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Phytochemicals and Cytotoxicity of Launaea procumbens on Human Cancer Cell Lines

© 2016 Pharmacognosy Magazine | Published by Wolters Kluwer ‐ Medknow S431 ABSTRACT Background: The plant Launaea procumbens belongs to the family Asteraceae and traditionally used in the treatment rheumatism, kidney, liver dysfunctions and eye diseases. In the present study Phytochemical analysis and fractions of methanolic extract of L. procumbens leaves were tested in vitro for their cytotoxicity. Objectives: Phytochemical analysis and cytotoxic activity of methanolic extract and fractions of Launaea procumbens against four cancer cell lines K562, HeLa, MIA-Pa-Ca-2 and MCF-2 by SRB assay. Materials and Methods: Powdered leaves of Launaea procumbens were extracted sequentially with hexane, ethyl acetate, butanol and water by cold extraction. Phytochemical analysis and cytotoxicity assay were carried out for these fractions using SRB assay against four human cancer cell lines, namely leukemia (K562), cervix (HeLa), pancreatic (MIA-Pa-Ca-2) and breast (MCF-7). Results: Ethyl acetate extract exerts potent cytotoxicity against human leukemia (K562), cervix (HeLa) and breast (MCF-7) cell lines IC50 value of 25.30±0.50, 19.80±0.10 and 36.90±4.90 μg/ ml respectively. Moderately cytotoxic effect found in hexane extract IC50 value of 41±8 and 48.20±0.50 μg/ ml against leukemia (K562), and breast (MCF-7) cancer cell line respectively. The Chemical composition analyzed by GC-MS showed considerable differences in solvent fractions of Launaea procumbens. Conclusion: This study revealed the cytotoxic potential of ethyl acetate and hexane fractions of L.procumbens leaves on different cancer cell lines.


INTRODUCTION
The family Compositae (Asteraceae) has the distinction of an extremely natural taxon, with its unique floral theme and micro morphological features. It has attracted fascinated and even repelled botanists for over two centuries. [1] Launaea procumbens is a herb belonging to Compositae (Asteraceae) family commonly known as jangali booti in Hindi and Al-Hewa in Arabic. [2,3] It is found as a weed throughout the plains of India and up to an altitude of 2400 m in the Himalayas. [4] It has been used as a food supplement and as a washing agent [5] in rheumatism and galactogogues. [6] It is used in the folk medicines in the treatment of tumors, skin problems and dysentery. [7] Ayurvedic and herbal preparations of this plant are used in wound healing, longevity, [5] painful urination, and reproductive diseases. [8] It also possesses antipyretic, [9] insecticidal and antifungal properties. [10] Asteraceae family consists of more than 4000 sesquiterpenoids structures with more than 30 different skeletal type. These natural compounds are responsible for wide range of bioactivities, including toxicity for certain cancer cell lines by inhibition of nuclear DNA synthesis. [11]

Plant material
Plant samples were collected from local area of Lucknow (India) in the month of June, 2014 and identified by Dr. Anand Prakash, Principal Scientist, National Botanical Research Institute (NBRI), Lucknow. A voucher specimen (No. 216343) has been deposited in the herbarium of NBRI.

Plant extract
The air-dried powdered leaves of L. procumbens (580 g) were extracted from methanol. The methanolic extract was evaporated in a rotatory evaporator and dried by vacuum pump. The methanolic extract was suspended on water and extracted successively with hexane, ethyl acetate and butanol.

Cell lines and culture medium
The cytotoxic activity was performed in Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai. All the cell culture work was performed under sterile conditions and under standard cell culture conditions. Cell cultures were grown in well cultured microtitre plates (RPMI-1640 medium with 2 mM glutamine, pH 7.4 supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin and 100 units/mL penicillin). The targeted human cancer cell lines were grown in a tissue culture flask in carbon dioxide incubator at 37°C and 90% relative humidity to obtain enough number of cells. The cells were harvested by the treatment of trypsin -EDTA and single-cell suspension in complete growth medium.

In-vitro cytotoxicity assay
In-vitro cytotoxic activity against different cancer cell lines was performed using 96-well culture plates in triplicates. To each well of the 96 well microtitre plates 100 µL suspension was added. The cells were allowed to grow at 37°C for 24 h in 5 % carbon dioxide incubator. In the cell suspension, different concentrations of extract were added. The plates were further incubated for 48 h and 25 µL of 50% trichloroacetic acid added gently to stop cell growth by thin layering of trichloroacetic acid on test compounds. The plates were further incubated at 40°C for 1 h to fix the cells attached to the bottom of the wells. The plates were washed five times with distilled water to remove traces of medium, trichloro-acetic acid, sample, serum proteins, and then air dried. The cell growth in air dried plates was measured by staining with sulforhodamine B dye. The unbound dye was removed by dissolving Tris-base buffer (100 µL/ well, 0.01M, pH 10.4) and plates were stirred for 5 min on a mechanical shaker. The optical density was measured at 540 nm on ELISA reader.
Gas chromatography-mass spectrometry (gc-ms) GC-MS analysis was performed with a Thermo Fisher TRACE GC ULTRA coupled with DSQ II Mass Spectrometer instrument using a TR 50MS column (30m x 0.25mm ID x 0.25 µm, film thickness). Constant flow at 1 mL/min of carrier gas (Helium) was used for the analysis. The injector temperature of the instrument was 220°C and oven temperature was started from 50°C, (hold time 5.0 min) to 250°C with ramp of 4°C/ min (hold time 5 min). Sample was injected in split mode (1:50) with injection volume of 1 µL. The ion source temperature was set at 220°C and transfer line temperature was at 300°C. The ionization of the sample was performed in electron impact mode at an ionization voltage of 70 eV. Mass range was used from m/z 50 to 650 amu.

Statistical analysis
The individual data values are presented as the arithmetic mean ± SD (standard deviation). The statistical significance of the results obtained from in vitro studies was evaluated by the ANOVA at P < 0.05, using STASTICAL software.

Cytotoxicity of the extracts
The cytotoxicity study were carried out for different fractions LPH (n-Hexane), LPE (Ethyl acetate), LPW (Water) and LPB (n-Butanol)  Table 2. Adriamycin drug has been used as a standard in all above human cell lines. Among the different extracts only ethyl acetate and hexane fraction exhibited the property to inhibit the growth of cancer cell lines (Figure 1 and Figure 2) except pancreatic cell line. Ethyl acetate fraction was found active against cervix (HeLa), leukemia (K562) and breast (MCF-7) cancer cell lines with IC 50 value of 42, 56.70 and 64 µg/ mL, respectively. Cytotoxic activity was found in hexane extract against leukemia (K562) cell lines with IC 50 of 69.10 µg/mL. The pancreatic cell line (MIA-Pa-Ca-2) was found resistant against these extracts. Water and butanolic fractions were not found to be active against these human cancer cell lines.

CONCLUSION
Our results indicate that ethyl acetate and hexane fractions prepared from methanolic extract of L. procumbens leaves were found active against specified human cancer cell lines. On the basis of literature pyrazole, imidazole and triterpenes show activity against cancer. Thus, anticancer potency in specified fractions against tested cell lines may be due to one of these or mixture of these as our study revealed high amount of these in plants.