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Sunkara Yashvanth et al.

/ Journal of Pharmacy Research 2010, 3(8),1775-1778
Research Article
Available online through
ISSN: 0974-6943
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

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

and culture were maintained in a humidified atmosphere with 5% CO2 .30±3. 1x104 Cells (counted by Trypan blue exclusion dye method) in 96.2 pg/ml (Figure 1). ( 1 0 µ g / m l ) µ g / m l) L P S µ g / m l) ml penicillin. 8 Concentration of nitrite (µm) pound for 48 hrs at 370 C in DMEM/MEM with 10% FBS (Fetal bovine 7 serum). T.61 Journal of Pharmacy Research Vol. However. Doxorubicin was used as standard. 3(8). % viability in treated cell lines Treated and normal cancer cells were observed in phase contrast mi- croscope ( Nicon TS-100 Eclipse). 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.39±2.81 45.Issue 8.01 6 0 0 * 10µg/ml+LPS 54.0 ware (version 2. n=3 MDA-MB Test Compound 56. 200 µg/ml streptomycin.1775-1778 Table 1. n=3 -40.38 Doxorubicin 4. 1 Molecular devices) IC–50 values were determined from plot: % inhibition 0 (from control) versus concentration.93 8 0 0 (Fraction II) 50µg/ml+LPS 6. S am PS ne LP d lipr alo tL un ith Ro ou po W dia M e a n Absorbance of Sample om ith Me W stc % Viability = X 100 Te Mean Absorbance of control *P<0. it could express the TNF alpha (309. 20. India.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.0 (ANOVA) followed by Dunnet’s test for sub-group comparison using Prism Soft. Pune.1 pg/ml. with LPS (50µg/ml).0 Statistical Analysis 100. Sunkara Yashvanth et al.0 0.0 0.0 Extrapolated to 100 % surviving cells the concentration of TNF alpha expressed was 563. Concn. Nitric oxide inhibition levels.0 120.87 1 0 0 0 25µg/ml+LPS 89. ( 1 0 L P S ( 5 0 W i t h o u t T . Reports are available on the 40.3. spectra max. IC-50 values of compounds on various Cell lines.50µg LPS.77±7.0 % Viability 80.2 pg/ml) with 55 % surviving cells when incubated with LPS (50 µg/ml). HT- 2 0 0 29 (Colon cancer).0 0 100 200 300 are Mean ±SD. Compound Concentration % Viability % Inhibition 1 2 0 0 (Average of duplicates) Concentration of TNF-alpha (pg/ml) Rolipram 50µg/ml+LPS 82. 100.0 Table 2.0 alone was 851.0 % Inhibition is 33. TNF-alpha inhibition levels. Hence. All values -20.3 93.05 . 40.01) wherever necessary.28 * 25µg/ml+LPS 7.0 Cell Line Compound IC-50µg/ml Concentration (µg/ml) HT-29 Test Compound 109.0 Statistical evaluations were performed using analysis of variance 60.3%. At a concentration of 10 µg/ml. 2mM L-glutamine. Percent viability was calculated using the following formula. – Rolipram.38 Doxorubicin 49. / Journal of Pharmacy Research 2010.93 -2. the percent inhibition of TNF alpha by the test compound 60.55 A549 MDA-MB HT-29 Doxorubicin 2.5 92.August 2010 1775-1778 . The amount of TNF alpha expressed due to LPS (50µg/ml) induction 80. formazan product. of test compound -10µg/ml Microscopic Observations Figure 3.38±6. Rol. there was 45 % cell death. 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.80 All values are Mean ±SD. cell lines were grown as adherents in DMEM. 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 2.13 17.The absorbance at 3 2 570nm was measured on a spectrophotometer (Plate reader.05 vs.20 6.C. Whereas. there after the above media was replaced 4 with 200µl of DMSO and incubated at 370 C for 10min.33±0.76%. C . 140.04 Test Compound 10µg/ml+LPS 102.Cytotoxicity of test *P<0.0 20.well plates were incubated with various concentrations of the test com. 100 µg / R o l . % Inhibition in treated cell lines assay of the compound in RAW cells is depicted in Table 1. Rolipram showed an inhibition of 38. A549 (Lung cancer). MTT Figure 4.95 A549 Test Compound 54. where as MDA-MB (Breast cancer cell line) was grown in Minimum 0 Essential Media (MEM) supplemented with 10% fetal bovine serum. MTT assay of compounds in RAW Mouse Macrophase cells Figure 1.19 4 0 0 * were obtained from National Center for Cell Science (NCCS).60±0.

dihydrocostunolide and methoxy derivatiives of costunolide and ß-cyclocostunolide on the cancer cell lines – Colo-205. Singh.. (2007) have reported the inhibition of TNF. caused by recep- tor-mediated pathway and inhibited telomerase activity in NALM-6 cells (Human B cell leukemia). However.. S.. Current Sci. 2005). Most normal human cells have no detectable telomerase activity. Ethnobotany of Kashmir-I. tone(2). (2006). Buth. and cytotoxic agent for MDA-MB cell lines. in particular. Park et al. G. but it is present in most cancer cells (Mori et al.29 after treatment(100µg) Zhang et al. Nair and I.. Virjee. 1994.type-P53) and MDA-MB (mutant P 53) cells treated with costunolide in a concentration and time- dependent manner. Kachroo and G. The inhibition of telomerase causes a progressive and critical reduction of telomers. Botany 5. B. since the test compound appears to be toxic to RAW cells at a concentration of 10 µg/ml.17ìM/ml (with 4. P. Park et al.. activity of the test compound was nearly comparable to that of the activity of Doxorubicin. The observed sub-cellular changes in the present study could be an indication of telomerase inhibition. Zhang et al. and M.(1999) and Matsuda et al. New Delhi1 1956 3. CSIR. S. Costunolide induced apoptic cells were anti-inflammatory activity of costunolide. various SLs have been demonstrated to execute their anti-cancer capability via inhibition of inflammatory responses.. MCF-7. 177-183 (1999). 789-799 (2004). Of studies are required at low concentrations since the test compound exhibited the reported six compounds in the present study. 1999). Chopra. Ethnomedicobotany of Indian Trans Himalaya. Whereas. The concentration of Nitric Oxide expressed was 5. Pae et al. H. J. 86(6). R. (1994) have reported anticancer activity of costunolide. C. Cytotoxicity Studies Cytotoxic activity of the test compound against the three cell lines was in the following order . Robinson et al.. 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