To, - Shodhganga : a reservoir of Indian theses @...

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


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


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)


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

.


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


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


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


Chloroform in

No Spot ---- ----


Chloroform.

No Spot ---- ----


Chloroform.

No Spot ---- ----


Chloroform.

No Spot

---- ----


Chloroform.

2 Spot Light yellow 0.35, 0.28


Chloroform.

1 Spot Light yellow 0.34


Chloroform.

No spot ---- ----


Chloroform.

No spot ---- ----


Chloroform.

No spot ---- ----


Chloroform.

No spot ---- ----


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


1500


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


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


Single herbal Formulation-B

Parameters

0/ INITIAL

1st MONTH

2ND

MONTH

3RD

MONTH

RT 400c RT 40

0c RT 40

0c RT 40

0c


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


Redispersibility


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



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


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



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


Redispersibility



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.


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.


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


Radiography


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

R

ISSN 2231-4040

Journ

al o

f Ad

van

ced

Ph

arm

aceu

tical T

ech

no

log

y &

Researc

h • V

olu

me 2

• Issue 3

• Ju

ly-S

ep

tem

ber 2

011

• Pages 1

35-***

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.



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



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



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



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.

REFERENCES

1. Ekambaram S, Perumal SS, Subramanian V. Evaluation of antiarthritic activity of Strychnos potatorum Linn seeds in Freund’s adjuvant induced arthritic rat model. BMC Complement & Altern Med 2010;10:56.

2. Axford SJ. Joint and bone infections. Rehum Int J 2006;34:405-12.3. Guidelines for the management of rheumatoid arthritis: 2002

Update. Arthr Rheum 2002;46:328-46.4. Guidelines for the management of rheumatoid arthritis. American

college of rheumatology ad Hoe committee on clinical guidelines. Arthr Rheum 1996;39:713-22.

5. Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, et al. Efficacy of B- cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med 2004;350:2572-81.

6. Barbier A, Novarro JC, Brelliere Roncucci R. Biochemical and clinical changes in rats with the developing arthritis. Inflam Res 1984;15:103-5.

7. Ward JR, Cloud RS. Comparative effect of antirheumatic drugs on adjuvant induced polyarthritis in rats. J Pharmacol Exp Ther 1966;152:116-21.

8. The Ayurvedic Pharmacopoeia: A Dictionary of Indian raw materials and industrial product. New Delhi: Council of scientific and industrial research; 1998. p. 43-5.

9. Kapoor LD. A Hand book of ayurvedic medicinal plants, Vol. 1, 1st ed. New York, USA: CRS Press; 2005. p. 1130-1.

10. Sharma PV. Dravyaguna-Vijana, Vol. 1, 2nd ed.Varansi, India: Chaukhambha Bharati Academy; 2001. p. 295.

11. Wilson B, Abraham G, Manju VS, Mathew M, Vimala B, Sundaresan S, et al. Antimicrobial activity of Curcuma zedoaria and Curcuma malabarica tubers. J Ethnopharmacol 2005;99:147-51.

12. Joshi S, Singh AK, Dhar DN. Isolation and structure elucidation of potential active principle of Curcuma zedoaria rhizome. Herb Hungar 1989;28:95-8.

13. Ansari MH, Ahmad S. Screening of some medicinal plants for antiamoebic action. Fitoterapia 1991;62:171-5.

14. Navarro Dde F, de Souza MM, Neto RA, Golin V, Niero R, Yunes RA, et al. Phytochemical analysis and analgesics property of Curcuma zedoaria grown in Brazile. Phytomedicine 2002;9:427-32.

15. Pamplona CR, de Souza MM, Machado Mda S, Cechinel Filho V, Navarro D, Yunes RA, et al. Seasonal variation and analgesic properties of different parts from Curcuma zedoaria Roscoe (Zingiberaceae) grown in Brazil. Z Naturforsch C 2006;61:6-10.



16. Matsuda H, Tewtrakul S, Morikawa T, Nakamura A, Yoshikawa M. Antiallergic principles from Thi zedoary structural requirements of curcumanoids for inhibition of degradation and effect on the release of TNF-a anIL-4 IN RBL-2H3 cells. Bioorg Med Chem 2004;12:5891-8.

17. Gupta RP, Majed Md, Eranna D1, Setty R. Evaluation of antiulcer effect of root of Curcuma zedoaria in rats. Ind J of Trad know 2003; 2:375-7.

18. Lee H, Lin JY. Antimutagenic activity of extracts from anticancer drug in Chinese medicines. Mutat Res 1988;204:229-34.

19. Seo WG, Hwang JC, Kang SK, Jin UH, Suh SJ, Moon SK, et al. Suppressive effect of Curcuma zedoaria rhizome on pulmonary metastasis of B16 melanoma cells. J Ethnopharmacol 2005;101:249-57.

20. Kokate CK. Practical Pharmacognosy, 3rd ed. Delhi, India: Vallabh Parkashan; 1994. p. 105-7.

21. Tripathi S, Sahoo SP, Pradhan D, Sahoo H, Satapathy DK. Evuation of antiarthritic potential of Hybanthus Enneasspermus. Afr J Pharm Pharmacol 2009;3:111-4.

22. Guidance for Industry, Clinical development program for drug, device and biologic products for the treatment of rheumatoid arthritis (RA) U.S. A: Food and Drug administration. Available form: http://www.fda.gov/eder/guidance/index.htm. [Last updated on 2006 Nov 6; cited on 1999].

23. OECD. Guideline for testing of chemicals 425 Ministry of health

and Family Welfare, New Delhi: Available from: http://www.epa.gov/endo/pubs/uterotrophic. [Last cited on 2001 Dec 17].

24. Butler SH, Godeyfroy F, Besson JM, Weil-Fugazza J. A limited arthritic model for chronic pain studies in the rat. Pain 1992;48:73-81.

25. Dimitrijevic M, Laban O, Djuric VJ, Stanijevic S, Miletic T, Kovacevic-Jovanović V, et al. Behaviour and severity of adjuvant-arthritis in four strains. Brain Behav Immun 2001;15:255-65.

26. Kulkarni SK. Hand book of experimental pharmacology, 3rd ed. New Delhi, India: Vallabha prakashan; 2002. p. 28-131.

27. De Castro Costa M, De Sutter P, Gybel J, Van Hess J. Adjuvant induced arthritis in rats A possible animal model of chronic pain. Pain 1981;10:173-85.

28. Chitme HR, Patel NP. Antiarthritis activity of Aristolochia bracteata extract in experimental Animals. Open Nat Prod 2009;2:6-15.

29. Carlson RP, Datko LJ, O’Neill-Davis L, Blazek EM, DeLustro F, Beideman R, et al. Comparison of inflammatory change in established type-II collagen and adjuvant-induced arthritis using outbred wistar rats. Int J Immunopharmacol 1985;7:811-26.

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

Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

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,


Madan L. Kaushik et al: Evaluation of Anti-Inflammatory effect of Ethanolic and Aqueous extracts of Curcuma Zedoaria Rosc Root

Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

361

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



Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

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.



Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

363

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

References:

1) Ravi VI, Saleem TSM, Patel SS, Ramamurthy J,

Gauthaman KI. Anti-Inflammatory effect of

methanolic extract of Solanum nigrum Linn

Berries. Int J App Rese Nat Prod 2009; 2: 33-36.

2) Wilson B et al. Antimicrobial activity of Curcuma

zedoaria and Curcuma malabarica tubers. J

Ethanopharmacol 2005; 1: 147-151.

3) Nadkarni KM. Indian Materia Medica. Mumbai,

India, Popular Prakashan,1999.

4) Prajapati ND et al. Agro’s Dictionary of Medicinal

Plants. India: ed 1, 2003.

5) The Ayurvedic Pharmacopoeia, Government of

India Ministry of health and family welfare. V-IV:

Ayush, New Delhi: 43-45.

6) 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.



Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

364

7) Vane J, Booting R. Inflammation and the

mechanism of action of anti-inflammatory drugs,

FASEB J 1987, 1: 89.

8) Higgs GA, Moncada S, Vane JR. Eicosanoids in

inflammation, Ann Clin Res. 1984; 16: 287-99.

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.

Drug Discovery and evaluation

Pharmacological Assays. New York: Springer, 1997,

PP 406–409.

11) Kokate CK. Practical Pharmacognosy. New Delhi:

Vallabh Prakashan, 1994, PP 107- 109.

12) Ministry of health and Family Welfare. OECD

Guideline for testing of chemicals. (425), New

Delhi; Government of India . [Online] 2001 Dec

17.

13) Ghosh MN. Fundamentals of Experimental

Pharmacology. Kolkatta, Scientific book agency,

1994, pp 153-158.

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

allergic diseases. N. Engl. J. Med, 2001, 11: 344-

347.

16) Vineger R, Trux JF, Selph, JH, Johnsstone PR,

Veenable AL, Mckenzie, KK. Pathway to

carrageenan-induced inflammation in the hind

limb of rat. Fed. Proc. 1987, 6: 118-126.

Int. J. Drug Dev. & Res., Jan-March 2011, 3 (1):360-365 Covered in Scopus & Embase, Elsevier


Fu

ll L

en

gt

h R

es

ea

rc

h P

ap

er

C

ov

er

ed

in

Of

fi

ci

al P

ro

du

ct

of

Els

ev

ie

r, T

he

Ne

th

er

la

nd

s

365

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

Kaushik et al. Asian J Pharm Clin Res, Vol 4, Issue 3, 2011, 9092

91

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

Kaushik et al. Asian J Pharm Clin Res, Vol 4, Issue 3, 2011, 9092

92

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

1. Wilson B et al. Antimicrobial activity of Curcuma zedoaria and Curcuma alabarica tubers. J Ethnopharmacol 2005;99 :147–151.

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.

5. Higgs GA, Moncada S, Vane JR. Eicosanoids in inflammation, Ann Clin Res. 1984;16 :287‐99.

6. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin‐like drugs. Nat New Biol. 1971; 231‐232, 235.

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.

8. Vogel GH, Vogel WH. Anti‐inflammatory activity. Drug Discovery and evaluation. Pharmacological Assays. 2nd ed. New York: Springer; 1997. p. 406–409.

9. Kokate CK. Practical Pharmacognosy, 3rd ed. New Delhi,Vallabh Prakashan; 1994. p. 107‐109.

10. Ministry of health and Family Welfare. OECD Guideline for testing of chemicals. (425), New Delhi; Government of India . [Online] 2001 Dec 17.

11. Kulkarni SK. Hand book of experimental pharmacology. 3rd ed. New Delhi: Vallabha prakashan; 2002. p. 28, 131.

12. Ghosh MN. Fundamentals of Experimental Pharmacology, 2nd ed. Kolkata, Scientific book agency; 1994. p. 153‐158.

13. Winter CA, Risley EA, Silber RH. Antiinflammatory activity of indomethacin and plasma corticosterone in rats. J Pharmacol Exp Ther 1968; 162,196‐201.

14. Devib P, Meera R. Study of antioxidant, anti‐inflammatory and wound healing activity of extracts of Litsea glutinosa J Pharm Sci & Res. 2010; 2: 155‐163.

15. Singh H, Ghosh MN. Modified plethysmometer for measuring foot volume of unanesthetized rats. J Pharm Pharmacol.1968;20 :316‐17.

16. Vinegar R, Schreiber W, Hugo RJ. Biphasic development of carrageenin edema in rats. J Pharmacol Exp Ther. 1969;166 :96‐103.

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.

Kaushik et al / International Journal of Pharmaceutical Research 2012 4(1) 77-81

78 | IJPR | January-March |

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.

Test groupsETE-1: FCAETE-II: FCAAQE-I: FCAQE-II: FC

Figure 5: EfCZ on anxrepresented when all growas consideZ indicates observation

Figure 6 EfCZ on anxValues are 0.05 was aP<0.001. Zduring obser Freund,s monoarthriInduction oBaseline prmineral oil all groups. thiopentoneAdjuvant wrats for indupaw was gr‘lateral mallneedle was ijoint percutsuperiorly f

Kaushik e

s: A+Ethanol etherA+Ethanol etherA+Aqueous extr

CA+Aqueous ext

fficacy of ethanoxiety behaviourmeans ± SEM a

oup compared wered as significan

0 value signifinot showed in f

fficacy of ethanoiety behaviour expressed as mconsidered as

Z indicates 0 valurvation not show

complate aitic in rats

of Mono-arthritisre-induction waand Freund,s CAll rats were

sodium intrapewas injected in luction of monoarasped and the leolus’ of the fibintroduced into ttaneously by dirfrom the midpoi

et al / Internatio

r extract 200 mgr extract 400 mgract 200 mg/kg (tract 400 mg/kg

olic and aqueousr of arthritic rat 0, 3, 7, 14, 21

with 0 day P valunt. cP<0.05, bP<cant decrease u

figure.

olic and aqueousof FCA induce

means ± SEM. Psignificant cP<

ue significant dewed in figure.

adjuvent (FC

s in Wistar femas made prior

Complete Adjuvaanaesthetized

eritoneally. Freeft ankle joint o

arthritis, the tarsfossa distal an

bula was palpatthe capsule of threcting it cephaint of the ‘infram

onal Journal of

g/kg (p.o) g/kg (p.o) (p.o) (p.o)

s root extracts orats. Values are1, 28, 35, 42 dayue less than 0.050.01, aP<0.001”

urinations during

s root extracts oed arthritic rats

P value less than<0.05, bP<0.01ecrease urination

CA) induced

male Wister ratsto injection o

ant on 0 day forwith 40 mg/kg

eund,s Completeof Wistar albinosial area of hindnd medial to theted. A 26 gaugehe tibiotarsal alad, mesiad andmalleolar fossa,

of Pharmaceuti

f e y 5 ”. g

f s. n , n

d

s, f r g e o d e e

d ’

until a dismm and cintracapsucharacteris[11]. BehavioraEach animinduction 28, 35, anblack gridcm). Illumabove the in darkneinitially pbehavioralparameterLatency tiseconds), III. Rearin(rubbing tUrination,of boluse)

Figure 7 Xaqueous roon 42 daygroup Standard-I(H) groupjoint. eachgroup comcompared RadiograpRadiograpradiographseverity oentire ankwere anaintraperitowere consof the lefevaluated destructionsurface; pnormal bo

ical Research 2

stinct loss of rescomplete adjuvaular injection, a stically felt afte

al observations-mal was subjectof arthritis on 0

nd 42 days. The d dividing the opmination was prcentre of the fie

ess and sound-placed in the cl parameters for s were selectedime to explore II. Ambulatoryng (Vertical locthe nose with (number of ur.

X- ray study shooot extracts of C

y. Photographs s(A) Normal, (BII, (E) ETE-I, (Fps round circlesh values are expmpared with norm

with control gro

phy phic evaluation hs of left anklef lesions were e

kle joint at the laaesthetized withonial injection. tantly kept on anft ankle joint. by blind scale tn of bony struceriosteal reactioone producing b

2012 4(1) 77-8

IJPR | Jan

sistance was felant or vehicle inj

firm resistanceer the injection o

-open field test ted to open fiel0 day and there floor was white

pen field into 84rovided by a bueld, while the res-attenuated rooentre of the fie5 minute [12-15

d for arthritic ra(exploring obs

y (Horizontal loccomotors activityits porepaws aination). VI. De

owed the efficacCZ on FCA indshowed left anklB) Control, (CF) ETE-II, (G) As showed inflam

pressed as meanmal group and doup.

was performede joint of rats. Tevaluated from rast day of the eh 40 mg/kg sOnce anaesthetn X-ray plate wThe following

tarso-metatarsal cture resulting

on, a fine ossifiebone thickening

81

nuary-March |7

lt approximatelynjected with a tre to injection wof 0.1 ml of flu

ld test before tafter 3, 7, 14, 2

e polyvinyl with4 squares (10 x ulb (60W) placst of the room wm. The rat weld and observ5] The behavioruats as follows: servations time comotors activityy), IV. Groominand preening), efecation (numb

cy of ethanolic anduced arthritic rale joint from ea) Standard-I, (D

AQE-I and AQEmmation of eas ± SEM. Contr

drug treated grou

d on the basis The presence anradiographs of texperiment all raodium thiopenttized, the anima

with the projectioparameters weregion, erosion,in irregular bo

ed line, paralleling; increase in so

79

y 4 rue

was uid

the 21, h a 10 ed

was was ved ual

I. in

y), ng V.

ber

nd ats

ach D) -II

ach rol

ups

of nd the ats tal als

ons ere , a ne ng oft


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,

et al. Suppression of ongoing experimental arthritis by a Chinese Herbal Formula (Huo-Luo-Xiao-Ling Dan)


IJPR | January-March |81

involves changes in antigen -induced immunological and biochemical mediators of inflammation. Eve-Bas Comp and Alt Med 2010; 1-10.

2. Kehlen A, Pachnio A, Thiele K, Langer J. Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patient with rheumatoid arthritis: upregulation proteins TSG-6. Arth Res Ther 2000; 28(5):184-185.

3. Zhang RX, Fan AY, Zhou AN, et al. Extract of the Chinese herbal formula Huo Luo Xia Ling Dan Inhibited adjuvant arthritis in rats. J of Ethnopharmacol 2009; 121(3): 366–371.

4. Dobrila N, Robert W, Brittberg M, et al. Cartilage tissue engineering for degenerative joint Disease. Adv Drug Deliv Rev 2006; 58(2): 300-322.

5. Alexander D, Crawford, Sandra LZ, et al. Bioassay-Guided Natural Product Discovery: Isolation of Angiogenesis Inhibitors from East African Medicinal Plants. PLoSONE 2011; 6(2): 146-94.

6. Wilsion E, Rajamanickam GV, Yas NA, Agarwal GP, Dubey. A review herb used in siddha medicine for arthritis. Ind J trad Knowl 2007; 6(4): 678-687.

7. Sharma PV. Dravyaguna-Vijana. Chaukhambha Bharati Academy: Varansi; 2001.p. 295.

8. Loboa R, Prabhua SK, Shirwaikara A A review of its chemical pharmacological and ethnomedicinal properties. J of Pharm and Pharmacol 2009; 61(1) 13-21.

9. Kokate CK. Practical Pharmacognosy. Vallabh Parkashan: Delhi; 1994.

10. Kaushik ML, Jalalpure SS. Evaluation of anti- inflammatory effect of ethanolic and aqueous extracts of curcuma zedoaria Rosc root. Int J Drug Dev & Res 2011; 3(1): 360-365.

11. Butler SH, Godeyfroy F,Besson JM, Jeaner WF A limited arthritic model for chronic pain studies in the rat. Pain 1992; 48(1):73-81.

12. Kulkarni SK. Hand book of Experimental Pharmacology. Vallabha prakashan: New Delhi; 2002. p. 28-131.

13. Dimitrijevic M, Laban O, Djuric VJ, et al. Behaviour and severity of adjuvant–arthritis in four strains. Bra Beh Immuni 2001; 15 (3): 255-65.

14. Costa MD, Sutter PD, Gybel J, Vanhess J. Adjuvant induced arthritis in rats: A possible animal model of chronic pain. Pain, 1981; 10: 173- 185.

15. Chitme HR, ar extract in experimental Animals. The Open Nat Prod 2009; 2: 6-15.

16. Carlson RP, Datko LJ, Lynn ON, Frank D, Beideman R, Lewis AJ. Comparison of inflammatory changes in established part-II collagen and adjuvant-induced arthritis using outbred wistar rats. J Immunopharmacol 1985; 7 (6): 811-826.

17. Calvino B, Bernard MO, Bars DL. Parallel clinical and behavioural studies of adjuvant- induced arthritis in the rats: possible relationship with chronic pain. Beh Bra Res 1987; 24: 11-29.

18. Aletaha D, Nell VK, Stamm T, et al. Acute phase reactants add little to composite disease activity indices for heumatoid arthritis: validation of clinical activity score. Arth Res Therp 2005; 7 (7): 796-806.

To, - Shodhganga : a reservoir of Indian theses @...

Documents

Transcript of To, - Shodhganga : a reservoir of Indian theses @...