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Apoptotic and Antioxidant Activities of Methanol Extract of Mussaenda roxburghii Leaves Farhadul Islam 1*, Obayed Raihan2, Dipjoy Chowdhury3, Mahbuba Khatun1, Natasha Zuberi1, Laboni Khatun1, Afrina Brishti4, Entaz Bahar3 and Jahan Ara Khanam1 1 Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh 2 Department of Pharmacy, Jessore Science and Technology University, Jessore, Bangladesh 3 Department of Pharmacy, International Islamic University Chittagong, Chittagong, Bangladesh 4 Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh *Address for correspondence: Md. Farhadul Islam, Assistant professor, Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh E-mail: [email protected]/[email protected] Tel. +880-1712974596 Fax: +880-721750064 Abstract The use of plants and their active substances increases day by day for the discovery of therapeutic agents due to their versatile applications. Current research is directed towards finding naturally-occurring antioxidants having anticancer properties since oxidants play a crucial role in developing various human diseases. The present study was designed to investigate the anticancer and antioxidant activity of methanol extract of Mussaenda roxburghii (designated as MMR). Anticancer activity of MMR has been carried out against Ehrlich ascites carcinoma (EAC) cells with three different doses (20, 40 and 60 mg/kg/day) by monitoring the parameters like tumor weight measurement, survival time of tumor bearing mice, growth inhibition of cancer cells, morphological changes and nuclear damage of EAC cells etc. whereas antioxidant capacity was determined by measuring total antioxidant capacity (TAC), DPPH free radical scavenging capacity, ferrous reducing capacity assay. The extract showed highest anticancer activity at the dose of 60 mg/kg/day (i.p.). It caused 81.4% (P < 0.01) cells growth inhibition and reduced tumor burden significantly (78.5%; P < 0.001) when compared with control. It also increased the life span of EAC bearing mice significantly (73.5 %; P < 0.01). High LD50 value (600 mg/kg) of MMR indicated its low host toxic effects. MMR treated EAC cells showed membrane blebbing, chromatin condensation, nuclear fragmentation (apoptotic feature) in Hoechst 33342 staining under fluorescence microscope. DNA fragmentation assay in agarose gel (1.5%) electrophoresis also rectified that it causes EAC cells death by apoptosis. MMR also showed moderate antioxidant properties compare to standard antioxidant catechin and ascorbic acid in a dose dependent manner. M. roxburghii possess significant anticancer and moderate antioxidant properties. Thus, this plant may therefore be considering a good source for natural chemopreventive drugs as well as a possible pharmaceutical supplement. Key Words: Apoptosis, Intrinsic pathway, anticancer agents, antioxidant, Mussaenda roxburghii, EAC cells, Chemoprotective drugs etc. Introduction Plants have been serving an important role for mankind’s medicine from the very beginning of human history and there is an incredible historical legacy in folklore uses of plant preparations in medicine [1]. Scientific research endeavor on plants used in ethno-medicine led to the discovery of many valuable drugs such as bleomycin, taxol, camptothecin, vincristine and vinblastine [2]. Active phytochemical principle such as flavonoids, polyphenols, proanthocyanidin etc. act as strong free radical scavenger. Free radicals including superoxide radicals, hydroxyl radicals, singlet oxygen and hydrogen peroxide (reactive oxygen species; ROS) are often generated as byproduct of biological reactions, or from exogenous factors. These ROS may be very damaging as they can induce oxidation of lipids causing membrane damage, decrease membrane fluidity and lead to cancer via DNA mutation [3]. Cancer is a non-communicable disease and due to lack of effective drugs/treatment modality it is rating the top second cause of death [4]. A potent scavenger of ROS may serve a possible prevention against the free radicals-mediated diseases like cancer [5]. Antioxidants are radical scavengers and protect the human body from free radicals. Antioxidant based drugs or formulations for the prevention and treatment of cancer engaged many researchers worldwide to explore the therapeutic potential of medicinal plants in nature. Mussaenda species member of the Rubiaceae (coffee family) are native to the old World tropics, from West Africa through the Indian sub-continent, South East Asia and into Southern China. They have been used in Chinese, Fijian and Indian traditional medicine for various ailments such as diuretic, antiphlogistic, antipyretic and also used to detoxify mushroom poisons and terminate early pregnancy [6]. Active ingredients from these family plants also have potential cytotoxic, antibacterial, antiviral, anti-RSV activity [7-10]. Exploration of an indigenous medicinal plant, Mussaenda roxburghii (family: Rubiaceae) commonly known as Himalayan Mussaenda which is a shrub and distributed in moist shady places of Bangladesh, Bhutan and Myanmar is under investigation. Root paste of the plant is applied to the tongue to treat boils [11], and a recent study showed that it inhibits the growth of both Staphylococcus aureus and Escherichia coli effectively [12]. As there is no further pharmacological report of this plant elsewhere, we first here present the anticancer activity against Ehrlich ascites carcinoma (EAC) cells and antioxidant activity to find out effective chemopreventive drugs. Materials and Methods Chemicals and reagents All the chemicals and reagents used throughout the investigation were of reagent grade and purchased from BDH, UK, E’MERK, Germany and Sigma Aldrich, US. Test animals Adult male Swiss albino mice, five to six weeks old (25±3 gm body weight) were collected from animal resource branch of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR’B) and used throughout the studies. Animals were housed in polypropylene cages containing sterile paddy husk as bedding material under hygienic conditions with a maximum of six animals in a cage. They were maintained under controlled conditions (12:12 h light-dark), temperature (25 ± 5○ C). The mice were fed with standard mice food-pellets (collected from ICDDR’B) and water was given in ad libitum. Cell lines EAC cells were obtained by the courtesy of Indian Institute of Chemical Biology (IICB), Kolkata, India. The cells were maintained as Ascites tumour in swiss albino mice by intraperitoneal inoculation (bi-weekly) of 2×106 cells/mouse. Ethical clearance Protocol used in this study for the use of mice as animal model for cancer research was approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources (225/320IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh. Plant materials The collected leaves of the plant were shade dried for several days with occasional sun drying and reduced to coarse powder. The dried powder was extracted with methanol (yield 8.5 %) at room temperature for 14 days with occasional shaking and stirring. The combined extract was filtered through cotton and then Whatman No.1 filter papers and was concentrated with a rotary evaporator under reduced pressure at 45° C. The crude extract designated (MMR) then dried and stored in a vacuum container for further use. The crude extract was dissolved in 2% (V/V) dimethylsulfoxide (DMSO) for the experiments. Phytochemical screening of crude extract The phytochemical components of the plant were screened using the standard methods [13]. The components analyzed for were saponins, saponin glycosides, steroid, glycosides, proanthocyanidin, anthraquinones, tannins, flavonoids, alkaloid, volatile oils, phenols and balsam (gum). Determination of median lethal dose (LD50) Methanol extract of M. roxburghii (MMR) was dissolved in 2 % (v/v) DMSO and were injected intraperitoneally to seven groups of mice (n=4) at different doses [200mg/kg (i.p.), 400 mg/kg (i.p.), 500 mg/kg (i.p.), 550 mg/kg (i.p.), 600 mg/kg (i.p.), 650 mg/kg (i.p.), 700 mg/kg (i.p.), and 750 mg/kg (i.p.)]. The LD50 value was then calculated by the procedure described in the literature [14]. Determination of cell growth inhibition Cells growth inhibition with the extract was performed by the standard method [15]. For this experiment, five groups of swiss albino mice (n=6) weighing 25±3 gm were used. For therapeutic evaluation 2×106 EAC cells in every mouse were inoculated into each group on day “0”. Treatments with MMR and bleomycin (standard clinically used anticancer drug) were started after 24 hours of tumor inoculation and continued for five consecutive days. Here group one to three received MMR at the doses of 20 mg/kg (i. p.), 40 mg/kg (i. p.), and 60 mg/kg (i. p.) per day respectively. Group four treated with bleomycin at the dose of 0.3 mg/kg (i. p.) whereas group five was used as control receiving solvent only. Mice from every group were sacrificed on day six and total intraperitoneal tumor cells were harvested by normal saline (0.98%). Viable tumor cells were first identified with trypen blue and then counted by a haemocytometer under inverted microscope (XDS-1R, Optika, Italy). Total numbers of viable cells in every animal of the treated groups were compared with those of control (EAC bearing only) mice. Bioassay of EAC cells The effect of MMR on transplantability/bioassay of EAC cells was studied by the method described in the literature [16]. For this experiment, two groups of mice (n=6) were inoculated with 17×105 EAC cells on day “0”. Group 1, was treated with MMR at the dose of 60 mg/kg/day (i.p.) for five consecutive days and group 2 used as control. On day 7, tumor cells from the treated mice (group 1) were harvested in cold (0.9 %) saline, pooled, centrifuged and reinoculated into two fresh groups of mice (n=6) as before. No further treatment was given on these mice. On day 5 of tumor re-inoculation, they were sacrificed and viable tumor cells/mouse were estimated. Determination of average tumor weight and survival time A brief description of the method used by Sur et al [15], is given bellow. For this experiment, five groups of swiss albino mice (6 in each group) were used. On day ‘0’, 2 ×106 EAC cells per mouse were inoculated to each group of mice for therapeutic evaluation. Treatment with MMR as well as bleomycin was started after 24 hours of tumor inoculation and continued for 10 days. Tumor growth was monitored by recording daily weight change. Host survival was recorded and expressed as mean survival time (MST) in days and percent increase of life span was calculated by using the following formula: Ʃ Survival time (days) of each mouse in a group Mean survival time (MST) = Total number of mice Percent increase of life span (ILS) % = MST of treated group MST of control group -1 × 100 Effect of MMR on normal peritoneal cells Effects of MMR on normal peritoneal cells on normal mice were determined by counting total peritoneal cells and number of macrophages [17]. Normal mice (n=6) were treated with MMR (i. p.) at the dose of 20 mg/kg, 40 mg/kg and 60 mg/kg/day for three consecutive days. The untreated group used as control. After 24 hours of last treatment, the animals were sacrificed after injecting 5 ml of normal saline (0.98%) into the peritoneal cavity of each mouse. The number of intraperitoneal exuded cells and macrophages were counted with 1% neutral red by haemacytometer under microscope. Morphological changes and nuclear damage of EAC cells Cellular apoptosis induced by the extract (MMR) was studied by the method described earlier with little modification [18]. Morphological observation of cells, in absence and presence of MMR (60 mg/day) were studied using a fluorescence microscope (Olympus IX71, Korea). At first EAC cells were collected from culture plates receiving MMR and saline (none treated control plates) and stained with 0.1 µg/ml of Hoechst 33342 at 37oC for 20 min. Then the cells were washed with phosphate buffer saline (PBS) and re-suspended in PBS for observation of morphological changes under fluorescence microscopy. In addition, to determine the necrotic or late apoptotic cell death, EAC cells were further washed by 0.01% Sodiun azide containing 0.9% NaCl and then stained with Propidium Iodide (PI). Effect of caspase inhibitors In order to find out the involvement of caspases in the MMR-induced cell death, the cells were also incubated in CO2 incubator with Z-DEVD-FMK (caspase-3 inhibitor, 2µmol/ml) and ZIETD-FMK (caspase-8 inhibitor, 2 µmol/ml) for 1 h, and then the cells were treated with the extract and kept for another 24 h [19]. DNA fragmentation assay DNA fragmentation assay in agarose gel electrophoresis was determined by the method described previously [20]. EAC cells obtained from mice treated with and without extract (1× 106/ml) at the dose 60 mg/kg/day for five consecutive days. The cells were washed with PBS and re-suspended again in PBS. The total DNA was isolated by using a DNA extraction kit (Promega, USA) and analyzed by electrophoresis on 1.5% agarose gel containing 0.1 µg/ml ethidium bromide and visualized under UV illuminator. Determination of antioxidant capacity Antioxidant potentiality of methanol extract of the M. roxburghii was evaluated by determining total antioxidant capacity, determining DPPH radical scavenging activity and ferrous reducing capacity assays. Determination of total antioxidant capacity Total antioxidant capacity (TAC) of MMR and catechin (CA) was determined by the standard method with some modifications [21]. For this experiment, 0.5 ml of MMR/CA (standard) at different concentrations was mixed with 3 ml of reaction mixture containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 1% ammonium molybdate into the test tubes. The test tubes were incubated at 950C for 10 minutes to complete the reaction. The absorbance was taken at 695 nm using a spectrophotometer against blank after cooling at room temperature. A typical blank solution contained 3 ml of reaction mixture and the appropriate volume of the same solvent used for the MMR/CA were incubated at 950C for 10 minutes and the absorbance was taken at 695 nm. Increased absorbance of the reaction mixture indicated increase total antioxidant capacity. Ferrous reducing antioxidant capacity assay The ferrous reducing antioxidant capacity (FRAC) of MMR/Ascorbic acid (AA) was measured by the method of Oyaizu [22]. For this purpose, 0.25 ml MMR/AA solution at different concentrations, 0.625 ml of potassium buffer (0.2 M) and 0.625 ml of 1% potassium ferricyanide [K3Fe (CN)6] solution were added into the test tubes. The reaction mixture was incubated for 20 minutes at 500 C to complete the reaction. Then 0.625 ml of 10% Trichloro acetic acid (TCA) solution was added into the test tubes. The total mixture was centrifuged at 3000 rpm for 10 minutes. After which, 1.8 ml supernatant was withdrawn from the test tubes and was mixed with 1.8 ml of distilled water and 0.36 ml of 0.1% FeCl3 solution. The absorbance of the solution was measured at 700 nm using a spectrophotometer against blank. Increased absorbance of the reaction mixture indicated increase reducing capacity. DPPH radical scavenging assay Free radical scavenging activity was determined by DPPH radical scavenging assay (DRSA) as reported by Choi et al [23]. For this experiment, a solution of 0.1 mM DPPH in methanol was prepared and 2.4 ml of this solution was mixed with 1.6 ml of extractives in methanol at different concentrations. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for half an hour. The absorbance of the mixture was taken spectrophotometrically at 517 nm. AA was used as reference standard. Percentage DPPH radical scavenging activity (% DRSA) was calculated by the following equation: (% DRSA) = {(Ao – A1)/Ao} × 100 Where, A0 is the absorbance of the control, and A1 is the absorbance of the extract/standard. Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated. Statistical Analysis The experimental results are expressed as the mean ± SEM (Standard Error of Mean). Data have been calculated by one way ANOVA followed by Dunnett ‘t’ test using statistical package for social science (SPSS) software of 10 version. Results Phytochemical screening of crude extract Phytochemical screening of crude extracts of M. roxburghii indicated that the plant had polyphenols, flavonoids, steroids, glycoside and also alkaloids. The components; anthraquinones, hydrolysable tannin, saponins, glycosides etc. were not detected in the crude methanol extracts of the plant (Table 1). Determination of median lethal dose (LD50) Lethal dose of MMR was found to be 600 mg/kg, for intraperitoneal treatment in male Swiss albino mice. The experimental animals showed toxicity at this dose regarding body weight, food intake as well as general appearance. Determination of cell growth inhibition Effects of MMR and bleomycin on EAC cells growth on day six of tumor inoculation are shown in Figure 1. Treatment with MMR resulted in maximum cell growth inhibition at the doses of 60 mg/kg (i. p.) and 40 mg/kg as evident from 81.4% and 68.7% respectively whereas bleomycin showed cell growth inhibition by 86.62% (0.3 mg/kg/day). Bioassay of EAC cells Transplantability of EAC cell receiving MMR decreased remarkably as 51.4% reduction of EAC cell growth was observed when EAC cells from MMR treated mice (at the dose 60 mg/kg i. p.) were re-inoculated into fresh mice and sacrificed and compared with control on day 5. Determination of average tumor weight and survival time All effective anticancer drugs show a significant impact on survival time of tumor bearing mice. The effect of MMR and bleomycin on survival time at different doses has been shown in Figure 2. It has been found that tumor induced mice treated with MMR at doses 20mg/kg, 40mg/kg and 60mg/kg resulted a significant increased of life span, which were 41%, 60.3%, 73.5% respectively compared to that of control mice. On the other hand, bleomycin increased life span by 88.7 % when compared to that of control. Effect of MMR at doses 20mg/kg (i.p.), 40mg/kg (i.p.), 60mg/kg (i.p.) and the antitumor drug bleomycin (0.3 mg/kg) on the average tumor weight owing to tumourgenesis is shown in Figure 3. Supplementation of the extract of mice previously inoculated with EAC cells resulted in the inhibition of tumor burden. In the case of control (EAC bearing) group, the body weight increased by 68.5% on 20th day when compared to the normal. Mice treated with MMR at doses 20 mg/kg (i.p.), 40 mg/kg (i.p.), 60 mg/kg (i.p.) the body weight increased by 37.6%, 28.52% and 21.5% respectively on the 20th day. In contrast the use of bleomycin as standard at the doses of 0.3 mg/kg (i. p.) the body weight was increased by 19.3 % on the 20th day. Effect of MMR on normal peritoneal cells The average number of peritoneal exudates cells of normal mice receiving MMR were found to be (13 ± 1.2) × 106 whereas the macrophage count was (3.6 ± 0.5) × 106 at 60 mg/kg/day respectively. Treatment with the extract at a dose of 60 mg/kg/day for three consecutive days significantly enhanced the number of macrophages. Results are shown in Figure 4. Morphological changes and nuclear damage of EAC cells Morphological changes of EAC cells were examined by Hoechst 33342 staining after culturing the cells with MMR and without extract (60 µg/ml) for 24 hrs. EAC nuclei were round, regular and homogeneously stained with Hoechst 33342 in control group as shown in Figure 5A. Whereas MMR treated EAC cells showed manifest fragmented DNA in nuclei as shown in Figure 5B. Apoptotic morphologic alterations such as membrane and nuclear condensation were also observed clearly by fluorescence microscopy. These results indicate that MMR could induce apoptosis of EAC cells. Necrotic or late apoptotic cell death caused by extract was also observed by staining with PI as shown in Figure 5C. Here the numbers of necrotic cells were found to be very low. Effect of caspase inhibitors Caspase inhibitors Z-DEVD-FMK (caspase-3 inhibitor) and Z-IETD-FMK (caspase-8 inhibitor) were used to understand which pathway was switched on with the MMR treatment. The cytotoxicity of extract towards Z-IETD-FMK-pretreated EAC cells was significantly reduced to 56.95 %, whereas Z-DEVD-FMK-pretreated cells did not any reduction of cytotoxicity in comparison to control (Figure 6). The activation of the endogenous Ca2+/Mg2+-dependent endonuclease is the most distinctive biochemical hallmark of apoptosis. DNA fragmentation assay This activated endonuclease-mediated the cleavage of inter-nucleosomes and generate oligonucleotide fragments of about 180-200 bp length or their polymers. A DNA ladder bands were obtained in agarose gel electrophoresis of DNA preparation extracted from MMR treated EAC cells which is characteristic feature of apoptosis induction, whereas in the control group, smear-like DNA degradation was obtained, which was shown in Figure 7. Determination of total antioxidant capacity The total antioxidant capacity (TAC) of MMR was shown in Figure 8. The plant M. roxburghii showed potent antioxidant activity like reference standard CA at all the concentrations. The absorbance of MMR and standard CA were 1.95and 2.82 respectively at 800 mg/ml. The extract was found to increase the total antioxidant activity with the increasing concentration. Ferrous reducing antioxidant capacity assay Figure 9 represent the reductive capabilities of the plant extract compared to Ascorbic acid which was determined using the potassium ferricyanide reduction method. The reducing power of the extract was moderately strong while increasing dose it showed remarkable increment. DPPH radical scavenging assay The DPPH radical scavenging activity of M. roxburghii is shown in Figure 10. This activity was found to increase with increasing concentration of the extract. The DPPH radical contains an odd electron, which is responsible for the absorbance at 515–517 nm and also for a visible deep purple color. The IC50 value of the extract was 120.4μg/ml while the IC50 value of ascorbic acid was 18.52μg/ml. These results indicate the moderate antioxidant activity of the methanol extract of M. roxburghii. Discussion Reduction of tumor burden, enhancement of life span of cancer bearing mice, tumor cell growth inhibition is the important contributing factors of a potential anticancer agent. The efficiency of MMR as anticancer agent was compared with data obtained by running parallel experiments with a known clinically used anticancer drug, bleomycin at the dose of 0.3 mg/kg (i.p.) and also with those found in similar type of extracts available in the literature [4, 24]. The average tumor weight/burden reducing capacity of this extract has been examined. For tumor bearing mice, body weight was found to be increased gradually with time period. Treatment of such mice with the extract reduced the grown up remarkably. The extract, MMR also inhibited the cell growth rate effectively; as more than 71 % inhibition was found at the dose of 60 mg/kg (i.p.) which is quite comparable to that of bleomycin (76.6%; at 0.3 mg/kg). Supplementation of MMR increased the life span of tumor bearing mice very effectively and the potency was found to increase with the enhancement of dose. In the present experimental design, a dose up to 60 mg/kg (i.p) was used and further enhancement of the life span will therefore be expected using higher doses. It is noted that the enhancement of life span has been assigned as a very important parameter for judging the suitability and efficacy of a compound as anticancer agent [25]. The high LD50 value (600 mg/kg) of this crude extract indicated little toxicity to the host. It has been found that the extract of M. roxburghii has significant effect on the enhancement of normal peritoneal cells in normal mice. Treatment with MMR at a dose of 60 mg/kg (i.p) increased the number of macrophages to some extent. This is being considered to be a very important parameter for acquiring self destroying ability of the animals or living beings towards cancer cells [26]. It is highly desirable to have compounds that can cause cancer cell death via apoptosis. Apoptosis eliminates malignant or cancer cells without damaging normal cells and surrounding tissues [27]. Apoptosis is characterized by cell morphological changes, chromatin condensation, DNA cleavage, and nuclear fragmentation. Typical morphological features of apoptotic cells can be observed through microscopic studies such as those using the inverted phase contrast and fluorescence microscope. Other features such as chromatin condensation and nuclear fragmentation can be better observed through the double staining with Hoechst 33342 and Propidium Iodide (PI) using fluorescence microscopic analysis. This is a convenient and rapid assay, widely used to identify live and dead cells. Hoechst 33342 is a blue fluorescing dye that stains chromatin DNA. The red fluorescing dye PI is only permeable to dead cells and cannot enter the intact plasma membrane of living cells. Thus, the staining pattern which resulted from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic, and dead cells population by fluorescence microscopy [28-31]. EAC cells treated with extract (MMR) shown nuclear condensation, fragmentation, cell membrane blebbing, apoptotic bodies etc. under the fluorescence microscope which implies that the extract induce EAC cells apoptosis. PI staining of EAC cells also indicates the late phase apoptosis of induction after treatment with the extract. The integrity of the DNA was also assessed by agarose gel electrophoresis. DNA isolated from cells showed a ‘‘ladder’’ pattern in apoptosis [20]. Genomic DNA isolated from treated and untreated cells shown apoptotic pattern in agarose (1.5 %) electrophoresis. This characteristics ladder like DNA band in the gel, further conform the induction of apoptosis in EAC cells to the treatment of MMR. There are two main apoptotic pathways-the extrinsic (death receptor) pathway is activated by binding of tumor necrosis factor (TNF) ligands such as TRAIL to corresponding cell surface receptors, for example, TRAIL receptors, triggering the assembly of the death-inducing signaling complex (DISC) that drives caspase-8 activation[27]. The intrinsic (mitochondrial) pathway involves mitochondrial outer membrane permeabilization accompanied by the release of cytochrome C and second mitochondria-derived activator of caspases (Smac) into the cytosol104. Cytochrome C initiates the formation of a caspase-9, caspase-3 activation platform, that is, the apoptosome, while Smac relieves the inhibition of caspases by neutralizing inhibitor of apoptosis (IAP) proteins[32]. To understand the involvement of specific signaling pathway trigger by MMR in EAC cells, the specific inhibitors of candidate molecules such as caspase-8 and caspase-3 were used. Data obtained from our experiment shows that EAC cells pretreated with caspase-8 inhibitor (caspase-8 blocked/inactivated and caspase-3 remain active) showed 56.95 % growth inhibition in comparison to control whereas, cells pretreated with caspase-3(caspase-3 inactive and caspase-8 active) shown very little or no cells growth inhibition. This result leads one to conclude that caspase-3 mediated signaling pathway is involved in EAC cells apoptosis induced by the treatment of M. roxburghii. Our present study results also reflected that the same extract of M. roxburghii showed moderate antioxidant activity. Numerous animal studies have been published demonstrating decreased tumor size and/or increased longevity with the combination of chemotherapy and antioxidants [33]. Resistance to chemotherapeutic agents is thought to be due to reduced accumulation into the tumor cell [34]. Recent research has focused on the ability of flavonoids type antioxidant compound to increase the concentration of chemotherapeutics in tumor cells. New findings within the past few years have revealed that the binding of selective glycosides compound to Na+, K + -ATPase results in complex but well-documented changes in cell signaling events [35]. This “signalosome” complex includes the enzyme, Na+, K + -ATPase as well as Src, phosphoinositide-3 kinase (PI3K), and phospholipase C each of which, in turn, sets into action complex signaling events that can result in tumor cell death through either apoptosis or autophagy-related mechanisms [36-37]. Apoptotic cell death mediated by glycosides has been demonstrated in a number of cell lines [38]. 2006) for example oleandrin produced an increase in expression of Fas and TumorNecrosis Factor Receptor 1 (TNFR1), resulting in potentiation of apoptosis in tumor cells but not in normal primary cells. As such, they represent a promising form of targeted cancer chemotherapy [39]. On the other hand, alkaloids derived from plants; block cell division by preventing microtubule function. There is increasing evidence showing that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting cell cycle progression at mitosis and eventually leading to apoptotic cell death. Though the presence of flavonoids, glycosides and alkaloids of the extract could be attributed to the anticancer effect of M. roxburghii through apoptosis but still there is no consensus about which substances are exactly responsible for these effects. So much more investigation including identification specific active compound involved has to be carried out against various other cancer cell lines and higher animal models, in order to confirm the plant as a potent anticancer drug reservoir. Conclusions In the light of above observations, it can be concluded that the methanol extract of M. roxburghii showing potential anticancer activity through intrinsic mitochondrial pathway and might be considered as one of the promising resources in cancer chemotherapy. 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Phytochemical components of crude methanol extract of M. roxburghii Phytochemical components E. camadulensis Alkaloids + Saponins - Saponin glycosides - Tannins - Hydrolysable tannins - Phlobatannins - Anthraquinones - Glycosides + Cardiac Glycosides - Flavonoids + Steroid + Volatile oils - PolyPhenols + +: Present; -: Absent Figure 1. Effects of MMR on EAC cells growth inhibition 100 *** ** * % of cell growth inhibition 80 60 40 20 0 Bleomycin (0.3mg/k) MMR (60mg/kg) MMR (40mg/kg) MMR (20mg/kg) Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P < 0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC bearing control mice (EAC bearing only). Figure 2. Effects of MMR on survival time of tumor bearing mice 50 *** ** Meas Survival Time (MST) in days 40 ** * 30 20 10 0 Control Bleomycin (0.3 mg/kg) MMR (20mg/kg) MMR (40mg/kg) MMR (60mg/kg) Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P < 0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC bearing control mice (EAC bearing only). Figure 3. Tumor weight of EAC bearing mice treated with MMR and bleomycin 25 20 Tumor weight in gm 15 * 10 ** *** 5 *** 0 0 5 10 15 Control Bleomycin (0.3 mg/kg) (40mg/kg) MMR -5 MMR (60mg/kg) 20 25 MMR (20mg/kg) Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P < 0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC bearing control mice (EAC bearing only). Figure 4. Effects of MMR on the enhancement of macrophages and peritoneal cells 16 * Number of cells (10 6) 12 *** * 8 ** ** 4 * 0 Control MMR (20mg/kg) Total peritoneal cell MMR(40mg/kg) MMR (60mg/kg) Macrophage Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P < 0.05, ** P < 0.01 and *** P < 0.001 when (Normal+ MMR) treated mice compared with control mice. Figure 5. Fluorescence microscopic view of control and treated EAC cells B A C A) EAC of normal mice shown no apoptotic feature. B) EAC cells treated with extract shown nuclear condensation, fragmentation (black arrow), cell membrane blebbing (red arrow), and apoptotic bodies (white arrow) etc. C) Cells undergone late apoptosis (black arrow) and normal dead (white arrow) shown in PI staining. Figure 6. Effect caspases inhibitors on EAC cells Results are shown as mean ± SEM (Standard Error of Mean). Figure 7. In vivo effects of extract on DNA fragmentation of EAC cells B A DNA run and detected on 1.5% agarose gel electrophoresis. A) DNA from control EAC cells, B) DNA from MMR treated EAC cells (DNA fragmentation detected from treated EAC cells). Figure 8. Determination of total antioxidant capacity of M. roxburghii 3.5 3 Absorbance (700 nm) 2.5 2 1.5 1 0.5 0 0 100 200 300 400 500 600 Concentration (m g/m l) MMR CA Results are shown as mean ± SEM (Standard Error of Mean). 700 800 Figure 9. Determination of ferrous reducing antioxidant capacity of M. roxburghii 3.5 Absorbance (695 nm) 3 2.5 2 1.5 1 0.5 0 0 200 400 600 Concentration (m g/m l) MMR AA Results are shown as mean ± SEM (Standard Error of Mean). 800 Figure 10. Determination of DPPH radical scavenging activity of M. roxburghii 100 90 % of scavenging 80 70 60 50 40 30 20 10 0 0 50 100 150 200 250 Concentration (microgram/ml) MMR AA Results are shown as mean ± SEM (Standard Error of Mean). 300 350 400