Sunkara Yashvanth et al.

/ Journal of Pharmacy Research 2010, 3(8),1775-1778

Research Article
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Anti-inflammatory and cytotoxic activity of chloroform extract of roots of Saussurea lappa Clarke
Sunkara Yashvanth1 , Robinson A1 , Suresh Babu, K 1 , Naidu, V G M 2 ,Vishnuvardhan, M V P S 2 , Ramakrishna, S 2 , Madhavendra S S 3* and Rao, J M 1
1.Natural Product Chemistry Division
2.Pharmacology Division
3.Electron Microscopy Center
Indian Institute of Chemical Technology ,Hyderabad-500007, AP., India
Received on: 15-04-2010; Revised on: 12-05-2010; Accepted on:13-06-2010

ABSTRACT
Anti-inflammatory activity of chloroform extract of the roots of Saussurea lappa was evaluated on RAW mouse macrophase cells by monitoring the TNF-α and
Nitric Oxide levels. Cytotoxicity studies of the extract were carried out on three cancer cell lines - HT-29 (Colon cancer), A549 (Lung cancer) and MDA-MB
(Breast Cancer). Cytotoxic activity on breast cancer cell lines (MDA-MB) was nearly comparable to that of the standard compound, doxorubicin. However, it
was not significant on the other two cell lines (HT-29 and A549) studied. The test compound exhibited significant effect on TNF-α levels. Impact on nitric oxide
was on par with Rolipram.

Key words: Anti-inflammatory, Cytotoxicity, HT-29, A549, MDA-MB, Saussurea lappa, TNF-α, NO
INTRODUCTION
Natural pharmaceuticals (naturoceuticals) are of great impor- extract was filtered and concentrated.The concentrated extract (residue)
tance as a reservoir of chemical diversity aimed at new drug discovery was subjected to column chromatography over silica gel (60-
and are explored for antimicrobial, cardiovascular, immuno suppressive 120mesh).The column was eluted with solvents of increasing polarity
and anticancer agents. Natural products including plants and animals (EtOAc in Hexane; 0.5-3.0%). Resolution of the components was
have been the basis of treatment of human diseases (Patwardhan et al., monitored by TLC and similar types of fractions were combined.Total
2004). Medicinal uses of Saussurea lappa are umpteen – used as antis- 28 fractions were collected, first thirteen fractions obtained with 0.5%
pasmodic, cures cough, asthma, cholera, chronic skin diseases and rheu- EtOAc in hexane were showing freak spots in TLC. They are pooled
matism (Chopra et al., 1956; Dhar et al., 1984); dysentery, ulcer and and named as Fraction-I. Remaining 15 fractions obtained with 1-3%
stomachache (Kala and Manjrekar, 1999); cough and cold (Singh, 1999); EtOAc in hexane were pooled and designated as Fraction-II. Fraction-II
malaria, leprosy, persistent hiccups (Kapoor, 2001); rheumatism, as- showed a short UV active single spot (Rocket shaped) in TLC. The spot
tringent, toothache and typhoid fever (Nautiyal et al, 2003); rheuma- turned pink on developing with 10% methanol in H2 SO 4 . Fraction II was
tism (Jain et al., 1984); diuretic and antihelmenthic (Govindan and used for pharmaceutical activities. Procedures for isolation of various
Bhattacharya, 1977); Gout, erysipelas and promotes spermatogenesis compounds in Fraction II were reported earlier (Robinson et al., 2010).
(Pandey et al., 2007); epilepsy, piles, headache, general weakness and
scalp scabies (Bapalal, 1998); headache, throat infection, backache, chest In vitro Anti-inflammatory activity
pain, scanty urination, skin rashes formed after insect bites, exhaus-
tion, luster and growth of hair, pustules and fainting spells (Kumar and In vitro Anti-inflammatory activity was evaluated by monitoring the
Kumar, 1989). TNF-α levels (Pae, 2007) and Nitric Oxide (NO) levels (Culotta and Koshland,
1992) in mouse macrophage cells. RAW-264.2 mouse macrophage cells were
Aqueous, alcoholic and hexane extracts of Saussurea lappa cultured in T25 flasks in Dulbeccos Modified Eagles Medium (DMEM) with-
roots were employed in the previous reports for pharmaceutical evalu- out phenol red and 10% heat inactivated serum at 37o C temperature and 5%
ation. Much is lacking on chloroform extract. Hence, an attempt was CO2 with 90% relative humidity. After 85% confluence, cells were trypsinized
made in order to explore new chemicals/activity in the chloroform
with trypsin EDTA solution (Himedia) and plated in 12 well plate at a density
extract of the roots of Saussurea lappa. We have isolated six compounds
- Alpha-amyrin eicosanoate (1), a triterpenoid and the sesqueterpene of 1 x 105 cells to each well and incubated at 37o C for 24h.
lactones, Dehydrocostus lactone (2), Beta-cyclo costunolide (3),
Costunolide (4), Epoxyisozaluzanin C 4α 15-epoxide (5) and Estimation of TNF-a and NO Levels
Epoxyisozaluzanin C 11α 13-epoxide (6) (Robinson et al., 2010). Com-
pound one was reported for the first time in the roots of Saussurea After incubation, cells were pre exposed with Fraction II (test com-
lappa. Present study reports the anti-inflammatory and cytotoxic ac- pound) at a concentration of 50, 25 and 10 µg/ml in DMSO. Rolipram was used
tivities of the chloroform extract of Saussurea lappa roots. as a Standard. After pre exposure, media in each well were replaced with fresh
media and again cells were exposed with test compound at specified concentra-
MATERIALS AND METHODS tions along with lipopolysaccharide at a concentration of 50 µg/ml (from E. coli,
sigma) and cells were incubated at 37o C with 5% CO2 . After stimulation, super-
Plant Material and Extraction natant in each well was removed at 6 and 24 h for estimation of TNF-alpha and
Commercial sample of Sassurea lappa roots was obtained from Kishan nitric oxide respectively. Samples were stored at -80o C until further analysis.
Lal Dawasaz (Ayurveda & Unani), Ladd Bazar, Hyderabad. Powdered TNF-alpha in the supernatant was estimated by Sandwich ELISA method
root sample was cold extracted in chloroform for a week (Maceration).The using R&D Biosystems kits. NO was estimated using Griess reagent method.
*Corresponding author. Evaluation of Cytotoxicity
Madhavendra SS
Electron Microscopy Center Cytotoxicity was evaluated in three cancer cell lines Viz., HT-29
Indian Institute of Chemical Technology
Hyderabad-500007, AP., India (Colon cancer), A549 (Lung cancer) and MDA-MB (Breast Cancer). Cell lines
Tel.: + 91-040 27193180
Telefax: +91 040 27160921
E-mail:sakunthala@iict.res.in Journal of Pharmacy Research Vol.3.Issue 8.August 2010 1775-1778
 Sunkara Yashvanth et al. / Journal of Pharmacy Research 2010, 3(8),1775-1778
Table 1. MTT assay of compounds in RAW Mouse Macrophase cells Figure 1. TNF-alpha inhibition levels.
Compound Concentration % Viability % Inhibition
1 2 0 0
(Average of duplicates)

Concentration of TNF-alpha (pg/ml)

Rolipram 50µg/ml+LPS 82.13 17.87 1 0 0 0
25µg/ml+LPS 89.20 6.04
Test Compound 10µg/ml+LPS 102.93 -2.93 8 0 0
(Fraction II) 50µg/ml+LPS 6.3 93.28 *
25µg/ml+LPS 7.5 92.01 6 0 0 *
10µg/ml+LPS 54.81 45.19
4 0 0
*
were obtained from National Center for Cell Science (NCCS), Pune, India. HT-
2 0 0
29 (Colon cancer), A549 (Lung cancer), cell lines were grown as adherents in
DMEM, where as MDA-MB (Breast cancer cell line) was grown in Minimum 0
Essential Media (MEM) supplemented with 10% fetal bovine serum, 100 µg / R o l . ( 1 0 L P S ( 5 0 W i t h o u t T . C . ( 1 0
µ g / m l ) µ g / m l) L P S µ g / m l)
ml penicillin, 200 µg/ml streptomycin, 2mM L-glutamine, and culture were
maintained in a humidified atmosphere with 5% CO2 .Cytotoxicity of test *P<0.05 .50µg LPS, Rol. – Rolipram; T.C. Test Compound
vs
compound in cells was determined by MTT assay which was based on mito-
chondrial reduction of yellow MTT tetrazolium dye to a highly colored blue Figure 2. Nitric oxide inhibition levels.
formazan product. 1x104 Cells (counted by Trypan blue exclusion dye method)
in 96- well plates were incubated with various concentrations of the test com- 8

Concentration of nitrite (µm)

pound for 48 hrs at 370 C in DMEM/MEM with 10% FBS (Fetal bovine 7
serum). Doxorubicin was used as standard. Then the above media was replaced 6
*
with 90µl of fresh serum free media and 10 µl of MTT reagent (5mg/ml) and 5

plates were incubated at 370 C for 4h, there after the above media was replaced 4
with 200µl of DMSO and incubated at 370 C for 10min.The absorbance at 3
2
570nm was measured on a spectrophotometer (Plate reader, spectra max,
1
Molecular devices) IC–50 values were determined from plot: % inhibition
0
(from control) versus concentration. Percent viability was calculated using the
following formula.

am
PS
ne

LP

lipr
alo

tL

un
ith

Ro
ou

po
W
dia

M e a n Absorbance of Sample

om
ith
Me

stc
% Viability = X 100

Te
Mean Absorbance of control
*P<0.05 vs. with LPS (50µg/ml); Concn. of test compound -10µg/ml
Microscopic Observations
Figure 3. % viability in treated cell lines
Treated and normal cancer cells were observed in phase contrast mi-
croscope ( Nicon TS-100 Eclipse). 140.0
120.0
Statistical Analysis 100.0
% Viability

80.0
Statistical evaluations were performed using analysis of variance 60.0
(ANOVA) followed by Dunnet’s test for sub-group comparison using Prism Soft- 40.0
ware (version 2.01) wherever necessary. 20.0
0.0
RESULTS AND DISCUSSION
0 50 100 150 200 250
Concentration (µg/ml)
Anti Inflammatory Activity:
A549 MDA-MB HT-29
TNF-α Levels
All values are Mean ±SD, n=3
T h e test compound was found to be toxic to the RAW cells up to the lowest
concentration of 25 µg/ml. At a concentration of 10 µg/ml, there was 45 % cell death. MTT Figure 4. % Inhibition in treated cell lines
assay of the compound in RAW cells is depicted in Table 1. However, it could express the
TNF alpha (309.2 pg/ml) with 55 % surviving cells when incubated with LPS (50 µg/ml).
100.0
Extrapolated to 100 % surviving cells the concentration of TNF alpha expressed was 563.2
pg/ml (Figure 1). The amount of TNF alpha expressed due to LPS (50µg/ml) induction 80.0
alone was 851.1 pg/ml. Hence, the percent inhibition of TNF alpha by the test compound 60.0
% Inhibition

is 33.76%. Whereas, Rolipram showed an inhibition of 38.3%. Reports are available on the 40.0
20.0
0.0
Table 2. IC-50 values of compounds on various Cell lines. All values
-20.0 0 100 200 300
are Mean ±SD, n=3
-40.0

Cell Line Compound IC-50µg/ml Concentration (µg/ml)

HT-29 Test Compound 109.77±7.55 A549 MDA-MB HT-29

Doxorubicin 2.60±0.95
A549 Test Compound 54.38±6.38
Doxorubicin 4.33±0.80
All values are Mean ±SD, n=3
MDA-MB Test Compound 56.30±3.38
Doxorubicin 49.39±2.61

Journal of Pharmacy Research Vol.3.Issue 8.August 2010 1775-1778

 Sunkara Yashvanth et al. / Journal of Pharmacy Research 2010, 3(8),1775-1778
Figure 3. Micrographs of Cancer Cell lines (Magnification 200X)
a) A549 Control b) A549 after treatment (100µg)
c) HT-29 Control d) HT- 29 after treatment(100µg)
e) Formazan crystals after adding MTT in A549 (control cells)
anti-inflammatory activity of costunolide, one of the constituents of Fraction II used in the
present study (Castro etal., 2000 Park et al.,1996; Nam, 2006; Pae et al., 2007).Rangler et
al.,(1999) and Matsuda et al.,(2000,2003) have attributed the anti-inflammatory activity to
theα -methylene-α-butyrolactone (CH2-BL) moiety of costunolide. This moiety contains
electrophilic α, α-unsaturated carbonyl group that can react with neucleophiles, especially
cystiene groups of biological proteins by a Michael-type addition (Rangeler et al., 1999). Of
the reported six compounds in the present study, the compounds – Dehydrocostus lac-
tone(2), Beta-cyclocostunolide (3) and Costunolide (4) have the CH2-BL moiety and the
inhibition of TNF-α levels in the present study is significant and nearly comparable with
Rolipram. Pae et al. (2007) have reported the inhibition of TNF- α and interleuken-6 by
inducing hemoxygenase-1 in RAW 264.7 macrophases treated with costunolide and con-
firmed the role of CH2-BL moiety for nuclear factor-erythroid 2-related factor 2 (Nrf2 )
activation leading to Hemeoxygenase (HO-1) expression which inturn inhibit the TNF-α
and IL-6 levels. Since the test compound appears to be cytotoxic to RAW cells at a
concentration of 10 µg/ml, further studies are required at lower concentrations of the test
compound.
Nitric oxide Levels
The concentration of Nitric Oxide expressed was 5.17ìM/ml (with
extrapolation to 100%). The amount of Nitric oxide expressed due to LPS (50µg/
ml) induction alone was 5.99ìM/ml. Hence, the percent inhibition of Nitric Oxide
Journal of Pharmacy Research Vol.3.Issue 8.August 2010
by the test compound (Fraction II) was 13.70%. Whereas Rolipram showed an
inhibition of 12.5% (Figure 2). Activity of the test compound was on par with
Rolipram. However, further studies are required at lower concentrations of the test
compound, since the test compound appears to be toxic to RAW cells at a
concentration of 10 µg/ml. Park et al., (1996) and Matsuda et al., (2003) also
have reported the inhibitory role of costunolide on NO-Synthase activity.
Cytotoxicity Studies
Cytotoxic activity of the test compound against the three cell lines was
in the following order - MDA-MB (Breast Cancer) > A549 (Lung Cancer) > HT-
29 (Colon Cancer). Cytotoxic data of the three cell lines are given in figures 3,4
and table 2. In MDA-MB cell line, activity of the test compound was nearly
comparable to that of the activity of Doxorubicin. Choi et al., (2005) have
reported the inhibition of telomerase in MCF-7 (wild- type-P53) and MDA-MB
(mutant P 53) cells treated with costunolide in a concentration and time-
dependent manner. Telomerase is a specialised nucleoprotein which plays an
essential role in cell prolifiration as a protective mechanism against the end
replication Problem. Most normal human cells have no detectable telomerase
activity, but it is present in most cancer cells (Mori et al., 1994, Park et al., 2001).
Kanno et al.(2008) have reported costunolide induced apoptosis, caused by recep-
tor-mediated pathway and inhibited telomerase activity in NALM-6 cells (Human
B cell leukemia). Mori et al., (1994) have reported anticancer activity of costunolide.
Lee et al., (2001) suggested the mitochondrial dependent pathway for the
costunolide- induced apoptosis of HL-60 human leukemia cell line. The inhibition
of telomerase causes a progressive and critical reduction of telomers, leading to
potent signal for the blockage of cell prolifiration and the induction of apoptosis
(Carlangelo et al., 2005). Robinson et al., (1998) have reported the cytotoxic
effect of Isodihydrocostunolide, dihydrocostunolide and methoxy derivatiives of
costunolide and ß-cyclocostunolide on the cancer cell lines – Colo-205, MCF-7,
A-43 andA-549. They attributed the cytotoxicity to the exo-methylene group on
lactone part of sesuiterpenes, which was in agreement with the previous report by
Zhang et al.,(2005). Zhang et al., (2005) have assessed the experimental evidence
for the anti-cancer function of sesquiterpne lactones (SLs) obtained from both in
vitro cell culture and in vivo animal models; various SLs have been demonstrated
to execute their anti-cancer capability via inhibition of inflammatory responses,
prevention of metastasis and induction of apoptosis. They also have outlined the
molecular mechanisms involved in the anti-cancer activity of SLs, in particular,
the SL-thiols reaction, the effect of SLs on cell signaling pathways such as nuclear
transcription factor-kappaB (NF-kB) and mitogen-activated protein kinases
(MAPK) and indicated that many SLs are emerging as promising anti-cancer
agents with potential applications in both cancer chemotherapy and
chemoprevention.Phase contrast micrographs of control cells and treated cells
(a-d) and MTT treated cells with blue colored Formazan crystals (e) are given in
figure 3. Control cells (untreated) are normal with clear-cut nuclear membranes;
nuclei are elliptical. Whereas, the treated cells are showing distorted membranes
and nuclei with blebs. The observed sub-cellular changes in the present study could
be an indication of telomerase inhibition. Costunolide induced apoptic cells were
estimated by nuclear damage observation by Kanno et al., (2006); chromatin
condensation and formation of apoptosis bodies were also reported.
In conclusion, the test compound (Fraction II) was very effective as
anti-inflammatory agent; and cytotoxic agent for MDA-MB cell lines. Further
studies are required at low concentrations since the test compound exhibited
cytotoxicity even at low concentrations.
ACKNOWLEDGEMENTS
The authors thank Dr. J. S. Yadav, Director, IICT for constant encourage-
ment.
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