monoarthritic rats. This model was suitable for long testing anti-arthritic activity of CZ root
extracts. SHF-A, SHF-C and SHF-D formulation of CZ showed antiarthritic activity in FCA-
induced arthritis rats.
Acute toxicity study
No toxic effects were observed at a higher dose of 2000 mg/kg body weight. Hence, there were
no lethal effects in any of the groups.
CFA-induced rats paw edema
Significant (p<0.001) progressive increase in paw edema were observed in control groups as
compared to normal group. However, standard-I, and standard-II treated groups showed
significant (p<0.001) reduction in rat paw edema on day 3 to 42 days but formulations of CZ
administrated groups showed significant (p<0.01) reduction on 3 day and showed highly
significant (p<0.001) reduction in rats paw edema on 7 day to last days of study except SHF-B
treated groups Fig. 1.
Changes in body weight
Although the weights were almost identical in all groups of animals at 0 to 7 days during the
subsequent course of disease, the weight was always lower in arthritic rats at 14 day to 42 days.
In normal group increase body weight were observed in the subsequent days, whereas standard-I,
standard-II, and SHF-A, C and D treated groups slightly reduced the body weight at 3 day to 7
day and recovery were observed from 14 day to last day of the experiment. However, the body
weight of SHF-B treated groups showed near to control group.
Haematological profiles
CFA-induced arthritic rats at 42 days have shown slightly elevation in the total WBC count and
reduction in RBC. However, significant (p<0.001) increased ESR, Neutrophils and monocytes
while the haemoglobin and lymphocytes were significant (p<0.001) reduced compared with
control group and standard I, standard II, SHF-A, C and D treated groups recovery were
observed in RBC and WBC count however, significant (p<0.001) recovery in Hb content, ESR
level, neutrophils, monocytes and lymphocytes, compared with control group. SHF-B treated
groups showed no achieved in haematological aspects Fig. 2.
Biochemistry profile
No abnormalities were found in the biochemistry profile, all groups showed ALT, AST, BUN,
Cr, Uric acid and total proteins result were near to normal group. There was no significant
difference between treated group and control group.
Nitric oxide level
Serum nitric oxide (NO) levels were significantly (p<0.001) elevated to about twice the normal
level of control group compared with normal group which considered as 100% (Fig.1). But in
contrast, all drug treated groups showed significant (p<0.001) reduced in the level of nitric oxide
in arthritic rats compared to control group SHF treated group showed non inhibitory action
against nitric oxide. These finding confirms the presence and role of nitric oxide in rheumatoid
arthritis and inhibitory effect of treatment on nitric oxide synthesis that would explain the
possible mechanism of anti-arthritic activity Fig.3.
Vascular permeability
Evans blue extravasation showed significant (p<0.001) augmentation in extravasations in CFA-
injected ankle joints of control group compared with normal group. However, all drug treated
groups produced significant (p<0.001) inhibition of Evans blue extravasation in arthritic joint of
rats compared with control groups Fig.4..
Behavioral approach
Latency time to explore
Latency time to explore significant (P<0.001) increase in normal group during 7 to 14 days, but
recovery was observed near to initial value, was achieved during 21 to 42 days. However, control
group and SHF-B group showed significant (P<0.001) increase in latency time during 3 to 42
days compared with 0 day. But in Standard-I, Standard-II, SHF-A, SHF-C and SHF-D
administered groups were significant (P<0.001) increases in latency time during 3 to 7 days but
significant (P<0.001) decrease during 14 days to last day of the study compared with 0 day
[Figure 1].
Ambulatory
Ambulatory decrease in normal group was observed during 3 days and significant (P<0.01)
increase during 7 to 14 days and maintained again near to normal value at day 42. Control group
showed significant (P<0.001) decrease ambulatory during 3 days to last day of experiment
compared with 0 week. In drug-treated groups had decreased ambulatory during 3 to 7 days,
whereas, increased ambulatory during 14 to 28 days and achieved near to normal value during 35
to 42 days, except SHF-B groups compared with 0 day [Figure 2].
Rearing
Rearing decreased in all groups at 3 and 7 days. In normal group, rearing were increased during
14 to 42 days but in control group, rearing significantly (P<0.001) decreased throughout the
study. In all drug-treated groups, progressive recovery was observed during 14 to 28 days and a
return to near initial values was achieved during 35 to 42 days. Rearing was identical in SHF-B
and control group compared with 0 day [Figure 3].
Grooming
Increased grooming was found during 3 to 14 days in normal groups, but decreases during 14 to
28 days; furthermore, decrease in level of initial values was seen during 35 to 42 days compared
with 0 day. . In control group, grooming was increased during 3 to 28 days and reached near to
normal value at day 42 compared with 0 day. All drug-treated groups increased grooming during
3 to 21 days, but decreases during 28 to 42 days compared with 0 day [Figure 4].
Urinations
Urinations in all group of animals showed decreased at day 3, but there were increase in the
frequency of urine at 7 days of observation compared with 0 day. However, control group
showed highly significant increase in frequency of urinations in 7 days, but decrease in
frequencies of urine during 14 days to last day of study compared with 0 day. Standard-I, SHF-
A, SHF-C and SHF-D groups showed significant (P<0.001) decrease in frequency of urinations
during 7 to 42 days compared with 0 day. Standard-II group was (P<0.001) significant decrease
in urine frequency during 14 to 42 days. However, SHF-B did not show significant result
throughout study compared with 0 day [Figure 5].
Defecation
In all groups showed decrease defecations during 3 to 42 days compared with 0 day; however,
decreased significant (P<0.001) frequency of defecations were observed during 3 to 42 days in
normal group compared with 0 day. Standard-II, SHF-A, SHF-C, and SHF-D groups showed
decreased significant (P<0.05) defecation in frequency during 7 to 42 days; however, standard-I
showed significant (P<0.01) reduction during 21 to 42 day compared with 0 days.
Radiographic
Radiographic examination of FCA injected to left ankle joint of arthritic rats at 42 days revealed
the severe soft tissues swelling; however, there were no signs of narrowing of the joint spaces
and the subsequent destruction of the bones and cartilages in the ankle joint compared with
normal group [Figure 7 A, B]. In all drug-treated groups, the soft tissue swellings were reduced,
except SHF-B groups which showed near to control group [Figure 7 C-J].
Histopathology of joints
The tissue sections of CFA-injected left ankle joint of arthritis rats revealed the pathological
changes that can be correlated with arthritis as compared to the normal joint (Fig. 4). In control
group the joint showed mild protective effect. However, treatment with standard-I, standard-II,
SHF-A, SHF-C, and SHF-D groups showed marked protection against the injury to hind paw
tissue sections and most of the histological changes were minimized and found negligible as
compared to arthritis control group. Whereas the treatment with SHF-B showed slight protective
effect on FCA-induced arthritis in rats joint Fig. 5.
Organ body weight ratio
Analysis of organ weight in toxicology studies is an important endpoint for identification of
potentially harmful effects of chemicals.
In our present study we evaluated the organ weight, organ-to-body weight ratio at last days of
study. Significant changes were found, weight reduced in thymus and gain in spleen and adrenal,
but not significant changes in liver, stomach, kidney, lungs, heart and brain in control group
when compared with normal group. all drug treated groups not showed any significant changes
in vital organs these were near to normal group.
Conclusion
In conclusions, it can be stated, that the SHF-A, SHF-C, and SHF-D formulation of Curcuma
zedoaria roots has beneficial effects in long lasting reduction in rats paw edema, recovery in
hematological changes, inhibitory effects on nitric oxide synthesis and vascular permeability. Its
also showed protective effects of arthritic rats joints which is mediated via the inhibition of
prostaglandin synthesis as well as central inhibitory mechanism. But no significant effects were
observed in treatment with SHF-B, treated rats.
Accelerated stability study of formulations
According to ICMR guideline traditional remedies have short life, to increasing their stability
and shelf life, and control their batch to batch variation could be challenging task for modern
scientist. Stability study of all the formulation were performed as per guidelines, international
conference on harmonization (ICH)26
.
The results are shown in table on the basis of stability study all formulation were stable at room
temp and 40 OC up to three month studies.
VI. FUTURE PLAN OF WORK
Remaining research work Published
Preparation for second house presentation
Apply for patent
VII. REFERENCES (NEW & MORE RELEVANT)
VIII. IN BRIEF,
PREVIOUS PROGRESS REPORTS.
As per university experts evaluation all previous progress reports were satisfactory. In these
reports following content were as follow.
Pharmacognostic study of Curcuma zedoaria Rosc root
Pharmacology evaluation of crude extracts of Curcuma zedoaria Rosc
Selection of dose and Pharmacological screening of extracts using animal model
Acute Oral Toxicity
Following parameters were studied in FCA induced arthritic in rats
Paw edema (every week up to 42 days)
Body Weight (every week up to 42 days
Erythrocyte sedimentation rate
Hemoglobin content
Creatinine levels,
Complete blood cell count will be measured
Nitric oxide synthesis
Radiography
Vascular permeability
Histological Study
Open Field Test (every week up to 42 days)
Latency time,
Exploration,
Grooming,
Urination,
Defecation
Chronic toxicity
Local anti-inflammatory
11 Remarks of previous progress reports
Previous I, II, and III reports were
satisfactory
12 Institutional Ethical Committee Clearance for the
current study (date & Certificate)
(Resolution No. 31/7/2010-13).
Approved
13 Recommendation of the Guide
(Enclose additional sheet if required)
Signature of the Research Scholar:
Signature of the Guide:
Signature of the Principal
With seal
Forwarded to Director, Academic Affairs,
KLE University, Belgaum
Journal of Advanced Pharmaceutical
Technology & ResearchVol 2 / Issue 3 / Jul-Sept 2011
www.japtr.org
An Official Publication of Society of Pharmaceutical Education & Research JAPT
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ISSN 2231-4040
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170 Journal of Advanced Pharmaceutical Technology & Research | Jul-Sep 2011 | Vol 2 | Issue 3
Effect of Curcuma zedoaria Rosc root extracts on behavioral and radiology changes in arthritic rats
Abstract
The present study was conducted to evaluate the effects of petroleum ether, chloroform, and methanol root extracts of Curcuma zedoaria Rosc (Family: Zingiberaceae) on behavioral and radiology aspects of Freund’s Complete Adjuvant (FCA)-induced monoarthritis in left ankle joint of rats using open-field test. Traditionally, Curcuma zedoaria root has been used as anti-inflammatory and antiarthritic drug. Behavioral aspects include latency time to explore, ambulatory, rearing, grooming, urination, and defecation. Animals were divided into ten groups each of six rats, all the animals were subjected to open-field test before the induction of arthritis at 0 day and thereafter 3, 7, 14 21, 28, 35, and 42 days of postinoculation FCA injection. The rat was placed in an open field and observed all behavioral aspects for 5 minutes and radiography analysis was made on day 42. Selected doses were 10 mg/kg.i.p. Indomethacin 200 mg/kg.p.o. marketed herbal drug Rumalaya forte and 200 and 400 mg/kg.p.o. of each extracts, respectively. The results showed significant decrease in ambulation and rearing; however, increase in latency time to explore and grooming, urination, and defecation in control group, but in contrast, drug-treated groups showed significant recovery in all behavioral aspects except methanol groups. On the basis of radiography examination, control and methanol groups showed highest swelling compared with normal group; however, all drug-treated groups showed significant reduced swelling. Treatments with petroleum ether and chloroform extracts recovery were observed in behavioral and radiological aspects in arthritic rats.
Key words: Behavioral aspects, Curcuma zedoaria rosc, monoarthritis, open-field test, radiology
Madan L. Kaushik, Sunil S. Jalalpure
Faculty of Pharmacy, KLE University, J. N. Medical College Campus,
Belgaum, Karnataka, India
J. Adv. Pharm. Tech. Res.
oriGinal articlE
Access this article onlineQuick Response Code:
Website:
www.japtr.org
DOI:
10.4103/2231-4040.85537
INTRODUCTION
Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology. The disease is characterized by articular inflammation and by the formation of an
inflammatory and invasive tissue, rheumatoid pannus that eventually leads to the destruction of joints.[1] Recently, it has been reported that microorganism including bacteria, viruses, fungi, parasites, bacterial DNA, and bacterial toxin may exacerbate the inflammatory response at the joint and bone. Mycobacterium tuberculosis and Mycobacterium leprae are the most severe and more common Mycobacterium causing joint and bone diseases.[2] Anti-inflammatory drugs have not been used successfully in all cases due to side effects such as gastric lesions caused by nonsteroidal anti-inflammatory drug and steroids are used to suppress the symptoms, while disease-modifying antirheumatic drugs and biological therapy, all of these agents are associated with numerous side effects.[3,4] This limitation has necessitated the search for novel therapeutic products are directed toward traditional system of medicine for the discovery of drugs that are long-acting antiarthritic with minimum side effects. [5] Although there is no ideal animal model for RA at this time, rat adjuvant arthritis shares many features of human RA.[6] The sensitivity of this model to
Address for correspondence:
Mr. Madan L. Kaushik, Faculty of Pharmacy, KLE University, J. N. Medical College Campus, Nehru Nagar, Belgaum - 590 010, Karnataka, India. E-mail: [email protected]
Kaushik and Jalalpure: Effect of Curcuma zedoaria Rosc root extracts
171Journal of Advanced Pharmaceutical Technology & Research | Jul-Sep 2011 | Vol 2 | Issue 3
evaluate antiarthritic agents[7] and support the behavioral and radiology aspects during adjuvant arthritis is the best available model for RA to evaluate the new antiarthritic drug development. Curcuma zedoaria Rosc is commonly known as white turmeric, consist of dried pieces of rhizome,[8] a large perennial herb with underground tuberous root-stock, growing widely in eastern Himalayas and in most deciduous forest of the central region of Karnataka and Kerala, also cultivated throughout India.[9] Traditionally, the plant has been used as an analgesic, anti-inflammatory, antiarthritic, diuretic, antiallergic, antiulcer, and antiasthmatic.[10] Reported pharmacologically evaluation on Curcuma zedoaria Rosc are reported for antimicrobial,[11] antifungal activity,[12] antiamoebic activity,[13] analgesic activity, antinociceptive activity,[14,15] antiallergic activity,[16] antiulcer activity,[17] anticancer activity,[18] and hepatoprotective activity.[19] Freund’s Complete Adjuvant (FCA)-induced monoarthritis in rat model are widely used to evaluate potential antiarthritic drugs for clinical use. Therefore, morphological similarities to human disease and capacity of the model to predict efficacy in human beings are important criteria in model selection. RA with a proven track record of predictability for efficacy on behavioral and radiology aspects in rat adjuvant arthritis is important criteria to evaluate antiarthritic drugs. On the basis of literature survey, no scientific study was carried out on this plant or its preparation for its behavioral and radiography aspects on FCA-induced arthritis in rats. Hence, the present study was carried out to validate and substantiate its traditional use in the treatment of arthritis.
MATERIALS AND METHODS
Plant MaterialRoots of Curcuma zedoaria Rosc (CZ) were collected in the month of February, 2009 from Cochin, Kerala, India. Roots were identified by Dr. A. K. S. Rawat, Scientist-E, National Botanical Research Institute, Lucknow, India (Specification no [NBRI-SOP-202]). A voucher specimen of the root itself is deposited in the department for future reference.
Preparation of ExtractsThe root of CZ were washed in tap water, cut into small pieces, and then shade dried. The dried pieces were then pulverized with an electric blender, and a yellow powder obtained (25-45 mesh size). The powdered material was subjected to successive extractions using petroleum ether (40-60), chloroform, and methanol for 72 hours.[20,21] These obtained extracts were evaporated under vacuum by flash evaporator to give residues.
ChemicalsFCA was purchased from Sigma Aldrich.[22] Indomethacin obtained from GMH Pharmaceutical Karnal, Haryana, India. Antiarthritic herbal drug Rumalaya forte tablets were obtained from Himalayan drug store local market
Belgaum, India. All other chemicals used were analytical grade and were obtained from Qualigen Fine Chemicals, Mumbai (India).
AnimalsFemale Wistar rats (150-170 g) were used for the present study, which were obtained from K. L. E. University’s College of Pharmacy, Belgaum, India. Food and water were supplied (ad libitum) and kept under controlled temperature 27 ± 2° C with a 12-hour light-dark cycle. The experimental procedures were conducted in accordance to the direction of Institutional Animal Ethics committee, (CPCSEA), Government of India, resolution No. 31/7/2010-13). Due to painful condition imposed on animals, the numbers of subjects used were restricted to the minimum six per group that allowed reliable statistical analysis of the results.
Acute Toxicity StudiesThe acute oral toxicity studies were carried out according to the guidelines set by Organization for Economic Co-operation and Development (OECD) 425. Female Wistar rats (160-200) were used for this study. The roots of each extracts of Curcuma zedoaria Rosc, at different doses (175, 550, 1 500, 2 000, and 5 000 mg/kg), were administered orally to normal rats. During the first four hours after the drug administration, the animals were observed for gross behavioral changes such as hyperactivity, grooming, convulsions, sedation, hypothermia, body weight, and mortality up to 14 days.[23]
Preparation of Test Solutions and Selection of Animal GroupsAll extract were dissolved in normal saline in normal saline and trichurated with 2% tween 60 making a suspension. The dose calculation was based on w/w and calculated for each extract. Animals were divided into ten groups each of six animals. Group-I: treated with 5 ml/kg.p.o. normal saline + mineral oil kept as normal. Group-II: treated with 5 ml/kg.p.o. normal saline + FCA served as control. Group-III: treated with 10 mg/kg.i.p. indomethacin + FCA served as standard-I. Group-IV: treated with 200 mg/kg.p.o Rumalaya forte + FCA served as standard-II. Group-V: treated with 200 mg/kg.p.o petroleum ether extract + FCA served as PEE-1. Group-VI: treated with 400 mg/kg petroleum ether extract + FCA served as PEE-II. Group-VII: treated with 200 mg/kg.p.o chloroform extract + FCA served as CH-I. Group-VIII: treated with 400 mg/kg.p.o chloroform extract + FCA served as CH-II. Group-IX: treated with 200 mg/kg.p.o methanol extract + FCA served as ME-I. Group-X: treated with 400 mg/kg.p.o. methanol extract + FCA served as ME-II. Mineral oil was injected in normal group and FCA injected in control and drug treated groups through intra-articular injection in left ankle joint of rats on 0 day.
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Induction of MonoarthritisFor the induction of monoarthritis in female Wistar rats, all the rats were anesthetized with 40 mg/kg thiopentone sodium intraperitoneally. Mineral oil was injected in left ankle joint of normal group of animals. FCA were injected into left ankle joint of control and drug-treated group, the tarsal area of hind paw was grasped and the fossa distal and medial to the “lateral malleolus” of the fibula was palpated. A 26 G needle was introduced into the capsule of the tibiotarsal joint percutaneously by directing it cephalad, mesiad, and superiorly from the midpoint of the “inframalleolar fossa,” until a distinct loss of resistance was felt approximately 4 mm and complete adjuvant or vehicle was injected. With a true intracapsular injection, a firm resistance to injection was characteristically felt after the injection of 0.1 ml of FCA.[24]
Behavioral Observation (open-field test)For behavioral observations, all the animals were subjected to open-field test before the induction of arthritis and thereafter 3, 14, 21, 28, 35, and 42 days of postinoculation of FCA injection. Rat was placed in an open field in the sound-attenuated room. The floor was white polyvinyl with a black grid dividing open field into 84 squares (10 × 10). Illumination was provided by a bulb (60 W) placed above the center of the field, while the rest of the room was darkened. [25,26] The rat was initially placed in the corner or in center of the field and observed for 5 minutes for all behavioral tests. After each animal observations test, the open field was cleaned with wet sponge and tissue paper[27] and all observations were made between 18.00 and 20.00 hours.
Based on the previous scientific data on behavioral observations of normal rats, the following behaviors were quantified: (I) Latency time to explore: means that time taken “to start explore (second)” from insertion time; (II) Ambulatory behavior: means that the rat “crossed grid line” (horizontal locomotor activity); (III) Rearing, means that the “look for” sometime in air for this it elevates its head and forepaws, almost standing up, (vertical locomotor activity); (IV) Grooming behavior: means licking parts of the rat body or sometimes rubbing the ears, nose, and head or the snout with forepaws, and preening; (V) Urinations: (number of urine passes) considered as anxiety behavior of rat; (VI) Defecation: (number of boluses pass) also considered as anxiety behavior of rat during open-field observation.[28]
Radiography ExaminationAt the end of the experiments, all rats were anesthetized with 40 mg/kg sodium thiopental intraperitoneal injection. Once anesthetized, the animals were constantly kept on X-ray plate, the projections of the left ankle joint were taken at day 42. The following parameters were evaluated blind using the tarsometatarsal region: erosion, a destruction of bony structure resulting in irregular bone surface; periosteal reaction, a fine ossified line, paralleling normal bone producing bone thickening; increase in soft tissue which
was manifested as an increase in width of the soft tissue; and calcification. The parameters were using score which follows: 0, no sign; 1, mild; 2, moderate; and 3, severe.[29]
Statistical AnalysisAll values are presented as mean ± SEM. Differences between means were assessed by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test; P < 0.05 was considered significant.
RESULTS
No toxic effects were observed after treatment with root extracts of Curcuma zedoaria at higher dose of 5000 mg/kg body weight. Hence, there were no lethal effects in any of the groups. Two different doses (200 and 400 mg/kg) were chosen from each extracts for further experimentation.
Latency time to explore significant (P < 0.001) increase in normal group during 7 to 14 days, but recovery was observed near to initial value, was achieved during 21 to 42 days. However, control group and methanol group showed significant (P < 0.001) increase in latency time during 3 to 42 days compared with 0 day. But in Standard-I, Standard-II, and CZ extracts, administered groups were also showed significant (P < 0.001) increases in latency time during 3 to 7 days but significant (P < 0.001) decrease during 14 days to last day of the study compared with 0 day [Figure 1].
Ambulatory decrease in normal group at 3 day but significant (P < 0.01) increase during 7 to 14 days and maintained again near to normal value at day 42 were observed. Control group showed significant (P < 0.001) decrease ambulatory during 3 days to last day of experiment compared with 0 week. However, All drug-treated groups had decreased ambulatory during 3 to 7 days, whereas,
Figure 1: Each value is expressed as mean ± SEM. The levels of significance in latency behavioral changes were analyzed by Dennett’s multiple range comparisons tests, all groups compared with 0 day. P value less than 0.05 was considered as significant. cP < 0.05, bP < 0.01, aP < 0.001
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increased ambulatory during 14 to 28 days and achieved near to normal value during 35 to 42 days, except methanol groups compared with 0 day [Figure 2].
Rearing decreased in all groups at 3 and 7 days. In normal group, rearing were increased during 14 to 42 days but in control group, rearing significantly (P < 0.001) decreased throughout the study. In all drug-treated groups, progressive recovery was observed during 14 to 28 days and a return to near initial values was achieved during 35 to 42 days, except in methanol groups, rearing was identical in methanol and control group compared with 0 day [Figure 3].
Grooming was increased during 3 to 14 days in normal group, but decreases during 14 to 28 days; furthermore, decrease in level of initial values during 35 to 42 days compared with 0 day was observed. In control group, grooming was increased during 3 to 28 days and reached near to normal value at day 42 compared with 0 day. In all drug-treated groups increased grooming during 3 to 21 days, but decreases during 28 to 42 days compared with 0 day [Figure 4].
Urination in all groups of animals were decreased at day 3, but there were increase in the frequency of urine at 7 days of observation compared with 0 day. However, control group showed highly significant increase in frequency of urinations in 7 days, but decrease in frequencies of urine during 14 days to last day of study compared with 0 day. Standard-I and petroleum ether 200 mg/kg groups showed significant (P < 0.001) decrease in frequency of urinations during 7 to 42 days compared with 0 day. Standard-II and chloroform 200 mg/kg groups showed (P < 0.001) significant decrease in urine frequency during 14 to 42 days. Petroleum 400 and methanol 200 mg/kg groups showed significant
Figure 2: Quantification of ambulatory behavior is expressed as (horizontal locomotors). Values are represented as mean ± SEM and analyzed by one way ANOVA, Dennett’s multiple comparisons range test. All groups compared with 0 day. P value less than 0.05 was considered as significant. cP < 0.05, bP < 0.01, aP < 0.001
Figure 3: Rearing behavior elements are quantified by vertical locomotors. Each value expressed as mean ± SEM and analyzed by one way ANOVA, Dunnett’s multiplies comparison test. All groups compared with 0 day. P value less than 0.05 was considered as significant cP < 0.05, bP < 0.01, aP < 0.001
(P < 0.001) decrease in frequency during 21 to 42 days; however, methanol 400 mg/kg did not show significant result throughout study compared with 0 day [Figure 5].
Defecation results are shown in Figure 6; mostly all groups showed decrease defecations during 3 to 42 days compared with 0 day; however, decreased significant (P < 0.001) frequency of defecations were observed during 3 to 42 days in normal group compared with 0 day. Standard-II, petroleum ether and chloroform 400 mg/kg groups showed decreased significant (P < 0.05) defecation in frequency during 7 to 42 days; however, standard-I showed significant (P < 0.01) reduction during 21 to 42 day compared with 0 days. Chloroform 200 mg/kg and methanol group showed decrease in the significant (P < 0.01) frequency of defecation during 28 to 42 days compared with 0 days.
Radiographic examination of left ankle joint of rats at 42 day revealed the severe soft tissues swelling; however, there were no signs of narrowing of the joint spaces and the subsequent destruction of the bones and cartilages in the ankle joint compared with normal group [Figures 7A and B]. In all drug-treated groups, the soft tissue swellings were reduced, except methanol groups which showed near to control group [Figures 7C-J].
DISCUSSION
The present study was to investigate the effect of root extracts of Curcuma zedoaria Rosc on behavioral and radiology changes in monoarthritis rats. Behavioral approach to the arthritic rats which has been proposed as an animal model for chronic pain and radiography approach were made at day 42 for conformed status of disease. [29] Behavioral observations were made over the 42 days
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Figure 4: Values are represented as mean ± SEM analyzed by Dennett’s multiple comparisons range test. All groups compared with 0 day, P value less than 0.05 was considered as significant cP < 0.05, bP < 0.01, aP < 0.001
Figure 5: Anxiety behavior of arthritic rats were quantification in number of boluses. Values are represented as mean ± SEM. All groups compared with 0 day, P value less than 0.05 was considered as significant. cP < 0.05, bP < 0.01, aP < 0.001. Z indicates 0 value significant decrease urination during observation not showed in diagram
Figure 6: Values are expressed as mean ± SEM. All groups compared with 0 day, P value less than 0.05 was considered as significant cP < 0.05, bP < 0.01, aP < 0.001”. Z indicates 0 value significant decrease urination during observation not showed in diagram. Each groups compared with 0 day
Figure 7: (A-J) Radiograph showed effect of Curcuma zedoaria root extracts on FCA-induced monoarthritis in left ankle joint of rats on 42 day. Control joint compared with normal joint and drug-treated joint compared with control joint
postinoculation period; latency time to explore, ambulatory, rearing, grooming,[25,27] urination, and defecation. The latency time to explore in FCA-induced arthritic rats has shown gradual delay in exploration ability. Treatment with indomethacin and rumalaya forte has shown an appreciable and significant decrease in latency time to explore in open field during 14 to 42 days. Extract-treated groups improved the condition by decreasing latency time to explore in open field during 14 days to throughout the study. However, latency time to explore was identical in methanol groups and control group. Control group showed gradual decrease in ambulatory and rearing behavior during 3 to 42 days of the study. However, all drug-treated groups showed decline in ambulatory and rearing at 3 days and improvement in mobility and spontaneous condition during 7 to 28 days,
but complete reversal of arthritis antiambulatory behavior during 35 to 42 days. Grooming [Figure 4] was affected by arthritis during 3 to 14 days in normal group but in control group, it was affected during 3 to 28 days and recovery at day 42. In all drug-treated groups, grooming was affected by arthritis during 3 to 21 days, but strongly improved during 28 to 42 days.
The angiogenic and anxiolytic effects of drug treatment on FCA-induced arthritic rats were studied by considering
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the frequency of urination and defecation in five minutes of exploratory. The fear due to arthritis induces anxiety in the animals when placed in an open field. The ultimate manifestation of anxiety in the animals is exhibited by decrease in the motor activity. Anxiolytic agents are expected to increase the motor activity, which were measured by frequency of urine and defecation during observation.[26]
Urine frequencies were reduced in 3 days but elevated the frequency of urinations in 7 days observation period at 28 days. In control group, higher frequency of urine was recorded during observation period; however, drug-treated animal showed reduced urine frequency during 14 to 42 days, except methanol 400 mg/kg group [Figure 5].
Defecations frequency was higher in control group during observation period, but there was significant decrease in defecation frequency in standard-1 petroleum ether during 14 to 42 days; however, standard-II, chloroform and methanol drug-treated group showed decreased frequency of defecation during 21 to 42 days of study. In present study, the radiographic examination revealed the severe soft tissue swelling, but not the subsequent destruction of the bones, cartilages, and narrowing of the joint spaces in the ankle joint of control and methanol groups at day 42; however, standard-I, standard-II, petroleum ether and chloroform extract showed significant reduction in soft tissue swelling; among these extracts, petroleum ether 200 mg/kg showed high reduction in soft tissue swelling of arthritic joint near to indomethacin-treated group [Figure 6].
Based on present studies, we validate the traditional and folk claims of the use of root Curcuma zedoaria Rosc in the treatment of RA. Petroleum ether and chloroform extracts at both dose showed on behavior doses showed on behavior studies confirms its ability to overcome stress, anxiety, and abnormality in mobility and protective effect on arthritic rats joint at day 42. It is considered that investigation for these medicinal properties might give scientific authentication to traditional clam use of root Curcuma zedoaria Rosc in the therapy of RA.
CONCLUSION
It has also been suggested that FCA-induced RA has a widespread effect on physiological homeostasis due to the severe discomfort in animals. In present studies, the control group showed gradual decrease in ambulatory and rearing behavior and gradual increase in latency time to explore, grooming, urinations, and defecations were observed. However, in drug-treated groups recovery were observed in ambulatory and rearing behavior whereas decrease in latency time to explore grooming, urinations, and defecations. Whereas, methanol groups have failed in all accepts. Radiograph showed increase swelling in
control and methanol joints but reduction in drug-treated groups. These observations support the efficacy of extracts treatment in behavior modulation induced by arthritis by decreasing irritation, anxiety, increased intention to walk, and reduction swelling of rats joint. This shows the possible applicability of petroleum ether and chloroform root extracts of Curcuma zedoaria used in symptomatic treatment of arthritis.
ACKNOWLEDGMENTS
Authors are very grateful to Dr. F. V. Manvi, Dean Faculty of Pharmacy, K. L. E. University Belgaum-10, Karnataka, for providing all the facilities to carry out this work. The authors wish to thank Dr. Anjana Bgewadi for her help and Dr. Vasli Keluskar H.O.D., X-ray department of V. K. Institute of Dental, Science, Belgaum, India, for skilful technical assistance in radiology study.
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How to cite this article: Kaushik ML, Jalalpure SS. Effect of Curcuma zedoaria Rosc root extracts on behavioral and radiology changes in arthritic rats. J Adv Pharm Tech Res 2011;2:170-6.
Source of Support: Nil, Conflict of Interest: Nil.
Evaluation of Anti-Inflammatory Effect of Ethanolic And Aqueous extracts of Curcuma Zedoaria Rosc Root
Madan L. Kaushik*, Sunil S. Jalalpure
Faculty of Pharmacy, KLE University, J. N. Medical College Campus, Nehru Nagar, Belgaum-10 Karnataka.
Key words:
Curcuma zedoaria Rosc, Carrageenan, Histamine,
Anti-inflammatory.
How to Cite this Paper:
Madan L. Kaushik, Sunil S. Jalalpure “Evaluation of
Anti-Inflammatory effect of Ethanolic and Aqueous
extracts of Curcuma zedoaria Rosc Root”, Int. J.
Drug Dev. & Res., Jan-March 2011, 3(1): 360-365.
Copyright © 2010 IJDDR, Madan L. Kaushik
et al. This is an open access paper distributed under
the copyright agreement with Serials Publication,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
work is properly cited.
Introduction:
Nature has provided a complete store-house of
remedies to cure all aliments of mankind [I] Medicinal
plants are believed to be an important source of new
chemical substances with potential therapeutic
effects. The research into plants with alleged folkloric
use as pain relievers and anti-inflammatory agents
should therefore be viewed as a fruitful and logical
research strategy in the search for new anti-
International Journal of Drug Development & Research | Jan-March 2011 | Vol. 3 | Issue 1 | ISSN 0975-9344 |
Available online http://www.ijddr.in Covered in Official Product of Elsevier, The Netherlands
©2010 IJDDR
Abstract
The present study investigates the anti-
inflammatory activity of ethanolic and aqueous
extracts of Curcuma zedoaria Rosc roots in albino
rats by using Carrageenan and Histamine induced
hind paw edema method. The medicinal values of
Curcuma zedoaria root have been mentioned in
ancient literature as useful in disorders of
inflammation. Dried pulverized root of Curcuma
zedoaria was extracted with ethanol by using
soxhlet apparatus and aqueous extract was
prepared by cold maceration. The paw edema was
induced by subplantar injection of above
inflammagens and oedema volume was recorded
using a Plethysmometer. Ethanol 200 and 400
mg/kg root extracts showed significant p<0.001
anti-inflammatory activity on 2nd to 6th hours and
aqueous root extract showed non-significant anti-
inflammatory activity when compared with control
group using standard groups (Indomethacin 10
mg/kg.i.p and Rumalaya forte 200 mg/kg p.o). It
concludes that ethanolic root extract of Curcuma
zedoaria Rosc augments that it shows good anti-
inflammatory activity against carrageenan and
histamine induced paw edema in rats.
*Corresponding author, Mailing address: Madan L. Kaushik Faculty of Pharmacy, KLE University, J. N. Medical College Campus, Nehru Nagar Belgaum-590010 Karnataka, India [email protected] Mobile: 91+8088701770
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Article History:------------------------
Date of Submission: 23-10-2010
Date of Acceptance: 25-01-2011
Conflict of Interest: NIL
Source of Support: NONE
Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1): 360-365 Covered in Scopus & Embase, Elsevier
360
inflammatory drugs since excisting synthetic
molecule like nonsteroidal anti-inflammatory drugs
(NSAIDs) and selective COX-2 inhibitors that
increase the incidence of adverse cardiovascular and
hepatotoxicity effects[2]. So in order to overcome
there is a need to focus on the scientific exploration
of herbal drugs having fewer side effects. Curcuma
zedoaria Rosc (CZ) also known as white turmeric,
zedoaria or gajutsu[3-4] is a perennial rhizomatous
herb that belongs to the Zingiberaceae family. The
plant is indigenous to Bangladesh, Sri Lanka and
India. In India it is known by its several vernacular
names, the most commonly used one being Kaccura
(Sanskrit), Kacura (Hindi) and Kachura (Bengali)[5].
It is used traditionally for the treatment of menstrual
disorders, dyspepsia, vomiting [3] and cancer[6]. Rural
people use the rhizome for its rubefacient,
carminative, expectorant, demulcent, diuretic and
stimulant properties while, the root is used in the
treatment of flatulence, cold, cough, inflammation
and fever[3]. Curcuma zedoaria is a reach source of
essential oils, starch, curcumin arabin and gums etc.
The enzyme phospholipase A2, is known to be
responsible for the formation of mediators of
inflammation such as prostaglandins and
leukotrienes which act by adhering
polymorphonuclear leucocytes to the site of
inflammation would lead to tissue damage probably
by the release of free radicals. Phospholipase A2
converts phospholipids in the cell membrane into
arachidonic acid which is highly reactive and is
rapidly metabolized by cycloxygenase (prostaglandin
synthase) to prostaglandins which are major
components that induce pain and inflammation[7-8].
The early phase of acute inflammation involves
cellular influx associated with the release of
mediators like histamine and prostaglandins
(PGEs)[9]. All these mediators produce inflammation
when injected subcutaneously in the rat paw[10]. The
present study is therefore an attempt to assess the
efficacy of different root extracts of Curcuma
zedoaria on inflammation induced by carrageenin
and histamine in rat paw oedema model.
Materials and Methods:
Plant material:
The matured roots of Curcuma zedoaria (CZ) were
collected from Cochin, Kerala, India. The plant
materials were identified by Dr. A.K.S. Rawat,
Scientist-E National Botanical Research Institute
Lucknow, India. All the roots were shade dried at
room temperature until they were free from moisture
and pulverized in mechanical grinder. The powder
obtained was extracted by continuous hot extraction
process using soxhlet apparatus with universal
solvent ethanol. The aqueous extract was prepared by
cold maceration [11]. The extracts were concentrated
under reduced pressure and dried.
Experimental animals:
Albino rats of either sex (Wistar strain) weighing
150-170 g were used for the present study. Food and
water were supplied ad libitum and the animals were
kept in a 12-hour light-dark cycle. All the animals
were maintained under controlled temperature
(27±20C). The experiment was conducted in
accordance with the direction of Institutional Animal
Ethics Committee (IAEC), CPCSEA, Government of
India. (Resolution No. 31/7/2010-13). Due to painful
condition imposed on animals the numbers of
subjects used were restricted to the minimum six per
group that allowed reliable statistical analysis of the
results.
Acute toxicity studies: [12]
Albino rats (150±40 g) received per oral (p. o.)
extracts of Curcuma zedoaria in an increasing order
of 175, 550, 1500, 2000 and 5000 mg/kg and an
equivalent dose of vehicle was administrated to the
control group. Extract treated and control group (5
animals each) were observed for 24 h under normal
environmental conditions with free access to food
and water. The gross behavioral changes were
observed in animals such as hyperactivity, grooming,
Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1): 360-365 Covered in Scopus & Embase, Elsevier
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convulsions, sedation, hypothermia and mortality up
to 14 days.
Anti-inflammatory Activity:
Curcuma zedoaria extracts were evaluated for anti-
inflammatory activity against carrageenan10 and
histamine (inflamagens) induced rat paw edema
method.
Selection of animal groups were taken for
experiment is as follows: g
Control group:
Group-I: Control: Inflamagens+Normal Saline (p.o)
Standard groups:
Group-II (SD-I):Inflamagens + 10 mg/kg
Indomethacin (i.p)
Group-III (SD-II): Inflamagens +Rumalaya forte 200
mg/kg (p.o)
Test groups:
Group-IV (ETE): Inflamagens +Ethanol extract 200
mg/kg (p.o)
Group-V (ETE): Inflamagens +Ethanol extract 400
mg/kg (p.o)
Group-VI (AE): Inflamagens +Aqueous extract 200
mg/kg (p.o)
Group-VII (AE): Inflamagens +Aqueous extract 400
mg/kg (p.o)
The root extracts were tested for anti-inflammatory
activity by carrageenan and histamine induced rat
paw edema method. Overnight-starved Wistar albino
rats were divided into seven groups of 6 animals
each. Selected doses were 200 mg and 400 mg/kg for
each extracts. All the extracts and herbal drug were
given orally half an hour before the administration of
carrageenan (Sigma chemical co, St. Louis MO, USA)
suspension. The edema was induced by the
subplantar injection of 0.1ml of 1% solution of
carrageenan and the volume of the injected foot was
measured periodically. The paw was marked with ink
at the level of lateral malleolus and immersed in
mercury up to the mark in the plethysmometer13. The
paw volume was measured before and after (1h) of
carrageenan injection and then every hour up to 6
hours of each group. The difference between the
initial and subsequent reading revels the actual
edema volume. The average paw swelling is
calculated by comparing with control, standards and
all extract treated groups.
Percent inhibition of inflammation was calculated by
using the formula,
% inhibition = (Vt-Vc)/Vt x100)14-15.
Where ‘Vc’ represents edema in control.
‘Vt’ is the edema in group treated with extracts.
Histamine–Induced Paw Edema:
In the present study of Histamine–induced paw
edema in rats were treated exactly with the same
method as carrageenan induced model but instead of
carrageenan, here 0.1 ml of 1% w/w histamine in
normal saline was used10.
Results:
Acute toxicity
No toxic effects were observed at a higher dose of
extracts and standard herbal drug 5 g/kg body
weight. Hence, there were no lethal effects in any of
the groups.
Anti-inflammatory activity
The effect of ethanol and aqueous extracts of C.
zedoaria Rosc on carrageenan induced edema in
albino rat is shown in Table I. The results obtained
indicate that both ethanol 200 and 400 mg/kg
extract showed significant anti-inflammatory activity
in albino rats when compared with reference
standards (p< 0.001). The potency was found to be
inversely proportional to the time taken for reduction
in the paw volume. The ethanolic 200 and 400
mg/kg extract treated groups of C. zedoaria
reduced edema up to 43.62 and 44.48% respectively.
Aqueous 200 and 400 mg/kg extract shows inhibit
paw edema up to 18.66 and 18.16% respectively.
When compared with control group
Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1): 360-365 Covered in Scopus & Embase, Elsevier
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The effect of ethanolic and aqueous extract of C.
zedoaria root on histamine-induced edema in rats
is shown in Table II. The histamine induced
inflammation significantly (p< 0.001) inhibits (37.72
and 36.35%) the paw edema at 200 and 400 mg/kg
dose of ethanol extract and the aqueous extract of CZ
200 and 400 mg/kg, shows non significant inhibition
15.76 and 11.47% respectively when compared with
control group.
Discussion:
The present study investigates the inflammation
inhibitory effect of ethanolic and aqueous extract of
Curcuma zedoaria Rosc root. The most commonly
used animal model for acute inflammation is
carrageenan and histamine-induced rat paw edema.
The inflammation induced by carrageenan is biphasic
in nature. The initial phase of edema has been
attributed to release histamine and serotonin. The
edema maintained during the plateau phase,
attributes to kinin like substances [15] and the late
phase is mainly mediated by bradykinin,
leukotrienes, polymorphonuclear cells and
prostaglandin produced in the tissue macrophages [16-
17]. The knowledge of these mediators involved in
different phases is an important for interpreting
mode of drug action. In this study the ethanol
extract of CZ (200 and 400 mg/kg) showed
significant reduce the paw edema at 2 h or more after
carrageenan injection where as the aqueous extract
(200 and 400 mg/kg) showed non significant
reduction, suggesting that curcumin produces an
anti-edematous effect during the second phase,
similar to Indomethacin. In addition, the efficacy of
ethanolic extract of CZ was comparable to that of
Indomethacin with a longer duration of action
showed significant reduction in paw edema volume
equal to Rumalaya forte in carrageenan and
histamine induced inflammation. More prominent
inhibitory effect were observed in the late phase
reaction indicating that this anti-inflammatory
activity could be due to inhibitory effect on
bradykinin, leukotrienes, polymorphonuclear cells
and macrophage infiltration. These studies provide, a
basis for further detailed investigations on
therapeutic efficacy of the root extracts of this plant.
Further study in the direction of elucidating the
mechanism of anti-inflammatory activity should be
carried out in future.
Acknowledgements:
Authors are grateful to Dr. F. V. Manvi, Dean, Faculty
of Pharmacy, K.L.E. University, Belgaum-10,
Karnataka for providing all the facilities to carry out
this research work.
Table-I: Effect of C. zedoaria root extracts on carrageenan induced paw edema in rats
Paw edema (ml) Treatment Groups (n=6)
Dose mg/kg 1sthour 2ndhours 3rdhours 4thhours 5thhours 6thhours
Control saline 0.550±0.050 0.883±0.130 0.967±0.966 0.900±0.063 0.817±0.060 0.716±0.030
SD-I 10 0.250±0.022a 0.316±0.030 a 0.367±0.033 a 0.367±0.042 a 0.300±0.025 a 0.283±0.030 a
SD-II 200 0.267±0.021a 0.283±0.030a 0.366±0.021 a 0.383±0.016 a 0.367±0.028 a 0.333±0.033 a
ETE 200 0.400± 0.043c 0.550±0.047a 0.583±0.033a 0.467±0.021a 0.3670.033a 0.333±0.043a
ETE 400 0.383±0.0477c 0.533± 0.033c 0.600±0.036b 0.467± 0.0494a 0.367±0.0333a 0.317± 0.0307a
AE 200 0.475 ±0.149 0.650 ± 0.043 0.725± 0.048c 0.750± 0.036c 0.700± 0.077 0.600± 0.045
AE 400 0.525± 0.111 0.825 ± 0.020 0.683 ± 0.06c 0.675± 0.04c 0.650±0.033 0.550± 0.062c
Values represent the mean ± S.E.M. (N=6) ap<0.001, bp<0.01, c p<0.05 significant level and were analyzed by one way analysis of variation (ANOVA) followed by Dunnet’s test.
Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1): 360-365 Covered in Scopus & Embase, Elsevier
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Table-II: Effect of C. zedoaria root extracts on histamine induced paw edema in rats
Paw edema (ml) Treatment Groups
(n=6) Dose mg/kg
1sthour 2ndhours 3rdhours 4thhours 5thhours 6thhours
Control saline 0.567± 0.056 0.800±0.052 1.212±0.307 1.083±0.040 0.967±0.056 0.900±0.577
SD-I. 10 0.416± 0.016c 0.550±0.341b 0.683±0.047a 0.700±0.036 a 0.683± 0.065b 0.667±0.066c
SD-II 200 0.367±0.044c 0.416±0.030 a 0.583±0.054a 0.617±0.030 a 0.425±0.047 a 0.583±0.030 a
ETE 200 0.380± 0.037 0.520± 0.037a 0.680±0.020a 0.640± 0.040a 0.600± 0.032a 0.580±0.037a
ETE 400 0.450± 0.035 0.533± 0.022a 0.667± 0.033a 0.667± 0.033a 0.583± 0.040a 0.533± 0.021a
AE 200 0.5167± 0.477 0.783± 0.031 1.033± 0.088 0.800± 0.082 0.775± 0.086c 0.750± 0.057
AE 400 0.483± 0.047 0.85± 0.062 0.967± 0.1174 0.916± 0.101 0.883± 0.185 0.867± 0.067
Values represent the mean ± S.E.M. (N=6) ap<0.001, bp<0.01, c p<0.05 significant level and were analyzed by one way analysis of variation (ANOVA) followed by Dunnet’s test.
Figure no: 1 Root extracts of C. zedoaria percentage Protection against carrageenan and histamine
Percentage inhibition was calculated. C –C1/C × 100. C= Control group and C1= drug treated groups
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2) Wilson B et al. Antimicrobial activity of Curcuma
zedoaria and Curcuma malabarica tubers. J
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4) Prajapati ND et al. Agro’s Dictionary of Medicinal
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6) Etoh H et al. 9-Oxo-neoprocurcumenol from
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attachment inhibitor against the bluemussel,
Mytilus edulis galloprovincalis. Bio Sci Biotechnol
Biochem 2003; 67: 911–913.
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9) DiRosa M, Giroud JP, Willoughby DA. Studies on
the mediators of the acute-inflammatory response
induced in rats in different sites by carrageenan
and turpentine. J Pathol 1971; 1: 15–29.
10) Vogel GH, Vogel WH. Anti-inflammatory activity.
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11) Kokate CK. Practical Pharmacognosy. New Delhi:
Vallabh Prakashan, 1994, PP 107- 109.
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Guideline for testing of chemicals. (425), New
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13) Ghosh MN. Fundamentals of Experimental
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14) Kulkarni SK. Hand book of experimental
pharmacology. New Delhi, India, Vallabha
prakashan; 2002, p. 28-131
DiRosa M. Biological properties of carrageenan
(Review). J. Pharm. Pharmac. 1972, 24: 89-102.
15) Lan R, Marcy MD, Fred S. Rosen MD. Allergy and
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Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1):360-365 Covered in Scopus & Embase, Elsevier
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Research Article
ANTIINFLAMMATORY EFFICACY OF CURCUMA ZEDOARIA ROSC ROOT EXTRACTS
MADAN L. KAUSHIK*, SUNIL S. JALALPURE
Faculty of Pharmacy, K.L.E. University, J. N. Medical College Campus, Belgaum10 Karnataka, India E mail: [email protected]
ABSTRACT
Anti‐inflammatory activity of Curcuma zedoaria Rosc was studied in albino rats by using Carrageenan and Histamine induced hind paw edema method. The paw edema was induced by the subplantar injection of above inflammagens, and oedema volume was recorded using a plethysmometer. Curcuma zedoaria Rosc are traditionally used in treatment of inflammation. Petroleum ether, chloroform and methanol root extracts of C. zedoaria were administered orally half hour before inducing inflammation. All extracts showed significant p<0.001 anti‐inflammatory activity except methanol extract, when compared to control with standard drugs (Indomethacin 10 mg/kg.i.p and Rumalaya forte 200 mg/kg). Amongst these extracts petroleum ether 200 and chloroform 400 mg/kg extracts of Curcuma zedoaria showed maximum anti‐inflammatory activity on 2nd to 6th hours.
Key words: Curcuma zedoaria Rosc, Carrageenan, Histamine, Anti‐inflammatory.
INTRODUCTION
Curcuma zedoaria Rosc, also known as white turmeric, zedoaria or gajutsu1 is a perennial rhizomatous herb that belongs to the Zingiberaceae family. The plant is indigenous to Bangladesh, Sri Lanka and India. In India it is known by its several vernacular names, the most commonly used ones being: Dravi·a (Sanskrit), Kacura (Hindi) and Kachora (Kannada).2
It is used traditionally for the treatment of menstrual disorders, dyspepsia, vomiting3 and for cancer.4 Rural people use the rhizome for its rubefacient, carminative, expectorant, demulcent, diuretic and stimulant properties while the root is used in the treatment of flatulence, dyspepsia, cold, cough and fever.1
Curcuma zedoaria is a reach source of essential oils, starch, curcumin arabin and gums etc. The enzyme, phospholipase A2, is known to be responsible for the formation of mediators of inflammation such as prostaglandins and leukotrienes which by attracting polymorphonuclear leucocytes to the site of inflammation would lead to tissue damage probably by the release of free radicals. Phospholipase A2 converts phospholipids in the cell membrane into arachidonic acid, which is highly reactive and is rapidly metabolized by cycloxygenase (prostaglandin synthase) to prostaglandins, which are major components that induce pain and inflammation.5,6
The early phase of acute inflammation involves cellular influx associated with the release of mediators like histamine and prostaglandins (PGEs).7 All these mediators produce inflammation when injected subcutaneously in the rat paw.8 The present study is therefore an attempt to assess the efficacy of different extracts of Curcuma zedoaria Rosc root extracts on anti‐inflammatory activity induced by carrageenan and histamine in rat paw oedema model.
MATERIALS AND METHODS:
Plant material:
The matured roots of Curcuma zedoaria Rosc were collected from Odakali, near to Cochin, Kerala. The plant materials were authenticated from National Botanical Research Institute Lucknow (NBRI). All the roots were Shade dried at room temperature until they were free from moisture pulverized in electric grinder.
The powder was obtained and extracted separately by continuous hot extraction process using soxhlet extractor with different solvents in increasing order of polarity from petroleum ether, chloroform, and methanol.9 The extracts were concentrated under reduced pressure and dried.
Experimental animals:
Healthy albino rats of either sex (Wistar strain) weighing 150‐160 g were used for present study. The animals had free access to food and water and were maintained under controlled temperature (27±20C) and 12 h: 12 h light and dark cycle. Initial body weight of each animal was recorded.
Acute toxicity studies: 10
The roots of each extracts of Curcuma zedoaria Rosc, at different doses (175, 550. 1500, 2000 and 5000 mg/kg) were administered orally to normal rats. During the first four hours after the drug administration, the animals were observed for gross behavioral changes.
The parameters such as hyperactivity, grooming, convulsions, sedation, hypothermia, body weight and mortality were observed up to 14 days. No mortality observed with oral administration of all the extracts even at the highest dose (5000 mg/kg). Institutional Animal Ethical Committee (IAEC) had approved the experimental protocol and care of animals was taken as per the guidelines of CPCSEA, Department of animal welfare, Government of India.
Test for Antiinflammatory Activity:
These extracts were tested for anti‐inflammatory activity by carrageenan11 and histamine12 (inflamagens) induced rat paw edema method.
Different groups of animals were taken for experiment is as follows: group
Group‐I: Control: Inflamagens+Normal Saline (p.o)
Standard group:
Group‐II (SD‐I): Standard: Inflamagens +10 mg/kg Indomethacin (i.p) Group‐III (SD‐II): Inflamagens+ Rumalaya forte 200 mg/kg (p.o)
Test groups:
Group‐IV (PEE): Inflamagens+Petroleum ether extract 200 mg/kg (p.o) Group‐V (PEE): Inflamagens+Petroleum ether extract 400 mg/kg (p.o) Group‐VI (CE): Inflamagens+Chloroform extract 200 mg/kg (p.o) Group‐VII (CE): Inflamagens+Chloroform extract 400 mg/kg (p.o) Group‐VIII (ME): Inflamagens+Methanol extract 200 mg/kg (p.o) Group‐IX: (ME): Inflamagens+Methanol extract 400 mg/kg (p.o)
Asian Journal of Pharmaceutical and Clinical Research Vol. 4, Issue 3, 2011 ISSN - 0974-2441
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N=6, Values are Mean±S.E.M. ap<0.001, bp<0.01, c p<0.05 (significant) statistical analysis was done by one way analysis of variation (ANOVA) followed by Dunnet’s test.
N=6, Values are Mean±S.E.M. ap<0.001, bp<0.01, c p<0.05 (significant) statistical analysis was done by one way analysis of variation (ANOVA) followed by Dunnet’s test.
Fig. 1: Root extracts of C. zedoaria Rosc percentage inhibition against carrageenan and histamine induced paw edema
Percentage inhibitions were culculated. C –C1/C X 100. C= Control group and C1= drug treated groups
Table I: Effect of C. zedoaria Rosc root extracts on carrageenan induced paw edema in rats Treatment Groups (n=6)
Dose mg/kg
Paw edema (ml)
1sthour 2ndhours 3rdhours 4th hours 5th hours 6th hours
Control saline 0.550±0.050 0.883±0.130 0.967±0.966 0.900±0.063 0.817±0.060 0.716±0.030 SD‐I 10 0.250±0.022a 0.316±0.030 a 0.367±0.033 a 0.367±0.042 a 0.300±0.025 a 0.283±0.030 a
SD‐II 200 0.267±0.021a 0.283±0.030 a 0.366±0.021 a 0.383±0.016 a 0.367±0.028 a 0.333±0.033 a
PEE 200 0.250±0.022a 0.383±0.031 a 0.417± 0.031 a 0.367± 0.033 a 0.350± 0.023 a 0.317±0.031 a PEE 400 0.350± 0.034b 0.383±0.031 a 0.450 ± 0.034 a 0.417± 0.031 a 0.350± 0.022 a 0.317±0.017 a CE 200 0.333±0.022b 0.367±0.022 a 0.433±0.033 a 0.467±70.033 a 0.383±0.031 a 0.350±0.024 a
CE 400 0.317±0.030b 0.366±0.033 a 0.450±0.042 a 0.417±0.047 a 0.383±0.040 a 0.350±0.022 a
ME 200 0.433±0.021 0.700±0.044 0.883±0.147 0.816±0.030 0.783±0.166 0.600±0.044
ME 400 0.483±0.047 0.800±0.089 0.883±0.016 0.850±0.056 0.667±0.056 0.617±0.083
Table II: Effect of C. zedoaria Rosc root extracts on histamine induced paw edema in rats
Treatment Groups(n=6)
Dose mg/kg
1sthour 2ndhours 3rdhours 4th hours 5th hours 6th hours
Control saline 0.567± 0.056 0.800±0.052 1.212±0.307 1.083±0.040 0.967±0.056 0.900±0.577
SD‐I. 10 0.416± 0.016c 0.550±0.341b 0.6833±0.047a 0.700±0.036 a 0.683± 0.065b 0.667±0.066c
SD‐II 200 0.367±0.044c 0.416±0.030 a 0.583±0.054a 0.617±0.030 a 0.425±0.047 a 0.583±0.030 a PEE 200 0.417± 0.031 0.517±0.032 a 0.567± 0.043 a 0.567±0.042 a 0.483±0.047 a 0.483±0.048 a
PEE 400 0.3670.033c 0.467±0.042a 0.583±0.048a 0.617±0.040a 0.583±0.05a 0.517±0.031a
CE 200 0.383±0.030 0.483±0.040 a 0.566±0.033 a 0.616±0.040 a 0.516±0.047 a 0.433±0.021 a CE 400 0.417±0.054 a 0.533±0.056 a 0.650± 0.068 a 0.667±0.056 a 0.567±0.033 a 0.500±0.063 a
ME 200 0.517± 0.031 0.783± 0.047 1.017± 0.047b 0.983± 0.075 0.800±0.073 0.750± 0.042 ME 400 0.500±0.036 0.733±0.042 0.983±0.054b 1.033±0.080 0.900±0.036 0.800±0.077
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Before the experiment, food was withdrawn overnight but adequate water was given to the rats. Dose selected were 200 mg and 400 mg/kg for each extract. The animals were divided into nine groups of 6 animals each. All the doses were given orally half an hour before the administration of carrageenan (Sigma chemical co, St. Louis MO, USA) and histamine into the plantar side of the left hind paw. The paw was marked with ink at the level of lateral malleolus and immersed in mercury up to the mark in the plethysmometer.13
The paw volume was measured after (1 h) injection carrageenan and then every hour till 6 h of each group. The difference between the initial and subsequent reading gave the actual edema volume. The average paw swelling is calculated by comparing the normal group with control, Standards and all treated groups compared with the control, Percent inhibition of inflammation was calculated by using the formula,
% inhibition = 100 (Vt/Vc x100)14‐16.
Where ‘Vc’ represents edema in control.
‘Vt’ is the edema in group treated with extracts.
Histamine–Induced Edema:
For the study of Histamine – induced paw edema in the animals were treated exactly the same method as carrageenan induced model but instead of carrageenan, here 0.1 ml of 1% w/w histamine in normal saline was used.
RESULTS
Acute toxicity
The acute toxicity study revealed non toxic nature of all the extracts at a higher dose of 5 g/kg body weight.
Antiinflammatory activity
The effect of petroleum ether and chloroform extracts of C. zedoaria Rosc on carrageenan induced edema in albino rat is shown in Table 1. The results obtained indicate that both above extract had significant anti‐inflammatory activity in albino rats, when compared with reference standards (p< 0.001). The potency was found to be inversely proportional to the time taken for reduction in the paw volume. The petroleum ether extract of C. zedoaria Rosc reduced edema 56.70% near to standard group II induced by carrageenan on oral administration 200 mg/kg, when compared with untreated control group. Petroleum ether 400 and chloroform extracts 200, 400 mg/kg shows 52.18, 50.90 and 51.99% standard groups‐I and II shows 60.6 and 56.95% inhibition respectively.
The effect of petroleum ether and chloroform extract of C. zedoaria Rosc on histamine‐ induced edema in rats is shown in Table: 2. The histamine induced inflammation is significantly (p< 0.001) inhibits the paw volume at 200 mg/kg dose of petroleum ether extract. Petroleum ether extract 400, chloroform extracts 200 and 400 mg/kg shows 42.41, 44.47 and 38.41% inhibition respectively. Standard group‐I and II inhibits paw edema 34.13 and 44.86% respectively. Methanol groups showed non‐ significant results.
DISCUSSION
Due to the increasing frequency of intake of NSAID’s and their reported common side effects, there is need to focus on the scientific exploration of herbal drugs having fewer side effects. So, there is a continuous search for indigenous drugs, which can provide relief to inflammation. Carrageenan induced inflammation is a biphasic phenomenon.15 The first phase of edema is attributed to release of histamine and 5‐hydroxytryptamine. Plateau phase is maintained by kinin like substances and second accelerating phase of swelling is attributed to prostaglandin like substances.
The knowledge of these mediators involved in different phases is an important for interpreting mode of drug action, in this study the petroleum ether and chloroform extract of C. zedoaria Rosc, (200 and 400 mg/kg) showed significant reduction of paw edema at 2 h
or more after carrageenan injection, suggesting that curcumin produces an anti‐edematous effect during the second phase, similarly to indomethacin. Therefore, our results confirm that the mechanism of the anti‐inflammatory effect of curcumin involves reduction of prostaglandins through inhibition of cyclooxygenase. The antiedematous effect of methanol extract of C. zedoaria showed a delayed onset (6 h), In addition, the efficacy of petroleum ether and chloroform extract of C. zedoaria was comparable to that of Indomethacin and Rhumalya fort with a longer duration of action showed significant reduction in paw edema volume in carrageenan and histamine induced inflammation.
Thus it can be concluded that petroleum ether and chloroform root extracts of plant C. zedoaria possess significant anti inflammatory activity in rats. Further studies involving the isolation and purification of the chemical constituents of the plant and the investigations in the biochemical pathways may result in the development of a potent anti‐inflammatory agent with a low toxicity and better therapeutic index.
ACKNOWLEDGEMENT
Authors are grateful to Dr. F. V. Manvi, Dean of Faculty of Pharmacy, K. L. E. University Belgaum, Karnataka for providing all the facilities to carry out this work.
REFERENCES
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2. Nadkarni KM. Indian Materia Medica. 3rd ed. Mumbai, India: Popular Prakashan Private Limited, 1999.
3. The Ayurvedic Pharmacopoeia, Vol. IV, Government of India Ministry of health and family welfare. Ayush New Delhi, 1996, P. 43‐45.
4. Etoh H et al. 9‐Oxo‐neoprocurcumenol from Curcuma aromatic (Zingibereaceae) as an attachment inhibitor against the bluemussel, Mytilus edulis galloprovincalis. Bio Sci Biotechnol Biochem 2003;67 :911–913.
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7. Rosa MD, Giroud JP, Willoughby DA. Studies on the mediators of the acute‐inflammatory response induced in rats in different sites by carrageenan and turpentine. J Pathol. 1971;1 :15–29.
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IJPR | January-March | 77
International Journal of Pharmaceutical Research 2012, Volume 4, Issue 1. 77-81.
ISSN 0975-2366
Research Article
Comparative Efficacy of Root Extracts of Curcuma zedoaria Rosc on Behavioural and Radiological Changes in Arthritic Rats
Madan L. Kaushik, Sunil S. Jalalpure
Faculty of Pharmacy, KLE University, J. N. Medical College Campus, Belgaum- 10, Karnataka * Corresponding Author: Email: [email protected]
Received: 25/06/2011, Revised: 15/09/2011, 30/09/2011 ABSTRACT Preclinical evaluation is an essential step in the assessment of new anti-inflammatory and antiarthritic drugs. This study was to investigate the effect of ethanolic and aqueous extracts of Curcuma zedoaria Rosc (Zingiberaceae), Root on behavioural and radiological changes in arthritic rats. Freund,s Complete Adjuvant-induced arthritis was followed over 42 days of postinoculation period in left ankle joint of Wistar albino rats using an open field test. Behavioural observations were made on 0, 3, 7, 14, 21, 28, 35, and 42 days and radiography study was performed at 42 day after drug treatments. Behavioural observations included latency time, ambulatory, rearing, grooming, urination and defecation. Radiography parameters included, soft tissue swelling, erosions of bone extremities, and joint space narrowing. Rats were divided into eight groups (n = 6), selected doses were indomethacin 10 mg/kg.i.p, 200 mg/kg,p.o, marketed herbal drug Rumalaya forte and 200, 400 mg/kg.p.o of ethanol and aqueous extracts respectively. Quantified behavioural changes in arthritis rats were decreased ambulation and rearing, whereas increased latency time, grooming, urination and defecation were observed in the control group. The standard drugs and extract treated groups showed significant (p<0.001) recovery in behavioural changes except aqueous groups compared with 0 day. On the basis of radiography examination control and aqueous groups showed highest swelling in left ankle joint of rats as compared to normal group joint. Groups treated with standards and extracts showed significant reduction in swelling on 42 day. However both doses of ethanolic extract of Curcuma zedoaria showed highly effective and appreciable results by impairing intension for improvement in arthritis. KEY WORDS: Anti-arthritic, Curcuma zedoaria, open field test, behavioral study, radiography INTRODUCTION
Rheumatoid arthritis (RA) is a chronic systematic disease usually manifesting inflammation of multiple joints which is one of the major autoimmune diseases of global prevalence [1] the severity of disease varies from patient to patient and also changes the behaviours during disease period. RA is characterized by a relapsing and remitting course of joint inflammation by chronic proliferative synovitis, inflammatory immune cell infiltration into the synovial fluid and cartilage destruction [2]. The use of the anti-inflammatory, DMRD, steroids and biological drugs for the treatment of RA is associated with severe adverse reactions and toxicity [3]. Today’s means treating arthritis are symptomatic and are not been effective in treating joint destruction and degradation of cartilage and have no use in repairing and rejuvenating the lost tissues [4]. This has lead to develop an interest of the patient and public to opt for alternative system of medicines in the treatment of arthritis. Currently the bioassay guided fractionation and evaluation of known medicinal plant extract is looking the fact of strong synogism of active constituents in the crude extract, which may prove more potent and effective compound and this may also relieve the toxic effect of constituents [5]. This hypothesis has encouraged us to use the root of Curcuma zedoaria Rosc used as a natural herb for treatment of arthritis. According to siddha therapy, Curcuma zedoaria Rosc roots used in crippling arthritis and frozen joints [6] traditionally it has been used as an analgesic, anti-inflammatory, diuretic, antiallergic, anti-asthmatic, antiulcer, menstrual disorders, Vomiting and improves blood circulation [7,]. Recently, Curcuma zedoaria Rosc has been scientifically evaluated for hypnotics, sedative,
anti-inflammatory, antimicrobial, antifungal, antiamoebic, analgesic, antinociceptive, antiallergic, antiulcer, platelet activating, hepatoprotective, Cytotoxic, anticancer, and antioxidant activities [8]. Up till now very less number of plant extracts have been systematically studied on behavioral and radiological changes in arthritic rats.
However, based on the literature survey, there is no scientific report proving the effect on behavioural and radiological changes of Curcuma zedoaria in arthritic rats. Hence the CZ was selected to evaluate the effects on behavioural and radiological changes on Wister albino rats during long post inoculation period. MATERIALS AND METHODS Plant material
The matured roots of Curcuma zedoaria Rosc were collected in February 2009 from Cochin, Kerala, India and identified by Dr. A.K.S. Rawat, Scientist-E National Botanical Research Institute Lucknow, India (Specification no NBRI-SOP-202). The fresh roots were cleaned with distilled water to remove extraneous matter, shade dried until to get constant weight and then powdered. Extraction procedure
The dried root powder of Curcuma zedoaria was extracted with ethanol by soxhlet apparatus and water, these extracted by a cold maceration. The extracts were subjected to vacuum distillation under reduced pressure using flash evaporator to yield a semisolid residue [9]. The ethanol and aqueous extracts of CZ roots were chosen for present study.
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Animals Female Wistar rats (160±40g) were obtained from the
JNMC central animal house Belgaum, Karnataka, India. The animals were housed six per cage with free access to food and water, and were maintained on a 12 hour light-dark cycle at a temp 23±10C. All experimental procedures were approved by the Institutional Animal Ethical Committee of K.L.E’s College of Pharmacy, Belgaum, India. Preparation of test samples and animal groups
Each extract were dissolved in normal saline suspended with 2% tween 60, administered orally to rats by oral catheter. The drugs treatment schedule was started from 0 to 42 days. The animals were divided into eight groups of six animals in each. Mineral oil injected in left ankle joint of rats as normal group received normal saline and FCA injected group as control group also received normal saline, indomethacin was used as standard drug, rumalaya forte was used as herbal standard drug (Marketed preparation for arthritic disease,) ethanol and aqueous
Figure 1 Efficacy of ethanolic and aqueous root extracts of CZ on changes latency behaviour in FCA induced arthritic rats each value is expressed means ± SEM. The levels of significance in behavioural changes were analyzed by Dennett, s multiple range test. Different letter represent significant difference cP<0.05, bP<0.01, aP<0.001, all groups compared with initial value at 0 day.
Figure 2 Efficacy of ethanolic and aqueous root extracts of CZ on ambulatory behaviour in FCA-induced arthritic rats. Values are represented means ± SEM analyzed by Dennett, s multiple comparisons range test. Each group compared with 0 day. Different alphabets are representing difference significance level P value less than 0.05 was considered as significant. cP<0.05, bP<0.01, aP<0.001.
extracts of CZ were used as test drugs. Dose calculation was based on w/w of each extract and doses were selected on the basis of our previous study [10]. Selection of animals groups were taken for experiment is as follows Statistical analysis
The data are expressed as means ± SEM. The results were analyzed by one way ANOVA followed by Dunnett’s multiple comparison test; P value less than 0.05 (p <0.05) were considered significant Normal Group Normal: Mineral oil+Normal Saline (p.o) Control Group Control: FCA+Normal Saline (p.o) Standard groups: Standard-I: FCA+10 mg/kg Indomethacin (i.p) Standard-II: FCA+Rumalaya forte 200 mg/kg (p.o)
Figure 3 Efficacy of ethanolic and aqueous extract of CZ root extracts on rearing behaviour in FCA-induced arthritic rats. Values are expressed as mean ± SEM and analyzed by one way ANOVA, Dunnett’s multiplies comparison test. All groups compared with 0 day for coming conclusion P value less than 0.05 was considered as significant. cP<0.05, bP<0.01, aP<0.001
Figure 4: Efficacy of ethanolic and aqueous root extracts of CZ on FCA-induced grooming behaviour in FCA induced arthritic rats values are represented means ± SEM and analyzed by Dennett, s multiple comparisons range test. All group compared with 0 day for results P value less than 0.05 was considered as significant cP<0.05, bP<0.01, aP<0.001.
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Kaushik et al / International Journal of Pharmaceutical Research 2012 4(1) 77-81
80 | IJPR | January-March |
tissue swelling which was manifested as an increase in width of the soft tissue; and calcification. The score were used which follows: 0, no sign; 1 mild; 2 moderate; and 3 severe [16]. RESULTS AND DISCUSSION
This study was undertaken to develop a new mode of evaluation of drug effectiveness based on behaviour and radiology study indicating well-being in a rat model of chronic inflammatory. The present study evaluated the effect of Curcuma zedoaria Rosc root extracts of quantitative analysis of chronically observed behavioural of FCA-induced monoarthritic in rats during 42 days observation period as well as effects on radiological changes. The control group treated with saline shown and gradual decrease in ambulatory and rearing behaviour. Whereas, gradual increase in number of grooming and increased in frequency of urination and defecation along with increased latency time to explore were observed during open field test,
Latency time to explore, exploring activity tend to increase in all groups during 3 to 14 days compared with 0 day. However; control group showed significant (P<0.001) increase latency time throughout study but in standard-1 standard-II, and ethanol groups recovery were observed near to initial value at 14 day onwards compared with 0 day. AQE-I group showed significant increase exploring behaviour during 3 to 35 days however, AQE-II group showed increase exploring behaviour from 7 to 35 days (Fig. 1).
Ambulatory behaviour decrease during diseases assessments however, after treatments significant recovery were observed in behavioural accepts. Ambulatory decrease in all group during 3 day to 7 days compared with 0 days however, normal group significant increase ambulatory 7 to 14 day and reached near to initial value at 42 day. Control group showed significant decrease ambulatory throughout the study but in standard-I, standard-II, ethanolic, and AQE-I groups increased ambulatory during 14 to 28 days and achieved near to normal value during 35 to 42 days except AQE-II groups compared with 0 day (Fig. 2).
Rearing behaviour decrease in all groups during 3 to 7 days in normal group rearing was increase during 14 to 42 days but in control and AQE-II groups rearing significant (P<0.001) decrease throughout the study. Based on present data’s standard-I, standard-II, ethanol, and AQE-I groups progressive recovery were observed which achieved near to initial values during 35 to 42 days compared with 0 day (Fig. 3).
In arthritic disease conditions inflammation and irritation induced. Grooming behaviour was increases at 3 to 14 days in all groups but decreases during 14 to 28 days furthermore, decreases level of initial values during 35 to 42 days compared with 0 day. In control group grooming was increased during 3 day to 28 days and reached near to normal value at 42 days compared with 0 day. Drug treated groups increases grooming during 3 to 21 days however; decreases during 28 to 42 days compared with 0 day. (Fig. 4)
Urinations and defecations observations represent the anxiety behaviour during open field test. Anxiety behaviour in each group of animals was decrease urine frequency at 3 day except standard II groups of animals However; control group was showed highly significant increase frequency of urinations at 7 day but decrease of urinations during 14 day
to end of experiment. In standard-I, ETE-1, ETE-II, and AQE-I and AQE-II groups decrease unine frequency throughout the study compared with 0 day (Fig. 5). Defecation the results shown in Fig. 6 each groups were showed decrease defecations during 3 to 42 day however; the level of significant decrease urine frequency at different days compared with 0 day (Fig. 6).
Radiography are widely accepted as the gold standard in assessing structural joint damage associated with rheumatoid arthritis and therefore considered as essential parameter in evaluating the efficacy of experimental therapeutics. In the present study intra-articular injection of FCA in control rats caused swelling and an abnormality in the toes and digits of FCA injected joint are shown in Fig. 7 on the basis of radiography analysis of control group joint increase significant (P<0.001) swelling at 42 day compared with normal group joint however, reduced the swelling in standard-I, standard-II, and ETE-1 and ETE-II groups, radiological photographs indicating their applicability in the last phase of the arthritis and also improving its efficacy in protection against permanent deformation and swelling [15-17] however, there aqueous groups has been failed to reduced swelling of joint. On the basis of overall results analysis we found in these observations supports the efficacy of root extracts treatment in behaviour modulation induced by arthritis by decreasing irritation, anxiety, ability to bear the pressure on inflamed paw, increased intention to walk. This shows the possible applicability of the root in symptomatic treatment of arthritis [12-13,15].
Based on the present study we validate the traditional and folk claims on anti-arthritic activity of ethanolic extracts of Curcuma zedoaria Rosc root showed the significant effects on behavioral and radiography in the treatment of arthritis rats. It is considered that investigation for these medicinal properties may give scientific authentication to traditional clams. CONCLUSION
In this study, the effects of ethanolic and aqueous extracts of Curcuma zedoaria Rosc root on behavioural and radiological aspects of arthritic rats for regional limited form of stable arthritis for long term study of inflammation using open field test. Ethanolic extract groups significantly improved Latency time to explore, ambulatory and rearing behavior and also shown significant reduction in grooming and frequency of urinations and defecations. Radiography studies reveled the ethanolic extracts reduced the FCA-induced swelling of left ankle joint of rats at 42 day however, aqueous extract has been failed in all aspects. This shows the possible applicability of the ethanolic extracts in symptomatic treatment of arthritis. . ACKNOWLEDGEMENT
Author’s are very grateful to Dr. F. V. Manvi, Dean Faculty of Pharmacy, K. L. E. University Belgaum-10, Karnataka, for providing all the facilities to carry out this work. The authors wish to thanks Dr. Anjan Bgewadi for her help and Dr.Vasli Keluskar H.O.D. X-ray department of V. K. Institute of Dental, Science, Belgaum, for skilful technical assistance in radiology study. REFERENCE 1. Yang YH, Rajaiah R, David Y, Lee W, Ma Z, Yu H,
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