Phytomedicine 21 (2014) 497–505
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Antioxidant, hepatoprotective and cytotoxic effects of icetexanes isolated from stem-bark of Premna tomentosa Naidu V.G.M c , Hymavathi Atmakur b , Suresh Babu Katragadda b , Bhavana Devabakthuni a , Anudeep Kota a , Chenna Keshava Reddy S. a , Madhusudana Kuncha a , Vishnu Vardhan M.V.P.S. a , Prasad Kulkarni a , Madhusudana Rao Janaswamy b , Ramakrishna Sistla a,∗ a
Division of Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India Division of Natural Product Chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India c Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Hyderabad 500037, India b
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 16 August 2013 Accepted 29 September 2013 Keywords: Premna tomentosa Antioxidant Hepatoprotective Anticancer Icetexanes
a b s t r a c t The study investigates the antioxidant, hepatoprotective and antiproliferative effects of novel icetexane diterpenoids (ice 1–4) isolated from hexane extract of stem bark of Premna tomentosa. A549, HT29, MCF-7, MDA-MB-231, A431 cells were used to assess the antiproliferative activity by MTT assay. Cell death induced by apoptosis was determined by morphological assessment studies using acridine orange/ethidium bromide staining (dual staining), mitochondrial potential measurement by JC-1 staining, and cell cycle analysis by propidium iodide staining method by Muse cell analyser. Anti oxidant activity was investigated by in vitro assays such as DPPH, nitric oxide and superoxide scavenging activities. Hepatoprotective activity was determined in vitro with HepG2 cells and in vivo by tBHP induced hepatic damage mice model. Based on the in vitro cytotoxic assays and morphological assessment studies using fluorescence microscopic study (acridine orange and ethidium bromide double staining) and mitochondrial potential measurements, it was found that ice 2 and 3 possess good antiproliferative effect via mitochondrial mediated apoptosis in human lung and breast cancer cells. Results of in vitro antioxidant studies demonstrated that ice-4 has showed good antioxidant activity. The restoration of serum levels of SGOT, SGPT and ALKP, liver GSH status and reduction or inhibition of lipid peroxidation in liver of tBHP intoxicated mice after administration of ice-4 at dose of 250 mg/kg indicated its potential use for hepatoprotective activity. © 2013 Elsevier GmbH. All rights reserved.
Introduction Development or identification of anticancer agents with limited or few adverse effects is sustained thrust in the area of anticancer research. The development of new synthetic molecules possessing potential anticancer activity is highly expensive and time-consuming process. For this reason there is increasing search for herbs possessing potential anti-cancer activity. Recent review pointed out that, about 74% of anticancer compounds being either natural or natural product-derived products, indicating potency of these scaffolds (Newman et al., 2003). The unprecedented structures of these molecules make them excellent synthetic targets, and their potent activity against a broad number of therapeutic indications makes these natural products excellent drug lead candidates for new therapeutics (Newman, 2008).
∗ Corresponding author. Tel.: +91 40 27193753. E-mail addresses: [email protected], [email protected] (R. Sistla). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.025
The genus Premna (Verbenaceae) comprises a group of more than 200 different species, distributed in tropical and subtropical parts of the world (Kubitzki, 2004; Marles and Farnsworth, 1995). Premna tomentosa is a well known medicinal plant and all parts of this plant have been employed for the treatment of various ailments in Indian systems of medicine. As part of pharmacological–phytochemical integrated studies of medicinal plants from Indian flora, we are investigating the chemical composition of plants from the genus Premna (Verbenaceae) as well as their cytotoxic activities (Suresh et al., 2011a,b). In the course, we have recently reported some icetexane diterpenes from the P. tomentosa (Fig. 1) which has effectively induced cytotoxicity in different human cancer cell lines (Hymavathi et al., 2009). However, the mechanism of action of these compounds inducing cytotoxicity was not known. The potent biological activity coupled with its novel structural features prompted us to investigate their molecular mechanism studies. In the present communication, we have performed the apoptosis screening techniques to differentiate apoptosis and necrosis, including evaluation of the
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repeated silica gel (100–200 mesh) column chromatography with hexane–chloroform–acetone (5:3.2:1.8) yielded ice-3 (0.005 g). Fraction F2 was subjected to silica gel column chromatography eluting with hexane–chloroform–acetone (5:3.4:1.6) to yield ice-4 (0.002 g). Fraction F3 was subjected to silica gel column chromatography with the elution of hexane–chloroform–acetone (5:3:2) to yield 0.080 g of ice-1, and with hexane–chloroform–acetone (5:3.8:1.2) to get 0.005 g of ice-2 and their chemical structures was depicted in Fig. 1.
Fig. 1. Chemical structures of icetexanes (ice-1–4) isolated from hexane extracts of Premna tementosa.
anti-proliferative and antioxidant activity. In addition, we also evaluated the hepatoprotective activity of these compounds on the basis of their antioxidant activity. Materials and methods
Finger printing Chromatographic separation was achieved by reverse-phase chromatography using the isocratic mode of elution. With Synergy 4, Fusion-RP 80A 150 mm × 4.6 mm column. LC–MS studies were performed using an Agilent 1100 series online ion trap MSD mass spectrometer with ESI source in positive-ion mode equipped with a degasser (G1379A), binary pump (G1312A), autosampler (G1329A), autosampler thermostat (G1329B) and diode array detector (G1315B). All the systems were from Agilent Technologies (Waldbronn, Germany). The data were acquired and processed using LC/MSD trap software 4.2 (Bruker, Waldbronn, Germany). An isocratic elution with methanol and water (80:20, v/v) as mobile phase was pumped at a flow rate of 1.0 ml/min; the sample injection volume was 20 l with column temperature maintained at ambient conditions. Nitrogen was employed as the nebulizer gas. The ion source conditions were set as follows: temperature, 300 ◦ C; nebulizer gas, 30 psi; dry gas, 8.0 L/min; skimmer 40.0 V; capillary exit 128.0 V; trap drive 44.5; max accu time 200,000us; Icc target 20,000.
Cell lines Anti-cancer activity A549 cells (lung cancer), HT-29 cells (colon cancer), MCF-7 cells (breast cancer), MDA-MB cells (breast cancer), A431 cells (skin cancer) and HepG2 cells (hepatocellular carcinoma) were obtained from National Centre for Cell Science (NCCS), Pune, India. Chemicals and reagents DPPH (2,2-diphenyl-1-pycrylhydrazyl), ascorbic acid, Griess reagent, Silymarin (originated from the fruit of Silybum marianum, Cat # S0292), GSH, O-pthaldehyde, 1,1,3,3-tetramethoxypropane, thiobarbituric acid (TBA), tricarboxylic acid (TCA), sodium chloride, nitro blue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), phenazine methosulphate, sodium pyrophosphate, glacial acetic acid, n-butanol, potassium dihydrogen phosphate, dihydrogen sodium phosphate, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM), sodium bicarbonate, thiazolyl blue tetrazolium bromide, trypsin solution, glutamine and JC-1 were obtained from Sigma Co. (St. Louis, MO, USA). Antibiotic and antimycotic mixture and fetal bovine serum were obtained from GIBCO BRL (NY, USA). Serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT) and alkaline phosphatase (ALKP) were obtained from Bayers Diagnostics (Leverkusen, Germany). Extraction and isolation of compounds from P. tomentosa bark The extraction and isolation of compounds from P. tomentosa was performed as described in previous study (Hymavathi et al., 2009). The stem bark (1 kg) of P. tomentosa is collected and dried, which is then ground and extracted three times with hexane in a soxhlet apparatus. The extracts were combined and concentrated under vacuum. The active hexane extract (2.5 g) was subjected to column chromatography (silica gel, 60–120 mesh) using step gradient of hexane/EtOAc resulting three fractions F1, F2 and F3. On eluting Fraction F1 by
Cytotoxicity assay The cytotoxic effect of icetexanes was assessed and quantified in different cell lines A549, HT-29, MCF-7, MDA-MB and A431 after incubating for different durations of 24 h and 48 h by MTT assay. A549, HT-29, A431 cells were grown in DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 1 mM sodium pyruvate and 1.5 g/l sodium bicarbonate under 5% CO2 at 37 ◦ C, where as MCF-7 and MDA-MB-231 cells were grown in MEM supplemented with 10% FBS. 1 × 104 cells per well were seeded in a 96-well plate and after 24 h the media was replaced with fresh medium containing different concentrations of icetexanes 1, 2, 3 and 4 dissolved in DMSO and in control 1% DMSO was added. After 24 h and 48 h incubation, the media were replaced with 100 l fresh media and 10 l of MTT reagent in to each well. Further cells were incubated at 37 ◦ C for 4 h. Later, the formazan crystals formed were solubilized in DMSO. Finally absorption at 570 nm was read from ELISA reader. IC-50 values were calculated against control. Morphological analysis Acridine orange (AO) and ethidium bromide (EtBr) dual staining. Acridine orange (AO) and ethidium bromide (EtBr) dual staining method was used to assess the chromatin condensation and membrane integrity, respectively, which serves as markers for apoptosis. AO is a cationic dye which enters only live cells and stains nuclei green. EtBr stains DNA orange, which is mainly taken up by the cells when cell membrane integrity was lost. Dual staining allows enumeration of four categories of cells as follows; live-non-apoptotic cells (nuclei stains bright green with organized structure), live-apoptotic cells (nuclei stains bright green with condensed or fragmented chromatin), dead-apoptotic cells (bright orange chromatin that is highly condensed or fragmented) and dead-non-apoptotic cells (bright orange chromatin with organized structure). Around 1 × 105 cells per well were dispensed in to 12 well culture plate and 24 h after seeding, cells were incubated with
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Compounds 2 and 3 (50 and 100 g/ml) for 24 h. The cells are then washed with phosphate buffered saline (PBS), trypsinized and stained with 10 l of staining solution containing 100 g/ml of AO and 100 g/ml of EB in PBS. A volume of 5 l of cell suspension was immediately taken on to the microscope slide and then examined by fluorescence microscope (NiKon TS100, Topa-Eclipse). Mitochondrial potential measurement. Loss of mitochondrial membrane potential ( ) is indicative of apoptosis and can be detected by a unique fluorescent cationic dye 5,51 ,6,61 -tetrachloro1,11 ,3,31 -tetraethylbenzamidazolocarbocyanin iodide commonly known as JC-1. This reagent penetrates passively into cells and mitochondria. In non-apoptotic cell, the mitochondrial potential is unaltered and the dye enters the mitochondria forming aggregates of red colored fluorescence where as in apoptotic cells due to altered mitochondrial potential the dye does not accumulate inside the mitochondria. Instead, it is distributed throughout the cell as monomer giving green fluorescence. MCF-7 cells were seeded in 12-well plate at a concentration of 105 cells per well. After 24 h the cells are treated with 50 g/ml ice-2 and ice-3. After 24 h, the cells were washed with PBS and incubated for 30 min in 10% DMEM without Phenol red containing JC-1 at a concentration of 2 g/ml and fluorescence is observed with fluorescence microscope at wavelengths of 520 (ex ) and 594 (em ). Flow cytometric analysis MCF-7 cells were incubated with two different concentrations 50 g/ml and 100 g/ml of ice-2 for 24 h. Cells were processed, fixed and stained with cell cycle reagent kit. Cells were analyzed for the DNA content to determine the percentage of cells present in different stages of cell cycle. Fluorescence emitted from the propidium iodide–DNA complex was quantified by using Muse cell cycle analyser. Antioxidant activity determination DPPH method The antioxidant activity of P. tomentosa dried stem bark extract was assessed by determining its ability to scavenge DPPH radical. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical, due to odd electrons it shows absorption maximum at 517 nm. DPPH accepts electron or hydrogen radical from antioxidant molecule to become a stable diamagnetic molecule, which is confirmed by decrease in absorbance at 517 nm. Ascorbic acid was used as standard. Aliquots of 25 l of various concentrations (100–1000 g/ml) of icetexanes 1–4 were mixed with 200 l of Tris-buffer and 125 l of DPPH in a 96 well plate and absorbance was recorded at 517 nm using UV-Spectrophotometer (Biotek, Synergy 4) after incubation for 15 min in dark at 37 ◦ C. The percentage of scavenging activity is determined using the following formula:
Percentage of inhibition (%) =
Acontrol − Asample Acontrol
∗ 100
where, Acontrol – absorbance of DPPH, Asample – absorbance of DPPH with test compound. Nitric oxide scavenging activity Nitric oxide radical is generated from reaction mixture containing sodium nitroprusside (20 mM) in phosphate buffered saline (pH 7.4) when incubated at 25 ◦ C for 30 min under a visible polychromatic light source (25 W tungsten lamp) (Marcocci et al., 1994). The nitric oxide radical thus generated interacts with oxygen to produce nitrite ion, which is assayed by mixing with an
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equal amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) (Hsieh et al., 2010). The resulting absorbance was measured at 570 nm. Decrease in absorbance in the presence of different concentrations of test compounds (100–1000 g/ml) indicated the nitric oxide scavenging activity. Ascorbic acid was used as positive control. The percentage of Nitric oxide scavenging activity is determined using the following formula.
Percentage of inhibition (%) =
Acontrol − Asample Acontrol
∗ 100
where, Acontrol – absorbance of reaction mixture without test compound; Asample – absorbance of reaction mixture with various concentrations of test compounds. Superoxide scavenging activity Superoxide scavenging activity was assessed according to the method described by Beauchamp and Fridovich (1971) and Rai et al. (2006). The method is based on generation of superoxide radicals; the superoxide radical reduces the NBT (Nitroblue tetrazolium) to a blue colored formazan that can be measured at 560 nm. The reaction mixture consisted of 0.1 ml of various concentrations (50–2000 g/ml) of icetexanes, 1.2 ml of sodiumpyrophosphate (pH 8.3, 0.052 M), 0.1 ml of phenazinemethosulphate (186 M), 0.3 ml of nitrobluetetrazolium (NBT) (300 M), and 0.2 ml of NADH (750 M). The reaction was started with the addition of NADH and after incubation for 90 s at 30 ◦ C. The reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture is stirred vigorously with 4 ml of n-butanol and the color intensity was measured spectrophotometrically at 560 nm. Gallic acid was used as standard. Hepatoprotective activity In vitro method In vitro hepatoprotective activity was assessed by ability of ice4 to preclude the oxidative stress induced by tBHP in HepG2 cells (hepatocellular carcinoma). HepG2 cells were plated at a density of 1 × 104 cells per well in a 96 well plate along with 100 l of MEM supplemented with 10% FBS in each well. Various concentrations of Compound 4 (12.5 g/ml, 25 g/ml and 50 g/ml) were exposed to HepG2 cells along with tBHP (250 M) for 6 h and the viability of cells was assessed by MTT assay (Hae-Ung et al., 2005). In vivo method Comparative study of hepatoprotection in tertbutylhydroperoxide (tBHP) induced hepatotoxicity in mice model was carried for the ice-4 and compared with positive control, silymarin. Male Swiss albino mice weighing 18–24 g were obtained from NCLAS, NIN, Hyderabad and housed at animal house facility at IICT, Hyderabad. Animals were maintained on stock pellet diet and tap water ad libitum and maintained on 12/12 h light/dark cycle. All the procedures during the animal experiment were prior approved by IAEC. Mice were divided in to four groups of six mice each. Animals received four successive doses of ice-4 and standard (silymarin) drugs at 0, 24, 48 and 72 h followed by inducer (tBHP) at a dose of 1.8 mM/kg, in a volume of 0.5 ml/100 g body weight (Hae-Ung et al., 2005; Valentao et al., 2004). Control group animals received only vehicle and tBHP group animals received vehicle followed by inducer. Test group animals were administered with ice-4 at 250 mg/kg body weight whereas standard group animals were administered with silymarin 250 mg/kg body weight as oral suspensions, in 1% gum acacia. The blood samples were collected through retro orbital plexus after 24 h of tBHP administration. The samples were allowed to clot and serum was separated by
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and A431 cells was observed. It was observed that percentage viability decreased with increase in time of exposure of icetexane compounds in all cell lines. IC-50 values were expressed as g/ml and presented in Tables 1 and 2. Among the isolates, ice-2 and ice-3 were demonstrated significant cytotoxicity compared to ice-1 and 4. In particular, ice-2 and ice-3 have momentous cytotoxic effect on breast cancer (MCF-7) and lung cancer cells (A549).
Fig. 2. Finger printing of icetexanes isolated from hexane extract of Premna tomentosa.
centrifugation (1000 × g, 15 min) for estimation of SGOT, SGPT and ALKP (Auto blood analyzer Express plus, Bayer Corporation USA). Mice were sacrificed by CO2 asphyxiation, dissected and the liver was excised from the animals, weighed and used for quantification of extent of lipid peroxidation and glutathione contents. Lipid peroxidation assay. Lipid peroxidation was assessed by measuring the amount of malondialdehyde (MDA), a secondary product of lipid peroxidation (Lee et al., 2005). An improved thiobarbituric acid spectrofluorometric method employing 1,1,3,3tetramethoxyproprane as precursor has used for producing MDA in standard solutions through acid hydrolysis. Excised liver tissue was homogenized in 50 mM phosphate buffer (pH 7.4). To a volume of 1 ml of tissue homogenate, 2 ml of 10% trichloroacetic acid (TCA) was added and mixed well. After centrifugation at 10,000 × g at 4 ◦ C for 20 min, the protein free supernatant was collected and to 1 ml of 1% thiobarbituric acid (TBA) reagent was added and mixed well. This solution was incubated at 70 ◦ C for 40 min. The reaction mixture was cooled to room temperature and fluorescence was measured at 510 nm excitation and 553 nm emission wavelengths. The results were expressed as g MDA formation per g liver tissue. Results and discussion Finger printing From the results it was found that the hexane extract of P. tomentosa gave peaks at the following retention time values: 3.11, 3.56, 4.03, 4.36, 4.84, 5.88, 6.52, 6.61, 7.50, 8.88, 10.28, 11.75, 12.66 min. Based on the molecular weight and RT values for pure icetexanes (1–4) from LC–MS data (Supplementary Fig. 1), the corresponding peaks in finger printing were depicted as shown in Fig. 2. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed. 2013.09.025. Cytotoxicity MTT assay is most widely used to determine the cytotoxic potential of medicinal agents and other toxic materials. The reduction of yellow MTT to purple colored formazan takes place only when mitochondrial reductase enzymes are active and therefore the conversion can be directly related to number of viable cells. The cytotoxic effect of icetexane compounds after 24 h and 48 h of treatment in A549, MCF-7, MDA-MB-231, HT-29
Acridine orange and ethidium bromide dual staining Morphological abnormalities were studied under phase-contrast and fluorescence microscope. Cells treated with 50 g/ml of either ice-2 or ice-3 showed morphological changes such as chromatin condensation, fragmentation and formation of apoptotic bodies. However, control cells retained normal healthy shape with intact nuclei and without any abnormalities. The results of light microscope are consistent with that of fluorescence microscopy as shown in Figs. 3 and 4. Mitochondrial potential Induction of apoptosis in a cell can be determined with mitochondrial membrane potential. Mitochondrial membrane potential was assessed with dye JC-1. In this experiment, we observed that cells treated with ice-2 and ice-3 showed green fluorescence indicating the loss of mitochondrial potential, hence induction of apoptosis, whereas vehicle treated cells showed red fluorescence as shown in Fig. 5. Cell cycle analysis MCF-7 cells were analyzed for DNA content using flow cytometry. The percentage of cells in G2/M phase increased with increase in concentration of ice-2 compared to control cells. The study revealed that ice-2 displayed G2/M phase arrest which is concentration dependent (Fig. 6). Antioxidant activity DPPH method It is the simplest method developed to determine the antioxidant activity using a stable free radical, 1,1-diphenyl-2-picryl-hydrazyl(DPPH) with specific absorption maxima at 517 nm. The extent of free radical scavenging activity is determined by decrease in absorbance at 517 nm compared to blank. The results indicated that ice-2, 3 and 4 have dose dependent ability to quench free radical. Furthermore, it has been found that ice-4 has significantly higher free radical quenching capacity when compared to ice-2 and ice-3 as represented by its lower EC50 (Fig. 7). Nitric oxide scavenging activity The nitric oxide scavenging activity of icetexanes was determined based on the inhibition of nitric oxide radical generation from sodium nitroprusside in buffer saline and measured by Griess reagent. The activity is expressed as percentage inhibition of nitric oxide production and from that EC50 was calculated for standard and test compounds. The results have revealed that NO scavenging activity of ice-4 was on par with standard ascorbic acid as indicated by no significant difference between their EC50 (p < 0.001). On the contrary, NO scavenging activity of ice-2 and 3 are not significant (Fig. 8). Super oxide scavenging activity: The reduction of yellow color of NBT to blue formazan is measured spectrophotometrically at 560 nm. The photometric results showed that these compounds have dose dependent ability to
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Table 1 IC-50 values of icetexanes on human cancer cell lines after 24 h incubation. All the values are expressed as mean ± SD, n = 3. Compound
MCF-7 (g/ml)
Ice 1 Ice 2 Ice 3 Ice 4
350.13 25.5 42.75 81.46
± ± ± ±
27.03 4.64 4.89 21.05
MDA-MB 231 (g/ml) 118.97 35.56 98.24 95.07
± ± ± ±
3.99 4.07 2.20 1.21
A549 (g/ml) 98.4 75.67 32.7 133.04
± ± ± ±
0.61 3.78 3.20 8.37
HT-29 (g/ml) ± ± ± ±
A431 (g/ml)
4.54 4.67 7.33 2.7
128.32 ± 7.21 NA NA NA
HT-29 (g/ml)
A431 (g/ml)
16.21 ± NA 14.57 ± 41.04 ± 1.06 ±
123.1 ± 14.7 NA NA NA 0.81 ± 0.22
121.65 122.06 138.38 142.54
Table 2 IC-50 values of icetexanes on human cancer cell lines after 48 h incubation. All the values are expressed as mean ± SD, n = 3. Compound
MCF-7 (g/ml)
Ice 1 Ice 2 Ice 3 Ice 4 Etoposide
15.96 10.75 15.84 79.27 0.34
± ± ± ± ±
0.21 4.65 0.37 1.24 0.015
MDA-MB 231 (g/ml) 108.73 ± 3.97 31.43 ± 4.43 88.94 ± 2.58 95.46 ± 3.07 0.45 + 0.026
quench free radicals. It also revealed that ice-4 has shown superior superoxide scavenging activity than standard compound gallic acid. On the other hand, ice-2 and 3 were demonstrated with no significant SO scavenging activity (p < 0.001) (Fig. 9).
A549 (g/ml) 18.62 43.65 21.37 96.13 0.38
± ± ± ± ±
0.02 0.32 0.10 2.03 0.03
0.01 0.69 6.08 0.021
50 g/ml concentrations but not at 12.5 g/ml (p < 0.001) and maximum protection in tBHP induced oxidative damage was observed at 50 g/ml as shown in Fig. 10. The protective effect of ice-4 can be explained with its ability to quench free radicals generated by tBHP.
Hepatoprotective activity In vitro method Since the antioxidant activity is important in providing protection against hepatic damage (Poli, 1993), in the present study, we have attempted to demonstrate the hepatoprotective activity of the compounds through antioxidant activity. The hepatoprotective effect of ice-4 was analyzed by measuring cell viability by MTT assay. Based on the result of MTT assay, IC-50 values of ice-4 was found to be 149.84 g/ml (results not shown). From the obtained data, non-toxic doses 50, 25 and 12.5 g/ml of ice-4 was further used for testing the protective effect on tBHP induced oxidative damage in HepG2 hepatic cell line. The results clearly demonstrated that hepatoprotective activity of ice-4 was significant at 25 and
In vivo method Serum SGOT, SGPT and ALKP levels are established biomarkers for liver injury in vivo. Hepatic injury occurred in tBHP intoxicated mice resulted in significant elevation of serum SGOT, SGPT and ALKP compared to control group of mice (p < 0.001). Treatment with standard drug silymarin and ice-4 at a dose of 250 mg/kg, showed significant reduction in elevation of serum biochemical markers (p < 0.001) compared to tBHP control and this activity was comparable with silymarin treated group (p < 0.001) (Fig. 11). tBHP intoxication in mice results in oxidative stress which results in depleted serum levels and lipid peroxidation. MDA is the stable important intermediate in the process of lipid peroxidation and hence its levels indicate the extent of lipid peroxidation
Fig. 3. Phase contrast microscopic images (magnification 100×) of (i) A549 (lung cancer cells) and (ii) MCF-7 (breast cancer cells) after treatment with ice-2 and 3. (A) Control cells treated with vehicle (0.1% DMSO), (B) cells treated with Compound 2 (50 g/ml for 24 h) and (C). Cells treated with Compound 3 (50 g/ml for 24 h), Arrows indicated as change in morphology of the cells with membrane blebbing which is a characteristic feature of cell undergoing apoptosis.
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Fig. 4. Fluorescent microscopic images (magnification 100×) of (i) A549 (lung cancer cells) after treatment with ice 2 and 3. (A) Control cells treated with vehicle (0.1% DMSO), (B and C) cells treated with ice 2 (50 g/ml for 24 h) and (D and E) Cells treated with ice-3 (50 g/ml for 24 h). (ii) MCF-7 cells after treatment with (b) ice-2 (50 g/ml for 24 h), (c) ice-3 (50 g/ml for 24 h).
in liver. Liver tissues of tBHP treated group mice have shown significantly elevated MDA levels compared to that of vehicle group mice (p < 0.001). Increase in MDA levels in the liver tissues were restored to the normal levels up on treatment with silymarin as well as ice-4 indicated the protective effect of ice-4 on tBHP induced oxidative damage (Fig. 12).
Discussion Recent studies indicated that P. tomentosa possesses diuretic, hepatoprotective, antioxidant and immunomodulatory activities. Our previous studies reported on the bioactivity guided phytochemical analysis of hexane extract of P. tomentosa led to the
Fig. 5. Effect of icetexanes-2 and 3 on loss of mitochondrial potential in MCF-7 cells by JC-1 dye method. The images represent control cells treated with vehicle (A), cells treated with 50 g/ml of ice-2 (B), cells treated with 50 g/ml of ice-3. Altered mitochondrial potential results in inhibition of JC-1 accumulation in mitochondrial matrix and the dye remain as monomer with green fluorescence.
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Fig. 8. Antioxidant activity of Icetexanes (1–4) by NO radical scavenging method. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
Fig. 6. Effect of compound 2 on cell cycle after exposing MCF-7 cells at concentration of 50 and 100 g/ml for 24 h and subsequently analyzed by Muse cell cycle analyser. (A) Cell cycle analysis of control cells treated with vehicle. (B) Cells treated with ice-2 (50 g/ml) and (C) cells treated with ice-2 (100 g/ml).
Fig. 7. Antioxidant activity of Icetexanes (1–4) by DPPH radical scavenging method. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
isolation of new icetexane type diterpenes (ice 1–4). In search of novel biologically active compounds from P. tomentosa, an initial screening for hepatoprotective, antioxidant and anticancer activities were conducted with isolated new icetexane (ice 1–4) type diterpenes. In the present study, we have evaluated the antioxidant potential of newly isolated icetexane diterpenes (ice 1–4) using DPPH method, Nitric oxide scavenging activity, superoxide scavenging activity and we also investigated in vitro hepatoprotective activity of these compounds by determination of IC50 of Icetexane diterpenes against t-BHP induced hepatotoxicity in HepG2 cells. The mechanism of tBHP induced hepatic damage is due to generation of free radicals, decreased activity of antioxidant enzymes and lipid peroxidation. tBHP induced oxidative stress is by two distinct pathways. One pathway involves Cyp-450 induced metabolism of tBHP to toxic peroxyl and alkoxyl radicals which is responsible for lipid peroxidation and formation of covalent bonds with cellular molecules leading to liver damage (Lin et al., 2000). The second metabolic pathway concerns a detoxification reaction involving
Fig. 9. Superoxide radical scavenging activity of icetexanes (2–4). Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
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Fig. 10. In vitro hepatoprotective activity of ice-4 on tBHP induced hepatic damage in HepG2 cell line. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs tBHP. Data were analyzed by using one-way ANOVA fallowed by Dunnett test.
glutathione peroxidase which gives t-butanol and oxidized glutathione (GSSG), which in turn alters calcium homeostasis and increase the formation of ROS (Hwang et al., 2002; Jewell et al., 1986). The results have shown that ice-4 possesses significant free radical scavenging efficacy per se, and explains the in vitro hepatoprotective activity. In accordance with the in vitro results, we have moved forward with in vivo hepatoprotective activity for ice-4 and showed significant protective effect against tBHP induced hepatic damage. Preliminary cytotoxicity assay using MTT has shown that
Fig. 12. Effect of ice-4 on liver MDA levels in t-BHP induced hepatotoxicity in mice. Values were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs normal control, ###p < 0.001 significant vs t-BHP. Data were analyzed by using one-way ANOVA followed by Dunnett test.
ice-2 and 3 possess potential anticancer activity in A-549 (lung cancer) MCF-7, MDA-MB 231(breast cancer) cells. Programmed cell death (apoptosis) is a physiological and crucial process that is regarded as the preferred way to eliminate cancer cells. Apoptosis is characterized by several morphological changes such as membrane blebbing, cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies.
Fig. 11. Effect of icetexane 4 on serum biochemical parameters a) SGOT (b) SGPT (c) ALP levels in t-BHP induced hepatotoxicity in mice. Values were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs normal control, ###p < 0.001 significant vs t-BHP. Data were analyzed by using one-way ANOVA followed by Dunnett test.
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The induction of apoptosis ice 2- and ice-3 was evidenced from the morphological alteration such as severe membrane blebbing, DNA fragmentation, formation of apoptotic bodies that could be clearly visualized from fluorescence microscopic photographs. Uncontrolled cell proliferation is associated with the loss of cell cycle checkpoints that regulate the passage through cell cycle. These check points monitor the integrity of DNA and ensure that the genes are expressed in a co-ordinate manner. Cell cycle analysis of ice-2 on MCF-7 cells showed signification accumulation of cell population at G2/M phase indicated that icetexanes showed anticancer activity by arrest cycle at G2/M phase which further need to be explored to identify the molecular target proteins for icetexanes involved in G2/M phase by western blot analysis. Thus, present study revealed that the novel isolated icetexane diterpenes possess antioxidant, hepatoprotective and anti-proliferative activities. Use of many potential anti-proliferative compounds has been limited due to their free radical induced oxidative stress (Sangeetha et al., 1990). Therefore, combination of ice4 with such potential anti-cancer moieties, potentiates the anti-cancer activity (Hirst and Robson, 2010) precluding the substantial adverse effects (Menna et al., 2012) further need to be explored. Conclusion In the present investigation, we have evaluated the hepatoprotective, antioxidant and antiproliferative effect of novel icetexane diterpene isolated from P. tomentosa. Based on preliminary fluorescence microscopy study and mitochondrial potential measurement, the active constituents ice-2 and 3, showed anti-proliferative effect via mitochondrial-mediated apoptosis induction by cell cycle arrest at G2/M phase. On the other hand, ice4 showed protective activity against tBHP induced hepatic damage in HepG2 cells because of high free radical scavenging activity. Further studies are required to know the exact molecular mechanism by which the icetexane diterpenes induce apoptosis and protect liver from hepatotoxins. Acknowledgements Authors thank Dr. A. Kamal, Acting Director, IICT, Hyderabad and Project Director, NIPER, Hyderabad, for his kind support in extending the facilities of IICT and NIPER-Hyderabad.
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Antioxidant, hepatoprotective and cytotoxic effects of icetexanes isolated from stem-bark of Premna tomentosa Naidu V.G.M c , Hymavathi Atmakur b , Suresh Babu Katragadda b , Bhavana Devabakthuni a , Anudeep Kota a , Chenna Keshava Reddy S. a , Madhusudana Kuncha a , Vishnu Vardhan M.V.P.S. a , Prasad Kulkarni a , Madhusudana Rao Janaswamy b , Ramakrishna Sistla a,∗ a
Division of Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India Division of Natural Product Chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India c Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Hyderabad 500037, India b
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 16 August 2013 Accepted 29 September 2013 Keywords: Premna tomentosa Antioxidant Hepatoprotective Anticancer Icetexanes
a b s t r a c t The study investigates the antioxidant, hepatoprotective and antiproliferative effects of novel icetexane diterpenoids (ice 1–4) isolated from hexane extract of stem bark of Premna tomentosa. A549, HT29, MCF-7, MDA-MB-231, A431 cells were used to assess the antiproliferative activity by MTT assay. Cell death induced by apoptosis was determined by morphological assessment studies using acridine orange/ethidium bromide staining (dual staining), mitochondrial potential measurement by JC-1 staining, and cell cycle analysis by propidium iodide staining method by Muse cell analyser. Anti oxidant activity was investigated by in vitro assays such as DPPH, nitric oxide and superoxide scavenging activities. Hepatoprotective activity was determined in vitro with HepG2 cells and in vivo by tBHP induced hepatic damage mice model. Based on the in vitro cytotoxic assays and morphological assessment studies using fluorescence microscopic study (acridine orange and ethidium bromide double staining) and mitochondrial potential measurements, it was found that ice 2 and 3 possess good antiproliferative effect via mitochondrial mediated apoptosis in human lung and breast cancer cells. Results of in vitro antioxidant studies demonstrated that ice-4 has showed good antioxidant activity. The restoration of serum levels of SGOT, SGPT and ALKP, liver GSH status and reduction or inhibition of lipid peroxidation in liver of tBHP intoxicated mice after administration of ice-4 at dose of 250 mg/kg indicated its potential use for hepatoprotective activity. © 2013 Elsevier GmbH. All rights reserved.
Introduction Development or identification of anticancer agents with limited or few adverse effects is sustained thrust in the area of anticancer research. The development of new synthetic molecules possessing potential anticancer activity is highly expensive and time-consuming process. For this reason there is increasing search for herbs possessing potential anti-cancer activity. Recent review pointed out that, about 74% of anticancer compounds being either natural or natural product-derived products, indicating potency of these scaffolds (Newman et al., 2003). The unprecedented structures of these molecules make them excellent synthetic targets, and their potent activity against a broad number of therapeutic indications makes these natural products excellent drug lead candidates for new therapeutics (Newman, 2008).
∗ Corresponding author. Tel.: +91 40 27193753. E-mail addresses: [email protected], [email protected] (R. Sistla). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.025
The genus Premna (Verbenaceae) comprises a group of more than 200 different species, distributed in tropical and subtropical parts of the world (Kubitzki, 2004; Marles and Farnsworth, 1995). Premna tomentosa is a well known medicinal plant and all parts of this plant have been employed for the treatment of various ailments in Indian systems of medicine. As part of pharmacological–phytochemical integrated studies of medicinal plants from Indian flora, we are investigating the chemical composition of plants from the genus Premna (Verbenaceae) as well as their cytotoxic activities (Suresh et al., 2011a,b). In the course, we have recently reported some icetexane diterpenes from the P. tomentosa (Fig. 1) which has effectively induced cytotoxicity in different human cancer cell lines (Hymavathi et al., 2009). However, the mechanism of action of these compounds inducing cytotoxicity was not known. The potent biological activity coupled with its novel structural features prompted us to investigate their molecular mechanism studies. In the present communication, we have performed the apoptosis screening techniques to differentiate apoptosis and necrosis, including evaluation of the
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repeated silica gel (100–200 mesh) column chromatography with hexane–chloroform–acetone (5:3.2:1.8) yielded ice-3 (0.005 g). Fraction F2 was subjected to silica gel column chromatography eluting with hexane–chloroform–acetone (5:3.4:1.6) to yield ice-4 (0.002 g). Fraction F3 was subjected to silica gel column chromatography with the elution of hexane–chloroform–acetone (5:3:2) to yield 0.080 g of ice-1, and with hexane–chloroform–acetone (5:3.8:1.2) to get 0.005 g of ice-2 and their chemical structures was depicted in Fig. 1.
Fig. 1. Chemical structures of icetexanes (ice-1–4) isolated from hexane extracts of Premna tementosa.
anti-proliferative and antioxidant activity. In addition, we also evaluated the hepatoprotective activity of these compounds on the basis of their antioxidant activity. Materials and methods
Finger printing Chromatographic separation was achieved by reverse-phase chromatography using the isocratic mode of elution. With Synergy 4, Fusion-RP 80A 150 mm × 4.6 mm column. LC–MS studies were performed using an Agilent 1100 series online ion trap MSD mass spectrometer with ESI source in positive-ion mode equipped with a degasser (G1379A), binary pump (G1312A), autosampler (G1329A), autosampler thermostat (G1329B) and diode array detector (G1315B). All the systems were from Agilent Technologies (Waldbronn, Germany). The data were acquired and processed using LC/MSD trap software 4.2 (Bruker, Waldbronn, Germany). An isocratic elution with methanol and water (80:20, v/v) as mobile phase was pumped at a flow rate of 1.0 ml/min; the sample injection volume was 20 l with column temperature maintained at ambient conditions. Nitrogen was employed as the nebulizer gas. The ion source conditions were set as follows: temperature, 300 ◦ C; nebulizer gas, 30 psi; dry gas, 8.0 L/min; skimmer 40.0 V; capillary exit 128.0 V; trap drive 44.5; max accu time 200,000us; Icc target 20,000.
Cell lines Anti-cancer activity A549 cells (lung cancer), HT-29 cells (colon cancer), MCF-7 cells (breast cancer), MDA-MB cells (breast cancer), A431 cells (skin cancer) and HepG2 cells (hepatocellular carcinoma) were obtained from National Centre for Cell Science (NCCS), Pune, India. Chemicals and reagents DPPH (2,2-diphenyl-1-pycrylhydrazyl), ascorbic acid, Griess reagent, Silymarin (originated from the fruit of Silybum marianum, Cat # S0292), GSH, O-pthaldehyde, 1,1,3,3-tetramethoxypropane, thiobarbituric acid (TBA), tricarboxylic acid (TCA), sodium chloride, nitro blue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), phenazine methosulphate, sodium pyrophosphate, glacial acetic acid, n-butanol, potassium dihydrogen phosphate, dihydrogen sodium phosphate, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM), sodium bicarbonate, thiazolyl blue tetrazolium bromide, trypsin solution, glutamine and JC-1 were obtained from Sigma Co. (St. Louis, MO, USA). Antibiotic and antimycotic mixture and fetal bovine serum were obtained from GIBCO BRL (NY, USA). Serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT) and alkaline phosphatase (ALKP) were obtained from Bayers Diagnostics (Leverkusen, Germany). Extraction and isolation of compounds from P. tomentosa bark The extraction and isolation of compounds from P. tomentosa was performed as described in previous study (Hymavathi et al., 2009). The stem bark (1 kg) of P. tomentosa is collected and dried, which is then ground and extracted three times with hexane in a soxhlet apparatus. The extracts were combined and concentrated under vacuum. The active hexane extract (2.5 g) was subjected to column chromatography (silica gel, 60–120 mesh) using step gradient of hexane/EtOAc resulting three fractions F1, F2 and F3. On eluting Fraction F1 by
Cytotoxicity assay The cytotoxic effect of icetexanes was assessed and quantified in different cell lines A549, HT-29, MCF-7, MDA-MB and A431 after incubating for different durations of 24 h and 48 h by MTT assay. A549, HT-29, A431 cells were grown in DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 1 mM sodium pyruvate and 1.5 g/l sodium bicarbonate under 5% CO2 at 37 ◦ C, where as MCF-7 and MDA-MB-231 cells were grown in MEM supplemented with 10% FBS. 1 × 104 cells per well were seeded in a 96-well plate and after 24 h the media was replaced with fresh medium containing different concentrations of icetexanes 1, 2, 3 and 4 dissolved in DMSO and in control 1% DMSO was added. After 24 h and 48 h incubation, the media were replaced with 100 l fresh media and 10 l of MTT reagent in to each well. Further cells were incubated at 37 ◦ C for 4 h. Later, the formazan crystals formed were solubilized in DMSO. Finally absorption at 570 nm was read from ELISA reader. IC-50 values were calculated against control. Morphological analysis Acridine orange (AO) and ethidium bromide (EtBr) dual staining. Acridine orange (AO) and ethidium bromide (EtBr) dual staining method was used to assess the chromatin condensation and membrane integrity, respectively, which serves as markers for apoptosis. AO is a cationic dye which enters only live cells and stains nuclei green. EtBr stains DNA orange, which is mainly taken up by the cells when cell membrane integrity was lost. Dual staining allows enumeration of four categories of cells as follows; live-non-apoptotic cells (nuclei stains bright green with organized structure), live-apoptotic cells (nuclei stains bright green with condensed or fragmented chromatin), dead-apoptotic cells (bright orange chromatin that is highly condensed or fragmented) and dead-non-apoptotic cells (bright orange chromatin with organized structure). Around 1 × 105 cells per well were dispensed in to 12 well culture plate and 24 h after seeding, cells were incubated with
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Compounds 2 and 3 (50 and 100 g/ml) for 24 h. The cells are then washed with phosphate buffered saline (PBS), trypsinized and stained with 10 l of staining solution containing 100 g/ml of AO and 100 g/ml of EB in PBS. A volume of 5 l of cell suspension was immediately taken on to the microscope slide and then examined by fluorescence microscope (NiKon TS100, Topa-Eclipse). Mitochondrial potential measurement. Loss of mitochondrial membrane potential ( ) is indicative of apoptosis and can be detected by a unique fluorescent cationic dye 5,51 ,6,61 -tetrachloro1,11 ,3,31 -tetraethylbenzamidazolocarbocyanin iodide commonly known as JC-1. This reagent penetrates passively into cells and mitochondria. In non-apoptotic cell, the mitochondrial potential is unaltered and the dye enters the mitochondria forming aggregates of red colored fluorescence where as in apoptotic cells due to altered mitochondrial potential the dye does not accumulate inside the mitochondria. Instead, it is distributed throughout the cell as monomer giving green fluorescence. MCF-7 cells were seeded in 12-well plate at a concentration of 105 cells per well. After 24 h the cells are treated with 50 g/ml ice-2 and ice-3. After 24 h, the cells were washed with PBS and incubated for 30 min in 10% DMEM without Phenol red containing JC-1 at a concentration of 2 g/ml and fluorescence is observed with fluorescence microscope at wavelengths of 520 (ex ) and 594 (em ). Flow cytometric analysis MCF-7 cells were incubated with two different concentrations 50 g/ml and 100 g/ml of ice-2 for 24 h. Cells were processed, fixed and stained with cell cycle reagent kit. Cells were analyzed for the DNA content to determine the percentage of cells present in different stages of cell cycle. Fluorescence emitted from the propidium iodide–DNA complex was quantified by using Muse cell cycle analyser. Antioxidant activity determination DPPH method The antioxidant activity of P. tomentosa dried stem bark extract was assessed by determining its ability to scavenge DPPH radical. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical, due to odd electrons it shows absorption maximum at 517 nm. DPPH accepts electron or hydrogen radical from antioxidant molecule to become a stable diamagnetic molecule, which is confirmed by decrease in absorbance at 517 nm. Ascorbic acid was used as standard. Aliquots of 25 l of various concentrations (100–1000 g/ml) of icetexanes 1–4 were mixed with 200 l of Tris-buffer and 125 l of DPPH in a 96 well plate and absorbance was recorded at 517 nm using UV-Spectrophotometer (Biotek, Synergy 4) after incubation for 15 min in dark at 37 ◦ C. The percentage of scavenging activity is determined using the following formula:
Percentage of inhibition (%) =
Acontrol − Asample Acontrol
∗ 100
where, Acontrol – absorbance of DPPH, Asample – absorbance of DPPH with test compound. Nitric oxide scavenging activity Nitric oxide radical is generated from reaction mixture containing sodium nitroprusside (20 mM) in phosphate buffered saline (pH 7.4) when incubated at 25 ◦ C for 30 min under a visible polychromatic light source (25 W tungsten lamp) (Marcocci et al., 1994). The nitric oxide radical thus generated interacts with oxygen to produce nitrite ion, which is assayed by mixing with an
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equal amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) (Hsieh et al., 2010). The resulting absorbance was measured at 570 nm. Decrease in absorbance in the presence of different concentrations of test compounds (100–1000 g/ml) indicated the nitric oxide scavenging activity. Ascorbic acid was used as positive control. The percentage of Nitric oxide scavenging activity is determined using the following formula.
Percentage of inhibition (%) =
Acontrol − Asample Acontrol
∗ 100
where, Acontrol – absorbance of reaction mixture without test compound; Asample – absorbance of reaction mixture with various concentrations of test compounds. Superoxide scavenging activity Superoxide scavenging activity was assessed according to the method described by Beauchamp and Fridovich (1971) and Rai et al. (2006). The method is based on generation of superoxide radicals; the superoxide radical reduces the NBT (Nitroblue tetrazolium) to a blue colored formazan that can be measured at 560 nm. The reaction mixture consisted of 0.1 ml of various concentrations (50–2000 g/ml) of icetexanes, 1.2 ml of sodiumpyrophosphate (pH 8.3, 0.052 M), 0.1 ml of phenazinemethosulphate (186 M), 0.3 ml of nitrobluetetrazolium (NBT) (300 M), and 0.2 ml of NADH (750 M). The reaction was started with the addition of NADH and after incubation for 90 s at 30 ◦ C. The reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture is stirred vigorously with 4 ml of n-butanol and the color intensity was measured spectrophotometrically at 560 nm. Gallic acid was used as standard. Hepatoprotective activity In vitro method In vitro hepatoprotective activity was assessed by ability of ice4 to preclude the oxidative stress induced by tBHP in HepG2 cells (hepatocellular carcinoma). HepG2 cells were plated at a density of 1 × 104 cells per well in a 96 well plate along with 100 l of MEM supplemented with 10% FBS in each well. Various concentrations of Compound 4 (12.5 g/ml, 25 g/ml and 50 g/ml) were exposed to HepG2 cells along with tBHP (250 M) for 6 h and the viability of cells was assessed by MTT assay (Hae-Ung et al., 2005). In vivo method Comparative study of hepatoprotection in tertbutylhydroperoxide (tBHP) induced hepatotoxicity in mice model was carried for the ice-4 and compared with positive control, silymarin. Male Swiss albino mice weighing 18–24 g were obtained from NCLAS, NIN, Hyderabad and housed at animal house facility at IICT, Hyderabad. Animals were maintained on stock pellet diet and tap water ad libitum and maintained on 12/12 h light/dark cycle. All the procedures during the animal experiment were prior approved by IAEC. Mice were divided in to four groups of six mice each. Animals received four successive doses of ice-4 and standard (silymarin) drugs at 0, 24, 48 and 72 h followed by inducer (tBHP) at a dose of 1.8 mM/kg, in a volume of 0.5 ml/100 g body weight (Hae-Ung et al., 2005; Valentao et al., 2004). Control group animals received only vehicle and tBHP group animals received vehicle followed by inducer. Test group animals were administered with ice-4 at 250 mg/kg body weight whereas standard group animals were administered with silymarin 250 mg/kg body weight as oral suspensions, in 1% gum acacia. The blood samples were collected through retro orbital plexus after 24 h of tBHP administration. The samples were allowed to clot and serum was separated by
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and A431 cells was observed. It was observed that percentage viability decreased with increase in time of exposure of icetexane compounds in all cell lines. IC-50 values were expressed as g/ml and presented in Tables 1 and 2. Among the isolates, ice-2 and ice-3 were demonstrated significant cytotoxicity compared to ice-1 and 4. In particular, ice-2 and ice-3 have momentous cytotoxic effect on breast cancer (MCF-7) and lung cancer cells (A549).
Fig. 2. Finger printing of icetexanes isolated from hexane extract of Premna tomentosa.
centrifugation (1000 × g, 15 min) for estimation of SGOT, SGPT and ALKP (Auto blood analyzer Express plus, Bayer Corporation USA). Mice were sacrificed by CO2 asphyxiation, dissected and the liver was excised from the animals, weighed and used for quantification of extent of lipid peroxidation and glutathione contents. Lipid peroxidation assay. Lipid peroxidation was assessed by measuring the amount of malondialdehyde (MDA), a secondary product of lipid peroxidation (Lee et al., 2005). An improved thiobarbituric acid spectrofluorometric method employing 1,1,3,3tetramethoxyproprane as precursor has used for producing MDA in standard solutions through acid hydrolysis. Excised liver tissue was homogenized in 50 mM phosphate buffer (pH 7.4). To a volume of 1 ml of tissue homogenate, 2 ml of 10% trichloroacetic acid (TCA) was added and mixed well. After centrifugation at 10,000 × g at 4 ◦ C for 20 min, the protein free supernatant was collected and to 1 ml of 1% thiobarbituric acid (TBA) reagent was added and mixed well. This solution was incubated at 70 ◦ C for 40 min. The reaction mixture was cooled to room temperature and fluorescence was measured at 510 nm excitation and 553 nm emission wavelengths. The results were expressed as g MDA formation per g liver tissue. Results and discussion Finger printing From the results it was found that the hexane extract of P. tomentosa gave peaks at the following retention time values: 3.11, 3.56, 4.03, 4.36, 4.84, 5.88, 6.52, 6.61, 7.50, 8.88, 10.28, 11.75, 12.66 min. Based on the molecular weight and RT values for pure icetexanes (1–4) from LC–MS data (Supplementary Fig. 1), the corresponding peaks in finger printing were depicted as shown in Fig. 2. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed. 2013.09.025. Cytotoxicity MTT assay is most widely used to determine the cytotoxic potential of medicinal agents and other toxic materials. The reduction of yellow MTT to purple colored formazan takes place only when mitochondrial reductase enzymes are active and therefore the conversion can be directly related to number of viable cells. The cytotoxic effect of icetexane compounds after 24 h and 48 h of treatment in A549, MCF-7, MDA-MB-231, HT-29
Acridine orange and ethidium bromide dual staining Morphological abnormalities were studied under phase-contrast and fluorescence microscope. Cells treated with 50 g/ml of either ice-2 or ice-3 showed morphological changes such as chromatin condensation, fragmentation and formation of apoptotic bodies. However, control cells retained normal healthy shape with intact nuclei and without any abnormalities. The results of light microscope are consistent with that of fluorescence microscopy as shown in Figs. 3 and 4. Mitochondrial potential Induction of apoptosis in a cell can be determined with mitochondrial membrane potential. Mitochondrial membrane potential was assessed with dye JC-1. In this experiment, we observed that cells treated with ice-2 and ice-3 showed green fluorescence indicating the loss of mitochondrial potential, hence induction of apoptosis, whereas vehicle treated cells showed red fluorescence as shown in Fig. 5. Cell cycle analysis MCF-7 cells were analyzed for DNA content using flow cytometry. The percentage of cells in G2/M phase increased with increase in concentration of ice-2 compared to control cells. The study revealed that ice-2 displayed G2/M phase arrest which is concentration dependent (Fig. 6). Antioxidant activity DPPH method It is the simplest method developed to determine the antioxidant activity using a stable free radical, 1,1-diphenyl-2-picryl-hydrazyl(DPPH) with specific absorption maxima at 517 nm. The extent of free radical scavenging activity is determined by decrease in absorbance at 517 nm compared to blank. The results indicated that ice-2, 3 and 4 have dose dependent ability to quench free radical. Furthermore, it has been found that ice-4 has significantly higher free radical quenching capacity when compared to ice-2 and ice-3 as represented by its lower EC50 (Fig. 7). Nitric oxide scavenging activity The nitric oxide scavenging activity of icetexanes was determined based on the inhibition of nitric oxide radical generation from sodium nitroprusside in buffer saline and measured by Griess reagent. The activity is expressed as percentage inhibition of nitric oxide production and from that EC50 was calculated for standard and test compounds. The results have revealed that NO scavenging activity of ice-4 was on par with standard ascorbic acid as indicated by no significant difference between their EC50 (p < 0.001). On the contrary, NO scavenging activity of ice-2 and 3 are not significant (Fig. 8). Super oxide scavenging activity: The reduction of yellow color of NBT to blue formazan is measured spectrophotometrically at 560 nm. The photometric results showed that these compounds have dose dependent ability to
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Table 1 IC-50 values of icetexanes on human cancer cell lines after 24 h incubation. All the values are expressed as mean ± SD, n = 3. Compound
MCF-7 (g/ml)
Ice 1 Ice 2 Ice 3 Ice 4
350.13 25.5 42.75 81.46
± ± ± ±
27.03 4.64 4.89 21.05
MDA-MB 231 (g/ml) 118.97 35.56 98.24 95.07
± ± ± ±
3.99 4.07 2.20 1.21
A549 (g/ml) 98.4 75.67 32.7 133.04
± ± ± ±
0.61 3.78 3.20 8.37
HT-29 (g/ml) ± ± ± ±
A431 (g/ml)
4.54 4.67 7.33 2.7
128.32 ± 7.21 NA NA NA
HT-29 (g/ml)
A431 (g/ml)
16.21 ± NA 14.57 ± 41.04 ± 1.06 ±
123.1 ± 14.7 NA NA NA 0.81 ± 0.22
121.65 122.06 138.38 142.54
Table 2 IC-50 values of icetexanes on human cancer cell lines after 48 h incubation. All the values are expressed as mean ± SD, n = 3. Compound
MCF-7 (g/ml)
Ice 1 Ice 2 Ice 3 Ice 4 Etoposide
15.96 10.75 15.84 79.27 0.34
± ± ± ± ±
0.21 4.65 0.37 1.24 0.015
MDA-MB 231 (g/ml) 108.73 ± 3.97 31.43 ± 4.43 88.94 ± 2.58 95.46 ± 3.07 0.45 + 0.026
quench free radicals. It also revealed that ice-4 has shown superior superoxide scavenging activity than standard compound gallic acid. On the other hand, ice-2 and 3 were demonstrated with no significant SO scavenging activity (p < 0.001) (Fig. 9).
A549 (g/ml) 18.62 43.65 21.37 96.13 0.38
± ± ± ± ±
0.02 0.32 0.10 2.03 0.03
0.01 0.69 6.08 0.021
50 g/ml concentrations but not at 12.5 g/ml (p < 0.001) and maximum protection in tBHP induced oxidative damage was observed at 50 g/ml as shown in Fig. 10. The protective effect of ice-4 can be explained with its ability to quench free radicals generated by tBHP.
Hepatoprotective activity In vitro method Since the antioxidant activity is important in providing protection against hepatic damage (Poli, 1993), in the present study, we have attempted to demonstrate the hepatoprotective activity of the compounds through antioxidant activity. The hepatoprotective effect of ice-4 was analyzed by measuring cell viability by MTT assay. Based on the result of MTT assay, IC-50 values of ice-4 was found to be 149.84 g/ml (results not shown). From the obtained data, non-toxic doses 50, 25 and 12.5 g/ml of ice-4 was further used for testing the protective effect on tBHP induced oxidative damage in HepG2 hepatic cell line. The results clearly demonstrated that hepatoprotective activity of ice-4 was significant at 25 and
In vivo method Serum SGOT, SGPT and ALKP levels are established biomarkers for liver injury in vivo. Hepatic injury occurred in tBHP intoxicated mice resulted in significant elevation of serum SGOT, SGPT and ALKP compared to control group of mice (p < 0.001). Treatment with standard drug silymarin and ice-4 at a dose of 250 mg/kg, showed significant reduction in elevation of serum biochemical markers (p < 0.001) compared to tBHP control and this activity was comparable with silymarin treated group (p < 0.001) (Fig. 11). tBHP intoxication in mice results in oxidative stress which results in depleted serum levels and lipid peroxidation. MDA is the stable important intermediate in the process of lipid peroxidation and hence its levels indicate the extent of lipid peroxidation
Fig. 3. Phase contrast microscopic images (magnification 100×) of (i) A549 (lung cancer cells) and (ii) MCF-7 (breast cancer cells) after treatment with ice-2 and 3. (A) Control cells treated with vehicle (0.1% DMSO), (B) cells treated with Compound 2 (50 g/ml for 24 h) and (C). Cells treated with Compound 3 (50 g/ml for 24 h), Arrows indicated as change in morphology of the cells with membrane blebbing which is a characteristic feature of cell undergoing apoptosis.
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Fig. 4. Fluorescent microscopic images (magnification 100×) of (i) A549 (lung cancer cells) after treatment with ice 2 and 3. (A) Control cells treated with vehicle (0.1% DMSO), (B and C) cells treated with ice 2 (50 g/ml for 24 h) and (D and E) Cells treated with ice-3 (50 g/ml for 24 h). (ii) MCF-7 cells after treatment with (b) ice-2 (50 g/ml for 24 h), (c) ice-3 (50 g/ml for 24 h).
in liver. Liver tissues of tBHP treated group mice have shown significantly elevated MDA levels compared to that of vehicle group mice (p < 0.001). Increase in MDA levels in the liver tissues were restored to the normal levels up on treatment with silymarin as well as ice-4 indicated the protective effect of ice-4 on tBHP induced oxidative damage (Fig. 12).
Discussion Recent studies indicated that P. tomentosa possesses diuretic, hepatoprotective, antioxidant and immunomodulatory activities. Our previous studies reported on the bioactivity guided phytochemical analysis of hexane extract of P. tomentosa led to the
Fig. 5. Effect of icetexanes-2 and 3 on loss of mitochondrial potential in MCF-7 cells by JC-1 dye method. The images represent control cells treated with vehicle (A), cells treated with 50 g/ml of ice-2 (B), cells treated with 50 g/ml of ice-3. Altered mitochondrial potential results in inhibition of JC-1 accumulation in mitochondrial matrix and the dye remain as monomer with green fluorescence.
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Fig. 8. Antioxidant activity of Icetexanes (1–4) by NO radical scavenging method. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
Fig. 6. Effect of compound 2 on cell cycle after exposing MCF-7 cells at concentration of 50 and 100 g/ml for 24 h and subsequently analyzed by Muse cell cycle analyser. (A) Cell cycle analysis of control cells treated with vehicle. (B) Cells treated with ice-2 (50 g/ml) and (C) cells treated with ice-2 (100 g/ml).
Fig. 7. Antioxidant activity of Icetexanes (1–4) by DPPH radical scavenging method. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
isolation of new icetexane type diterpenes (ice 1–4). In search of novel biologically active compounds from P. tomentosa, an initial screening for hepatoprotective, antioxidant and anticancer activities were conducted with isolated new icetexane (ice 1–4) type diterpenes. In the present study, we have evaluated the antioxidant potential of newly isolated icetexane diterpenes (ice 1–4) using DPPH method, Nitric oxide scavenging activity, superoxide scavenging activity and we also investigated in vitro hepatoprotective activity of these compounds by determination of IC50 of Icetexane diterpenes against t-BHP induced hepatotoxicity in HepG2 cells. The mechanism of tBHP induced hepatic damage is due to generation of free radicals, decreased activity of antioxidant enzymes and lipid peroxidation. tBHP induced oxidative stress is by two distinct pathways. One pathway involves Cyp-450 induced metabolism of tBHP to toxic peroxyl and alkoxyl radicals which is responsible for lipid peroxidation and formation of covalent bonds with cellular molecules leading to liver damage (Lin et al., 2000). The second metabolic pathway concerns a detoxification reaction involving
Fig. 9. Superoxide radical scavenging activity of icetexanes (2–4). Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs positive control. Data were analyzed by using one-way ANOVA followed by Dunnett test.
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Fig. 10. In vitro hepatoprotective activity of ice-4 on tBHP induced hepatic damage in HepG2 cell line. Data were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs tBHP. Data were analyzed by using one-way ANOVA fallowed by Dunnett test.
glutathione peroxidase which gives t-butanol and oxidized glutathione (GSSG), which in turn alters calcium homeostasis and increase the formation of ROS (Hwang et al., 2002; Jewell et al., 1986). The results have shown that ice-4 possesses significant free radical scavenging efficacy per se, and explains the in vitro hepatoprotective activity. In accordance with the in vitro results, we have moved forward with in vivo hepatoprotective activity for ice-4 and showed significant protective effect against tBHP induced hepatic damage. Preliminary cytotoxicity assay using MTT has shown that
Fig. 12. Effect of ice-4 on liver MDA levels in t-BHP induced hepatotoxicity in mice. Values were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs normal control, ###p < 0.001 significant vs t-BHP. Data were analyzed by using one-way ANOVA followed by Dunnett test.
ice-2 and 3 possess potential anticancer activity in A-549 (lung cancer) MCF-7, MDA-MB 231(breast cancer) cells. Programmed cell death (apoptosis) is a physiological and crucial process that is regarded as the preferred way to eliminate cancer cells. Apoptosis is characterized by several morphological changes such as membrane blebbing, cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies.
Fig. 11. Effect of icetexane 4 on serum biochemical parameters a) SGOT (b) SGPT (c) ALP levels in t-BHP induced hepatotoxicity in mice. Values were expressed as mean ± SD, n = 5. ***p < 0.001 significant vs normal control, ###p < 0.001 significant vs t-BHP. Data were analyzed by using one-way ANOVA followed by Dunnett test.
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The induction of apoptosis ice 2- and ice-3 was evidenced from the morphological alteration such as severe membrane blebbing, DNA fragmentation, formation of apoptotic bodies that could be clearly visualized from fluorescence microscopic photographs. Uncontrolled cell proliferation is associated with the loss of cell cycle checkpoints that regulate the passage through cell cycle. These check points monitor the integrity of DNA and ensure that the genes are expressed in a co-ordinate manner. Cell cycle analysis of ice-2 on MCF-7 cells showed signification accumulation of cell population at G2/M phase indicated that icetexanes showed anticancer activity by arrest cycle at G2/M phase which further need to be explored to identify the molecular target proteins for icetexanes involved in G2/M phase by western blot analysis. Thus, present study revealed that the novel isolated icetexane diterpenes possess antioxidant, hepatoprotective and anti-proliferative activities. Use of many potential anti-proliferative compounds has been limited due to their free radical induced oxidative stress (Sangeetha et al., 1990). Therefore, combination of ice4 with such potential anti-cancer moieties, potentiates the anti-cancer activity (Hirst and Robson, 2010) precluding the substantial adverse effects (Menna et al., 2012) further need to be explored. Conclusion In the present investigation, we have evaluated the hepatoprotective, antioxidant and antiproliferative effect of novel icetexane diterpene isolated from P. tomentosa. Based on preliminary fluorescence microscopy study and mitochondrial potential measurement, the active constituents ice-2 and 3, showed anti-proliferative effect via mitochondrial-mediated apoptosis induction by cell cycle arrest at G2/M phase. On the other hand, ice4 showed protective activity against tBHP induced hepatic damage in HepG2 cells because of high free radical scavenging activity. Further studies are required to know the exact molecular mechanism by which the icetexane diterpenes induce apoptosis and protect liver from hepatotoxins. Acknowledgements Authors thank Dr. A. Kamal, Acting Director, IICT, Hyderabad and Project Director, NIPER, Hyderabad, for his kind support in extending the facilities of IICT and NIPER-Hyderabad.
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