Bangladesh Journal of Pharmacology Research Article Cytotoxic activity of isolated con-Cytotoxic activity of isolated con-Cytotoxic activity of isolated con- stituents from leaves of stituents from leaves of stituents from leaves of Premna Premna Premna serratifoliaserratifoliaserratifolia on MCFon MCFon MCF---7 and HT7 and HT7 and HT---29 29 29 cell linescell linescell lines BJP Introduction Premna serratifolia Linn., commonly known as Arni or Agnimantha, is a large shrub or a small tree common along the Indian peninsular and Andaman coast. Pharmacologically, it has been reported to have activities such as hepatoprotective (Vadivu et al., 2009), immunomodulatory, cardiac stimulant, antibacterial, cytotoxic, antiarthritic, anti-inflammatory (Biradi and Hullatti, 2013), anti-diabetic (Mujumder et al., 2014), anti-oxidant (Mali et al., 2014), anti-obesity and used in treatment of tumors (Cragg and Newman, 2005). Plant derived cytotoxic constituents have played an important role in the development of clinically useful anticancer agents (Cragg and Newman, 2005). Various cytotoxic constituents have been isolated from plants and which are used as anti-cancer agents. Among all cancer types breast cancer is the most common disease in women worldwide with high mortality rate (18%) and relative risk factors, including dietary factors (Ahmed et al., 2014). Colorectal cancer is the third most leading cause of cancer death in both men and women. About 96% of colorectal cancers are adenocarcinomas, which evolve from glandular tissue (Anonymous, 2013). However, the efficacy and effect of currently available drugs is very limited and are more toxic on normal cells. By increasing the use of existing knowledge with newer and established screening tests the majority of these cancers and deaths could be prevented. Although various extracts of P. serratifolia are reported to have cytotoxic activity in the literature (Selvam et al., 2009; Singh, 2011), there are no reports available on isolation based study. As part of ongoing investigation P. serratifolia plant has been selected for bioactivity guided isolation and here cytotoxic activity of two isolated constituents from the n-hexane soluble fraction (unsaponifiable portion) of methanolic extract of P. serratofolia leaves on human cancer cell lines. Materials and Methods Collection and authentication of plant material A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 205-208 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract Premna serratifolia (Syn: Premna integrifolia) is an important medicinal herb known as “Agnimantha” in Ayurveda and traditionally used for anticancer activity. The objective of present study was to isolate the cytotoxic phyto- constituents from the n-hexane soluble fraction of P. serratifolia leaf extract. Unsaponifiable portion of n-hexane soluble fraction was subjected to silica based column chromatography. The major constituents present in all the sub- fractions were identified by TLC and phytochemical tests. Two constituents were isolated and they were purified. Sub-fractions with isolates were tested for cytotoxic effect by BSL bioassay. Two isolates were found to be active and which were tested on cancer cell lines MCF-7 and HT-29 for their cytotoxicity. Among two isolates, one compound has shown significant cytotoxicity. From the results we conclude that the plant isolates showed cytotoxicity against selected human cancer cell lines. Article Info Received: 17 January 2015 Accepted: 3 March 2015 Available Online: 11 March 2015 DOI: 10.3329/bjp.v10i1.21658 Cite this article: Biradi M, Hullatti K. Cytotoxic activi- ty of isolated constituents from leaves of Premna serratifolia on MCF-7 and HT-29 cell lines. Bangladesh J Phar- macol. 2015; 10: 205-08. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Cytotoxic activity of isolated constituents from leaves of Premna serratifolia on MCF-7 and HT-29 cell lines Mahesh Biradi and Kirankumar Hullatti Department of Pharmacognosy, KLES College of Pharmacy, Nehru Nagar, Belagavi 590 010, India. Leaves of P. serratifolia were collected from the premises of Regional Medical Research Centre, (ICMR), Belgaum during January 2011 and authenticated by Dr. Harsha V. Hegde, Scientist ‘B’ RMRC, Belgaum, India. The voucher specimen is deposited in ICMR Herbarium repository (Accession Number RMRC-554). Extraction, fractionation and cytotoxicity by BSL assay The extraction and fractionation of P. serratofolia leaves has been done according modified Cos et al., 2006. Based on the results of previous study (Biradi and Hullatti, 2013), n-hexane soluble fraction was taken up for present study. Phytochemical investigation Phytochemical investigation of isolates was carried out according to the standard procedure (Trease and Evans, 2005; Sandjo and Kuete, 2013). Optimization of TLC system Different solvents systems were tried for developing a TLC system for identification of constituents in the extracts and the one with good resolution was selected as the mobile phase for the study. Toluene: Ethyl acetate: Glacial acetic acid (7:2:1) was found to be the most suitable solvent system for n-hexane fractions as it gave maximum number of bands. Isolation of the phytoconstituents Unsaponifiable portion of n-hexane soluble fraction was subjected to silica based column chromatography. The different fractions were generated using gradient elution technique using petroleum ether, chloroform and acetone with increasing the polarity. Five sub- fractions (SF) generated from unsaponifiable portion (i.e. PS/F1/USM). The chemical nature of all the sub fractions was established by TLC (Plate No. 1-3) and chemical tests (Table I). Processing of the isolated precipitates The sub fractions PS-01 and PS-02 were purified by washing with cold methanol followed by ethanol. The solid mass was weighed and collected in previously labelled eppendorf tubes and stored in a desiccator. In vitro cytotoxicity of the isolated compounds on selected cell lines Brine shrimp lethality (BSL) bioassay BSL assay was performed for all the five sub fractions to confirm the cytotoxic activity and only the isolates which shown the cytotoxicity were subjected for cell line studies. Cytotoxicity of the active compounds on MCF-7 and HT-29 cell lines The active fractions from BSL bioassay were subjected for in vitro cytotoxicity studies using human cancer cell lines MCF-7 (breast carcinoma) and HT-29 (adenocarcinoma). Cell lines and culture medium (Koba et al., 2009) HT-29 (adenocarcinoma) and MCF-7 (breast carcinoma) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Stock cells were cultured in 25 cm2 culture flasks in Dubelcco's Modified Eagle's Minimum Essential Medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS), penici- llin (100 IU/mL), streptomycin (100 mg/mL) and amphotericin B (5 mg/mL) in an humidified atmos- phere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). Preparation of test solutions Stock solution of 1 mg/mL concentration of each test drugs were prepared separately by dissolving in distilled DMSO and made volume with DMEM in 2% FBS and filtered. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies. Determination of cell viability by MTT assay The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/mL using DMEM containing 10% FBS. To each well of the 96-well microtitre plate, 0.1 mL of the diluted cell suspension (approximately 10,000 cells) was added. After 24 hours, supernatant was flicked off upon formation of partial monolayer. Then washed the monolayer once with medium and 100 mL of different test concentrations (62.5 to 1000 µg/mL) of test drugs were added on to the monolayer in microtitre plates. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere and observations were noted every 24 hours interval by microscopic examination. After 72 hours, the drug solutions in the wells were discarded and 50 mL of MTT in PBS was added to each well. The plates were gently shaken and incubated again for 3 hours. The supernatant was removed and 100 mL of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line. %Growth inhibition = (Mean OD of individual test group/Mean OD of control group) × 100 Results and Discussion The study was taken to identify the constituents 206 Bangladesh J Pharmacol 2015; 10: 205-208 responsible for cytotoxic activity of P. serratifolia leaf extract. The n-hexane soluble fraction (F1) has shown significant cytotoxicity in the earlier studies (Biradi and Hullatti, 2013). The unsaponifiable portion which was rich in steroids and triterpenoids was subjected for further fractionations using column chromatography. The column chromatography has yielded 5 sub- fractions (PS-01 to PS-05) with gradient elution. Out of these 5 sub-fractions, PS-01 and PS-02 were found single compound fractions as identified by TLC. The chemical tests and TLC has shown that all the 5 sub- fractions are triterpenoids in nature. The BSL bioassay of these sub-fractions has indicated that PS-01 and PS- 02 are more cytotoxic than other 3 sub-fractions LC50 values are depicted in Table II. Further after purifica- tion these 2 fractions were analysed on MCF-7 and HT- 29 cell lines. Result of this study has indicated that the sub-fractions PS-02 has significant cytotoxicity in both the cell lines which are depicted in Figure 1 and 2 (LC50 value 100.0 and 99.9 µg/mL respectively). Various authors have reported cytotoxic activity of number of triterpenoids such as ursolic acid, oleane and sterols from different plants (Da Silva Filho et al., 2009; Awasare et al., 2012). These data indicate that the certain triterpenoids of P. serratifolia may be useful as cytotoxic component that needs further characterization to identify the structure. Bangladesh J Pharmacol 2015; 10: 205-208 207 Table I Cytotoxicity screening of the column sub-fractions (BSL assay) Fractions LC50 PS-01 54.6a PS-02 30.8a PS-03 323.3 PS-04 143.6 PS-05 209.6 aNote: The fractions with LC50 values ≤100 μg/mL were considered as active fractions % G ro w th in hi bi tio n Concentration (µg/mL) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 120  120 100 80 60 40 20 0 0 200 400 600 800 1000 1200 Figure 1: Graphical representation of cytotoxic effect of drugs on MCF-7 cell line Plates 1-3: TLC of sub-fractions at different pre and post derivatization Plate. No 1: Visualization at UV 254 nm; Solvent system: Toluene: Ethyl acetate: Glacial acetic acid (7:2:1) Table II TLC profile of the column sub-fractions Fractions Gradient program Physical nature Number of spots observed Rf value PS-01 Petroleum ether Yellowish and amorphous 2 0.82, 0.62 PS-02 CHCl3:PE (1:4) White and amorphous 1 0.65 PS-03 CHCl3/PE (1:1) Yellowish and sticky 3 0.69, 0.49, 0.36 PS-04 CHCl3 Yellowish and sticky 2 0.69, 0.36 PS-05 Me2CO Brownish and sticky 2 0.69, 0.33 Plate. No 2: Visualization at UV 366 nm; Solvent system: Toluene: Ethyl acetate: Glacial acetic acid (7:2:1) Plate. No 3: Visualization after derivati- zation by ANS spray; Solvent system: Toluene: Ethyl acetate: Glacial acetic acid (7:2:1) PS-01 PS-02 From the present study, the triterpenoids of P. serratifolia provide a new insight into the anti-cancer approach. 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J Sci Res. 2009; 1: 145-52. 208 Bangladesh J Pharmacol 2015; 10: 205-208 Concentration (µg/mL) Figure 2: Graphical representation of cytotoxic effect of drugs on HT-29 cell line 0 200 400 600 800 1000 1200 0 20 40 60 80 100 120  120 100 80 60 40 20 0 0 200 400 600 800 1000 1200 % G ro w th in hi bi tio n PS-01 PS-02 Author Info Kirankumar Hullatti (Principal contact) e-mail: drhullatti@klepharm.edu Your feedback about this paper 1. Number of times you have read this paper 2. Quality of paper 3. Your comments undefined: undefined_2: undefined_3: undefined_4: undefined_5: undefined_6: undefined_7: undefined_8: undefined_9: undefined_10: DatePrinted: This article was downloaded by you on: Jul 18, 2017 Text2: Dropdown3: [Click] Dropdown4: [0]