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Antioxidant, cytotoxic and antineoplastic effects of Carissa carandas Linn. leaves
Experimental and Toxicologic Pathology xxx (xxxx) xxx–xxx
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Antioxidant, cytotoxic and antineoplastic effects of Carissa carandas Linn. leaves Marina Khatuna, M. Rowshanul Habiba, M. Ahasanur Rabbib, Ruhul Aminb, M. Farhadul Islama, ⁎ M. Nurujjamana, M. Rezaul Karima, M. Habibur Rahmana, a b
Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi 6205, Bangladesh Bangladesh Council of Scientific & Industrial Research (BCSIR) Laboratories, Rajshahi, Bangladesh
A R T I C L E I N F O
A B S T R A C T
Keywords: Carissa carandas Leaves Antineoplastic Cytotoxic Antioxidant
For scientific clarification of some traditional uses, this study was designed to explore the antioxidant, cytotoxic and antineoplastic properties of leaf extract of Carissa carandas Linn., a traditional medicinal plant of Bangladesh. The methanol extract of Carissa carandas leaves (MELC) was applied on DPPH and ABTS experiments to determine its antioxidant activity. In vitro the cytotoxic effect of MELC was evaluated against colonic adenocarcinoma cell lines (SW-480 and SW-48) whereas in vivo its antineoplastic property was tested against Ehrlich ascites carcinoma (EAC). The DPPH and ABTS assays revealed the antioxidant activity of MELC with IC50 10.5 ± 1.2 and 1.75 ± 0.3 μg/ml that was comparable to L-ascorbic acid. In vitro cytotoxic study, MELC reduced the viability of adenocarcinoma cells in dose dependent manner and in vivo, administration of MELC (25 mg/kg) resulted in a significant (p < 0.05) decrease in viable EAC cell count thereby increasing the life span of the EAC cell bearing mice. Restoration of hematological parameters such as red blood cells (RBC), hemoglobin and white blood cells (WBC) to normal levels in MELC-treated mice was also observed. Moreover, treatment with MELC induced apoptosis of EAC cells as observed in fluorescence microscopic view of DAPI (4,6diamidino-2-phenylindole) stained cells and also increased p53 gene expression MELC-treated cells in respect to untreated EAC control. In addition, the MELC was rich in polyphenol content and its GC–MS chromatogram confirmed the presence of some compounds all of which showed anticancer and cytotoxic activities in previous studies. In a word, this study supports the use of Carissa carandas in traditional medicine as well as highlights the need to further explore the potentials of MELC as an antineoplastic agent.
1. Introduction Cancer, a group of diseases characterized by uncontrolled growth and spread (invasion and metastasis) of abnormal cells, is considered as the second most common cause of death in the developed world (Tanaka, 1994). A similar picture has also arisen in the developing countries like Bangladesh. Both several external environmental factors (tobacco, chemicals, radiation, and infectious organisms) and/or internal factors (mutations, hormones, and altered immunity) are responsible for neoplastic transformation (Tanaka, 1997). As cancer chemoprevention, the use of natural or synthetic chemical agents is an important strategy to reduce the risk of cancer. Many previous studies have suggested the beneficial effects of various phytochemicals in reducing the risk of cancer (Kuno et al., 2012). So research on the medicinal plants for their antineoplastic effect bears a significant value. Carissa carandas Linn. commonly known as Karamcha, is a common
⁎
herb of Apocynaceae family and it is found throughout all over the Bangladesh. It has a long history of use in traditional system of medicine. The fruits, leaves, barks and roots of Carissa carandas have been used for ethnomedicine in the treatment of human diseases, such as diarrhea, stomachic, anorexia, intermittent fever, mouth ulcer and sore throat, syphilitic pain, burning sensation, scabies and epilepsy (Chanchal et al., 2013). Earlier studies have shown that the extracts of different parts of this plant possesses cardiotonic, antipyretic, anticonvulsant, antidiabetic and antiviral activities (Ya’u et al., 2008; Itankar et al., 2011; Chanchal et al., 2013). Chemical constituents of Carissa carandas include steroids, terpenes, tannins, flavonoid, benzenoids, phenylpropanoid, lignans, sesquiterpenes, and coumarins (Begum et al., 2013). Moreover, this plant is used by traditional practitioner of some districts in Bangladesh for the treatment of cancer. But, no scientific data is available to confirm this folkloric use. Here, the methanol extract of Carissa carandas dried leaves (MELC) was evaluated
Corresponding author. E-mail address: [email protected] (M.H. Rahman).
http://dx.doi.org/10.1016/j.etp.2017.03.008 Received 25 November 2016; Accepted 31 March 2017 0940-2993/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Khatun, M., Experimental and Toxicologic Pathology (2017), http://dx.doi.org/10.1016/j.etp.2017.03.008
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for antioxidant, cytotoxic and antineoplastic activities and its various phytochemical compounds were analyzed by GC–MS. 2. Materials and method 2.1. Plant materials Leaves of Carissa carandas (Linn) plant (Family: Apocynaceae) were used for this study. The plant samples were collected during the month of June-July 2015 from the relevant area of Rajshahi district. This medicinal plant was identified and authenticated by plant taxonomist of Department of Botany, University of Rajshahi, Rajshahi, Bangladesh. Voucher specimen (voucher specimen No. 1150) was deposited at Department of Botany, University of Rajshahi, for future reference. Fig. 1. Phenolic and flavonoid content of MELC.
2.2. Extraction Table 1 Chemical composition of MELC analyzed by GC–MS.
The leaves of Carissa carandas was shade dried and ground into powder. The powder (0.3 kg) was extracted with methanol (Sigma Aldrich, Germany) at room temperature for 7 days. The solvent was completely removed by rotary vacuum evaporator and the crude methanol extract of Carissa carandas leaves (4.4 g) (designated as MELC) was stored in a vacuum desiccator for further use. 2.3. Determination of total phenolic and total flavonoid content The Folin–Ciocalteu method modified by Ranilla et al. (2010) was applied to determine the total polyphenolic content of MELC. One milliliter of sample solution was transferred into a 10 ml volumetric flask and mixed with 6 ml of distilled water. 0.5 ml of Folin–Ciocalteu reagent (50%, v/v) (Labscan, Thailand) was added to sample and mixed. After 5 min, 1 ml of Na2CO3 (5%, m/v) (Sigma Aldrich, Germany) was added to the mixture and adjusted to 10 ml with distilled water. After standing for 60 min at room temperature, the absorbance was measured at 760 nm. Gallic acid (Sigma Aldrich, Germany) was used for constructing the standard curve. The total phenolic content was expressed as gallic acid equivalents (mg/g of dry weight of extract). Total flavonoids content of MELC were estimated using the method described by Ordonez et al. (2006). Here, catechin (Sigma Aldrich, Germany) was used as standard. 1.5 ml of methanol, 100 μl of 10% aluminum chloride (Sigma Aldrich, Germany), 100 μl of 1 M potassium acetate (Labscan, Thailand) solution and 2.8 ml of distilled water was added to 0.5 ml of sample/standard. After one hour 30 min of incubation at room temperature (RT), the absorbance was measured at 420 nm. The samples/standard was evaluated at a final concentration of 0.1 mg/ml. Total flavonoid contents were expressed in terms of catechin equivalent, (mg/g of dry weight of extract).
Sl
Retention time (min)
Compounds
Area (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
3.34 3.41 3.60 4.45 10.80 10.90 11.17 11.64 12.81 13.93 15.65 15.71 26.57 27.72 28.31 29.12 28.84 30.77
Isobenzofuran Ethylbenzene o-Xylene 1,2,4-trimethylbenzene, Card-20(22)-enolide Methyl 4-O-methyl-D-arabinopyranoside n-Decanoic acid 4-O-Methylmannose Hexadecanal Hexadecanoic acid 9,12-Octadecadienoic acid 8,11,14-Docosatrienoic acid Stigmasterol β-Asarone 24-Noroleana-3,12-diene Lupeol Urs-12-en-3-ol Lup-20(29)-en-3-ol Total area =
3.80 0.32 1.02 0.68 2.72 1.86 0.25 10.6 1.20 12.06 1.53 14.20 1.04 0.79 4.91 17.10 0.58 4.89 79.25
2.5. ABTS%+ scavenging capacity ABTS%+ assay was carried out according to the method described previously (Cai et al., 2004). The ABTS%+ solution was prepared by mixing 7 mM ABTS (Sigma Aldrich, Germany) and 2.45 mM potassium persulfate (Carl Roth, Germany) and incubating in the dark at room temperature for 12 h. The ABTS%+ solution was then diluted with water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. ABTS%+ solution (3 ml) was added to 0.1 ml of the test sample with various concentrations (0.625–3.75 μg/ml) and mixed vigorously. The absorbance was measured at 734 nm after standing for 6 min. The ABTS%+ radical scavenging activity of the samples was expressed as
2.4. DPPH% scavenging activity
ABTS%+ scavenging effect (%) = [1 − (Asample/Acont)] × 100
Free radical scavenging activity was determined by DPPH (Sigma Aldrich, Germany) radical scavenging assay as previously described method (Mohsen and Ammar, 2009) with some modification. A solution of 0.1 mM DPPH in methanol was prepared and 3 ml of this solution was mixed with 1 ml of extractives in methanol at different concentrations (6.25–37.5 μg/ml). The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as reference standard. Percentage of DPPH radical scavenging was calculated as DPPH% scavenging effect (%) = [1 − (Asample/Acont)] × 100 EC50 values (μg/ml), the effective amount of the sample needed to scavenge DPPH% by 50%, were determined from the plotted graphs of scavenging activity against the concentration of the extracts.
where Acont is the absorbance of the blank control (ABTS%+ solution without test sample) and Asample is the absorbance of the test sample.
2.6. GC-analysis The chemical composition of MELC was established by gas chromatography mass spectrometry/Quadropole detector analyses using GCMS-QP2010S (Shimadzu Kyoto, Japan) spectrometer. They equipped with a flame ionization detector and capillary column with HP-5MS (30 m × 0.25 mm × 0.25 μm). The temperature program for the column was from 120 °C (1 min) to 230 °C at a rate of 6 °C/min and then held at 200 °C for 35 min. Helium was used as a carrier gas at a flow of 14 psi (Split 1:10), and the injection volume of each sample was 1 μl. 2
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Table 2 Antioxidant properties of MELC. Sample
DPPH% scavenging activity
ABTS% + scavenging capacity
Concentration (μg/ml)
Scavenging effect (%)
EC50 (μg/ml)
Concentration (μg/ml)
Scavenging effect (%)
EC50 (μg/ml)
MELC
6.25 7.5 10 12.5 25 37.5
27.1 43.0 49.0 64.4 87.4 86.9
10.5 ± 1.2
0.625 1.25 2.0 2.5 3.0 3.75
27.1 35.8 57.0 63.2 76.5 88.5
1.75 ± 0.3
Vitamin-C
0.25 0.50 1.00 2.00 4.00 8.00
7.76 18.3 30.0 47.6 68.2 88.1
2.16 ± 0.1
0.160 0.315 0.625 1.25 1.875 2.5
10.8 27.5 41.0 77.0 74.4 97.4
0.78 ± 0.02
Fig. 2. GC–MS chromatogram of MELC. Table 3 Effect of MELC on viable EAC count, survival time and body weight gain of EAC cell bearing mice. Treatment
Viable EAC cells on day 6 after inoculation (x107 cells/mL)
MST (in days)
%ILS
Body weight gain (g) after 15 days
Untreated control EAC + MELC (25 mg/kg) EAC + Bleomycin (0.3 mg/kg)
4.21 ± 0.87 1.36 ± 0.24t
21.5 ± 2.97 30.2 ± 2.86t
– 41.8 ± 4.19
13.7 ± 3.0 8.75 ± 2.1t
0.35 ± 0.07t
39.0 ± 1.85t
81.4 ± 3.97
5.8 ± 0.57
t
Data are expressed as the mean ± S.E.M (n = 8); Significantly different from untreated group: tP < 0.05; MST: Mean survival time; %ILS: Percentage (%) increase in life span.
The database used for the identification of chemical compounds and measurements of peak areas obtained is that of NIST/EPA/NIH MS LIBRARY (NIST 05) and also AMDIS version 2.0 d.
2.7. In vitro cytotoxicity assay against SW480 and SW-48 SW-480 (ATCC CCL228; Duke’s class B/stage II, colonic adenocarcinoma) and SW-48 (ATCC CCL-231; Duke’s class C/stage III colonic adenocarcinoma) were maintained according to ATCC guidelines. 3
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Fig. 3. In vitro cytotoxic effect of MELC against SW-480 and SW-48 cell lines. Table 4 Effect of MELC on hematological parameters of EAC cell bearing mice. Parameters
Normal
Only EAC (Untreated control)
EAC + MELC (25 mg/kg)
EAC + Bleomycin (0.3 mg/kg)
Hgb (g/dL) RBC(×109 cells/mL) WBC(×106 cells/mL)
14.48 ± 1.47 6.67 ± 0.52 8.75 ± 1.12
9.86 ± 0.83* 1.57 ± 0.11* 46.2 ± 4.96*
12.28 ± 1.16t 3.33 ± 0.41t 26.6 ± 4.03t
14.57 ± 0.85t 4.80 ± 0.08t 9.27 ± 0.69t
Data are expressed as mean ± S.E.M for eight animals in each group. * P < 0.05: against normal group. t P < 0.05: against untreated control group.
perature 25 ± 2 °C; humidity 55 ± 5%) with 12 dark/light cycle. They were allowed free access to standard dry pellet diet (collected from ICDDR,B) and water ad libitum. The newly collected mice were given a week to get themselves adapted with the new environment of the laboratory before being employed in any experiment.
Colon cancer cell lines (SW-480 and SW-48) were maintained in Leibovitz’s L-15 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a CO2 incubator. Cytotoxicity assay was performed against SW-480 and SW-48 using a cell counting kit-8 (CCK-8; Sigma–Aldrich) (Amirghofran et al., 2011). Both SW-480 and SW-48 cells were first seeded in flat-bottom 96-well plates at 1 × 104 cells/well and the plate was pre-incubated for 24 h in a humidified incubator (e.g., at 37 °C, 5% CO2). Then cells were incubated with various concentrations (1, 100, 200 and 400 μg/mL) of MELC. Following 24 and 48 h of incubation, 10 μl of CCK-8 solution was added into each well and further incubated for another four hours. The absorbance was measured at 450 nm using a microplate reader. Here untreated cells were used as negative controls and Cisplatin was used as a positive reference standard. The percentage of inhibition was measured as [1 − (optical density of test/optical density of negative control)] × 100. The IC50 value (the concentration of 50% cell inhibition) was calculated from the graph of inhibition percentage against different extract concentrations.
2.9. Tumor cells EAC cells were obtained through the courtesy of Indian Institute for Chemical Biology (IICB), Kolkata, India and were maintained by weekly intraperitoneal (ip.) inoculation of 105 cells/mouse in the laboratory. 2.10. Acute toxicity study The acute toxicity study as previously described (Lorke, 1983) was conducted to determine the LD50 value of MELC in mice. In this method, a single intraperitoneal injection was applied on thirty six animals (6 in each group) at different doses (100, 200, 400, 800, 1600 and 3200 mg/kg body weight) and LD50 was evaluated by recording mortality after 24 h.
2.8. Animals
2.11. Study on in vivo EAC cell growth
This study was carried out using Swiss albino mice of either sex, 3–4 weeks of age, weighing between 20 and 25 gm. They were collected from the Animal Research Branch of the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B). The mice were grouped and housed in plastic cages with not more than eight animals per cage and maintained under standard laboratory conditions (tem-
In order to determine the effect of MELC on EAC cell growth, 24 mice were randomly divided into three groups (8 animals in each group) and 1.5 × 105 cells were inoculated (i.p.) into each mouse of each group on day 0. The group 1 was treated with vehicle (2% 4
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Fig. 4. Fluorescence microscopic view of EAC cells collected from the mice of untreated control (A) and MELC treated (B) groups. Arrows indicating apoptotic features (condensed chromatin and nuclear fragmentation).
2.12. Study on morphological appearance and p53 gene expression
Dimethylsulfoxide; DMSO) and it was considered as untreated control. Mice in group 2 and 3 received (i.p.) MELC (25 mg/kg b.w./day) and bleomycin (0.3 mg/kg b.w./day). Treatment was started and continued for 5 days. The mice were sacrificed on the 6th day after transplantation of EAC cells and cells were collected by repeated intraperitoneal wash with normal saline (0.98% NaCl). Viable EAC cells were counted with a haemocytometer using trypan blue (Sigma–Aldrich) and total number of viable cells per mouse of the treated group was compared with those of control (Osman et al., 2011). During studies on cell growth inhibition, EAC cells were also collected from mice of untreated and MELC treated group after sacrificing for observation of morphological changes of cell by 4,6diamidino-2-phenylindole (DAPI) (Carl Roth) staining and for studying the p53 gene expression by RT-PCR.
Collected EAC cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and then visual images were taken using fluorescent microscopy (Senthilkumar et al., 2008). Fragmented or condensed nuclei were defined as apoptotic cells. PCR specificity was determined using both a melting curve analysis and gel electrophoresis. To analyze the expression of p53 gene by RTPCR (Real time-PCR), TRIzol method was applied to extract total RNA from EAC cells of MELC-treated and untreated groups. Then 3 μg RNA was reversed transcribed into cDNA in a final volume of 20 μl containing100 pmol random hexamer, and 50 U of MuLV reverse transcriptase (New England Biolab) according to the manufacturer’s guideline. Expression of p53 gene was studied using this cDNA as template for PCR. β-actin was used as the housekeeping gene. A 25-μl reaction volume containing 25 pmol each of forwarded and reverse primer, 2.5 mM of each dNTP, and 0.25 U of platinum Taq polymerase 5
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3. Results 3.1. Total phenolic and flavonoid content The amounts of total phenolic and flavonoid content found in MELC are shown in Fig. 1. The total phenolic and flavonoid content of MELC were 137.2 ± 20.7 and 6.55 ± 0.47 mg/g of dry weight of extract, respectively. The results showed that MELC has higher total polyphenolic content. 3.2. Chemical composition of MELC The chemical composition of MELC was characterized by 18 constituents and these constituents were accounted for 79.25% of the total extract. The identified compounds and the chemical compositions of MELC are presented in Table 1 and Fig. 2. The other major components were lupeol (17.10%), 8,11,14-Docosatrienoic acid (14.2%), hexadecanoic acid (12.06%), 4-O-Methylmannose (10.6%), 24-Noroleana-3,12-diene (4.91%) and Lup-20(29)-en-3-ol (4.89%). In addition, MELC also contains card-20(22)-enolide (2.72%), methyl 4-Omethyl-D-arabinopyranoside (1.86%), isobenzofuran (3.80%) and βAsarone (0.79%). 3.3. Antioxidant properties The antioxidant activity of MELC was determined by the DPPH and ABTS test system. As demonstrated in Table 3, the MELC exhibited moderate DPPH% and ABTS%+ radical scavenging ability when compared with vitamin-C and the percentage scavenging effect of MELC was increased with the increasing of its concentration. In DPPH experiment, the EC50 values of MELC and vitamin-C was found to be 10.5 ± 1.2 and 2.16 ± 0.1, respectively, whereas it was 1.75 ± 0.3 and 0.78 ± 0.02 for MELC and vitamin-C in ABTS%+ experiment.
Fig. 5. Effect of MELC on p53 expression of EAC cells. Total RNA extracted from treated and untreated EAC cells were reverse transcribed using random hexamer and PCR reaction was carried out using primers specific for p53 and control gene, β-Actin. (A) Real-time PCR relative quantification of p53 levels in untreated control and MELC-treated EAC cells. (B) PCR reaction products separated on 1% agarose gel stainedwith ethidium bromide. L represents 1 kb DNA ladder; U represents RNA from EAC cells of untreated control; T represents RNA from EAC cells of MELC-treated mice.
(Tiangen, China), was prepared. A BioRad (USA) gradient thermal cycler was used for amplification. The cycling condition was initial PCR activation step of 5 min at 95 °C, followed by 35 cycles of 95 °C/min, 55 °C/min, 72 °C/min, and final 72 °C/10 min (elongation). All the PCR reactions were analyzed in 1.0% agarose gel and GeneRular 1 kb DNA ladder (Fermentus, USA) was used as marker. Moreover, we also measured relative quantities (RQ) of p53 tumour suppressor mRNA of control and treated EAC cells by quantitative real time PCR using a melting curve analysis.
3.4. In vitro cytotoxic activity In vitro cytotoxic assay, MELC had a noticeable activity against SW480 and SW-48 cell lines. Against SW-480, 140.6 ± 5.2 and 108.4 ± 8.3 μg/ml were resulted as IC50 values for MELC after 24 and 48 h treatment whereas 376.6 ± 15.0 and 290.0 ± 11.6 μg/ml were found against SW-48 cell line after 24 and 48 h treatment (Fig. 3.). However, Cisplatin showed the high inhibitory effect against these cell lines with low IC50 values as shown in Fig. 3.
2.13. Study on survival time and heamatological parameters
3.5. In vivo antineoplastic activity against EAC
Mice in three groups (8 animals per group) were inoculated with 1.5 × 105 cells/mouse on the day 0. After 24 h of inoculation, mice in group 1, 2 and 3 were treated (i.p) with 2% v/v DMSO, MELC (25 mg/ kg b.w./day) and bleomycin (0.3 mg/kg), respectively and continued for 10 days. On 15th day after tumour inoculation, hematological parameters (Hemoglobin, RBC, and WBC) were measured from tail vein blood of each mice of each group (Mukherjee, 1988). Then the average body weight changes and mean survival time of each group were noted. The mean survival time (MST) of the treated groups was then compared with that of the untreated control group and percentage increase in life span (%ILS) was calculated according to the previously described formula (Habib et al., 2010).
In acute toxicity study, intraperitoneal administration of graded doses of MELC to Swiss albino mice produced a LD50 of 2860.7 mg/kg body weight. Antineoplastic activity of MELC against EAC cell bearing mice was assessed by the parameters such as viable EAC cell count, mean survival time (MST), percentage (%) increase of life span (%ILS) and body weight gain. The average number of viable tumour cells per mouse of untreated group was found to be (4.21 ± 0.87) × 107 cells/mL. Treatment with MELC (25 mg/kg) and bleomycin (0.3 mg/kg) decreased the viable cells significantly (P < 0.05). The effect of MELC on the survival of EAC bearing mice is shown in Table 3. The MST of the untreated group was 21.5 ± 2.97 days, whereas it was 30.2 ± 2.86 and 39.0 ± 1.85 for the group treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg), respectively. The increase in the life span of EAC cell bearing mice treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg) was found to be 41.8% and 81.4%, respectively (table. 3). On 15th day of tumour cell inoculation, the average weight gain of only EAC cell bearing mice was 13.7 ± 3.0 g whereas it was 8.75 ± 2.1 and 5.8 ± 0.57 for the groups treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg),
2.14. Statistical analysis All values were expressed as mean ± S.E.M (Standard Error of Mean). Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test using SPSS statistical software of 16 version. P < 0.05 were considered to be statistically significant. 6
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Voller et al., 2010). MELC also contained lupeol and lup-20(29)-en-3-ol in rich amount. Lupeol and its derivatives have already been recognized as dietary chemopreventive compounds for cancer (Chaturvedi et al., 2008; Siddique and Saleem, 2011). So the observed activities of MELC are thought to be a result of the synergistic action of these compounds with antioxidative property.
respectively. In addition, when compared to normal mice, significant changes had been observed in hematological parameters of EAC cell bearing mice on the 15th day after EAC cell inoculation (Table 4). The total WBC count was found to increase with a reduction in the hemoglobin content and total RBC count. At the same time interval, treatment of MELC (25 mg/kg) could change these parameters near to normal. Fluorescence microscopic view of DAPI stained EAC cells demonstrated that MELC induced the alterations (i.e., nuclear condensation, fragmentation, membrane blabbing and apoptosis of cell) in the morphology of EAC cells (Fig. 4A and B). Moreover, the quantitative analysis of p53 mRNA level by RT-PCR showed that p53 gene expression was quite absent in untreated EAC control but it was increased in MELC treated EAC cells (Fig. 5A). The products of RTPCR for MELC treated EAC cells had also given clear band on agarose gel stained with ethidium bromide (Fig. 5B).
Results of this study conclude that the MELC was effective in inhibiting the growth of cancer cells in vitro and vivo conditions. The phytochemical and antioxidants studies were also supported its cytotoxic and antineoplastic properties. Therefore further studies are required to isolate and characterize the active principles of Carissa carandas Linn. leaves which can offer enhanced anti-cancer activity and to establish their mechanism of action.
4. Discussion
Funding
In this study, MELC showed in vitro cytotoxic effect against colonic adenocarcinoma (SW-480 and SW-48) cell lines and in vivo antineoplastic activity against EAC. Generally, prolongation of lifespan of the treated animal and decrease in WBC count of blood are considered as the reliable criteria for evaluating an anticancer drug (Jules Hirsch, 2006). Our results showed an increase in lifespan accompanied by a reduction in WBC count in MECL treated mice. Treatment with MELC produced a significant effect in increasing the life span of ascities tumour bearing animals and also reduced the viable EAC cells in animal models. In addition, MELC restored the hemoglobin content, RBC and WBC cell count to normal values and thereby improving the condition myelosuppression and anaemia which are major complications of cancer chemotherapy (Hogland, 1982). Moreover, fluorescence microscopic view of DAPI stained EAC cells of MELC treated mice demonstrated the apoptosis of EAC cells when compared with untreated EAC control (Fig. 4). Apoptosis (programmed cell death) plays a critical role in normal physiological functions, but there is a dysregulation of apoptosis in cancer. P53 which is one of the most important proapoptotic mediators, is lost or functionally inactivated in more than 50% of human cancers (Hanahan and Weinberg, 2000). Preneoplastic and malignant cells represent a protective mechanism against induction of P53 thereby promoting their proliferation (Mukhtar, 2012). In this study, we had also found that treatment of MELC induced the expression of p53 gene in respect to untreated control (Fig. 5). So this finding indicates that apoptosis induced in MELC treated EAC cell was probably mediated by p53-dependent pathway. However, this study also demonstrated the potent free radical scavenging activity using DPPH% and ABTS%+ experiments (Table 2). The broad range of effects of free radicals has drawn the attention of many scientists in the past decade. It has been proved that free radicals play an important role in the pathogenesis of certain diseases including cancer. (Formica and Regelson, 1995). Plant constituents like polyphenol compounds have shown free radical scavenging or antioxidant activity. Polyphenols are the major plant compounds with antioxidant activity. This activity is believed to be mainly due to their redox properties (Nitin et al., 2010) which play an important role in adsorbing and neutralizing free radicals. Moreover, several studies have showed the biological activities (Cytotoxic, anticancer, antitermitic, antibacterial, nematicidal and insecticidal) of polyphenol rich extracts from various plant species (Kaur and Arora, 2009; Mayaud et al., 2008; Pandey et al., 2009; Park et al., 2007; Seo et al., 2009). In this study, we found that the polyphenolic content of MELC was high in respect flavonoid (Fig. 1). In addition, the GC–MS profile of the MELC confirmed that it contained isobenzofuran, card-20(22)-enolide, methyl 4-O-methyl-D-arabinopyranoside, β-asarone and 24-noroleana-3,12diene all of which showed anticancer and cytotoxic activities in previous studies (Bayer et al., 2005; Wen et al., 2016; Li et al., 2015;
This study was financially supported by the Ministry of Science and Technology, Bangladesh (39.009.006.01.00.049.2013–2014/BS-105/ 105 and 39.009.006.01.00.049.2013–2014/BS-105/535).
5. Conclusions
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Seo, S.M., Kim, J., Lee, S.G., Shin, C.H., Shin, S.C., Park, I.K., 2009. Fumigant antitermitic activity of plant essential oils and components from Ajowan (Trachyspermum ammi), Allspice (Pimenta dioica), Caraway (Carum carvi), Dill (Anethum graveolens), Geranium (Pelargonium graveolens), and Litsea (Litsea cubeba) oils against Japanese termite (Reticulitermes speratus Kolbe). J. Agric. Food. Chem. 57, 6596–6602. Siddique, H.R., Saleem, M., 2011. Beneficial health effects of lupeol triterpene: a review of preclinical studies. Life Sci. 88, 285–293. Tanaka, T., 1994. Cancer chemoprevention by natural products (review). Oncol. Rep. 1, 1139–1155. Tanaka, T., 1997. Chemoprevention of human cancer: biology and therapy. Crit. Rev. Oncol. Hematol. 25, 139–174. Voller, J., Zatloukal, M., Lenobel, R., Dolezal, K., Béres, T., Krystof, V., Spíchal, L., Niemann, P., Dzubák, P., Hajdúch, M., Stmad, M., 2010. Anticancer activity of natural cytokinins: a structure-activity relationship study. Phytochemistry 71, 1350–1359. Ya’u, J., Yaro, A.H., Abubakar, M.S., Anuka, J.A., Hussaini, I.M., 2008. Anticonvulsant activity of Carissa edulis (Vahl) (Apocynaceae) root bark extract. J. Ethnopharmacol. 120, 255–258.
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.com/locate/etp
Antioxidant, cytotoxic and antineoplastic effects of Carissa carandas Linn. leaves Marina Khatuna, M. Rowshanul Habiba, M. Ahasanur Rabbib, Ruhul Aminb, M. Farhadul Islama, ⁎ M. Nurujjamana, M. Rezaul Karima, M. Habibur Rahmana, a b
Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi 6205, Bangladesh Bangladesh Council of Scientific & Industrial Research (BCSIR) Laboratories, Rajshahi, Bangladesh
A R T I C L E I N F O
A B S T R A C T
Keywords: Carissa carandas Leaves Antineoplastic Cytotoxic Antioxidant
For scientific clarification of some traditional uses, this study was designed to explore the antioxidant, cytotoxic and antineoplastic properties of leaf extract of Carissa carandas Linn., a traditional medicinal plant of Bangladesh. The methanol extract of Carissa carandas leaves (MELC) was applied on DPPH and ABTS experiments to determine its antioxidant activity. In vitro the cytotoxic effect of MELC was evaluated against colonic adenocarcinoma cell lines (SW-480 and SW-48) whereas in vivo its antineoplastic property was tested against Ehrlich ascites carcinoma (EAC). The DPPH and ABTS assays revealed the antioxidant activity of MELC with IC50 10.5 ± 1.2 and 1.75 ± 0.3 μg/ml that was comparable to L-ascorbic acid. In vitro cytotoxic study, MELC reduced the viability of adenocarcinoma cells in dose dependent manner and in vivo, administration of MELC (25 mg/kg) resulted in a significant (p < 0.05) decrease in viable EAC cell count thereby increasing the life span of the EAC cell bearing mice. Restoration of hematological parameters such as red blood cells (RBC), hemoglobin and white blood cells (WBC) to normal levels in MELC-treated mice was also observed. Moreover, treatment with MELC induced apoptosis of EAC cells as observed in fluorescence microscopic view of DAPI (4,6diamidino-2-phenylindole) stained cells and also increased p53 gene expression MELC-treated cells in respect to untreated EAC control. In addition, the MELC was rich in polyphenol content and its GC–MS chromatogram confirmed the presence of some compounds all of which showed anticancer and cytotoxic activities in previous studies. In a word, this study supports the use of Carissa carandas in traditional medicine as well as highlights the need to further explore the potentials of MELC as an antineoplastic agent.
1. Introduction Cancer, a group of diseases characterized by uncontrolled growth and spread (invasion and metastasis) of abnormal cells, is considered as the second most common cause of death in the developed world (Tanaka, 1994). A similar picture has also arisen in the developing countries like Bangladesh. Both several external environmental factors (tobacco, chemicals, radiation, and infectious organisms) and/or internal factors (mutations, hormones, and altered immunity) are responsible for neoplastic transformation (Tanaka, 1997). As cancer chemoprevention, the use of natural or synthetic chemical agents is an important strategy to reduce the risk of cancer. Many previous studies have suggested the beneficial effects of various phytochemicals in reducing the risk of cancer (Kuno et al., 2012). So research on the medicinal plants for their antineoplastic effect bears a significant value. Carissa carandas Linn. commonly known as Karamcha, is a common
⁎
herb of Apocynaceae family and it is found throughout all over the Bangladesh. It has a long history of use in traditional system of medicine. The fruits, leaves, barks and roots of Carissa carandas have been used for ethnomedicine in the treatment of human diseases, such as diarrhea, stomachic, anorexia, intermittent fever, mouth ulcer and sore throat, syphilitic pain, burning sensation, scabies and epilepsy (Chanchal et al., 2013). Earlier studies have shown that the extracts of different parts of this plant possesses cardiotonic, antipyretic, anticonvulsant, antidiabetic and antiviral activities (Ya’u et al., 2008; Itankar et al., 2011; Chanchal et al., 2013). Chemical constituents of Carissa carandas include steroids, terpenes, tannins, flavonoid, benzenoids, phenylpropanoid, lignans, sesquiterpenes, and coumarins (Begum et al., 2013). Moreover, this plant is used by traditional practitioner of some districts in Bangladesh for the treatment of cancer. But, no scientific data is available to confirm this folkloric use. Here, the methanol extract of Carissa carandas dried leaves (MELC) was evaluated
Corresponding author. E-mail address: [email protected] (M.H. Rahman).
http://dx.doi.org/10.1016/j.etp.2017.03.008 Received 25 November 2016; Accepted 31 March 2017 0940-2993/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Khatun, M., Experimental and Toxicologic Pathology (2017), http://dx.doi.org/10.1016/j.etp.2017.03.008
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for antioxidant, cytotoxic and antineoplastic activities and its various phytochemical compounds were analyzed by GC–MS. 2. Materials and method 2.1. Plant materials Leaves of Carissa carandas (Linn) plant (Family: Apocynaceae) were used for this study. The plant samples were collected during the month of June-July 2015 from the relevant area of Rajshahi district. This medicinal plant was identified and authenticated by plant taxonomist of Department of Botany, University of Rajshahi, Rajshahi, Bangladesh. Voucher specimen (voucher specimen No. 1150) was deposited at Department of Botany, University of Rajshahi, for future reference. Fig. 1. Phenolic and flavonoid content of MELC.
2.2. Extraction Table 1 Chemical composition of MELC analyzed by GC–MS.
The leaves of Carissa carandas was shade dried and ground into powder. The powder (0.3 kg) was extracted with methanol (Sigma Aldrich, Germany) at room temperature for 7 days. The solvent was completely removed by rotary vacuum evaporator and the crude methanol extract of Carissa carandas leaves (4.4 g) (designated as MELC) was stored in a vacuum desiccator for further use. 2.3. Determination of total phenolic and total flavonoid content The Folin–Ciocalteu method modified by Ranilla et al. (2010) was applied to determine the total polyphenolic content of MELC. One milliliter of sample solution was transferred into a 10 ml volumetric flask and mixed with 6 ml of distilled water. 0.5 ml of Folin–Ciocalteu reagent (50%, v/v) (Labscan, Thailand) was added to sample and mixed. After 5 min, 1 ml of Na2CO3 (5%, m/v) (Sigma Aldrich, Germany) was added to the mixture and adjusted to 10 ml with distilled water. After standing for 60 min at room temperature, the absorbance was measured at 760 nm. Gallic acid (Sigma Aldrich, Germany) was used for constructing the standard curve. The total phenolic content was expressed as gallic acid equivalents (mg/g of dry weight of extract). Total flavonoids content of MELC were estimated using the method described by Ordonez et al. (2006). Here, catechin (Sigma Aldrich, Germany) was used as standard. 1.5 ml of methanol, 100 μl of 10% aluminum chloride (Sigma Aldrich, Germany), 100 μl of 1 M potassium acetate (Labscan, Thailand) solution and 2.8 ml of distilled water was added to 0.5 ml of sample/standard. After one hour 30 min of incubation at room temperature (RT), the absorbance was measured at 420 nm. The samples/standard was evaluated at a final concentration of 0.1 mg/ml. Total flavonoid contents were expressed in terms of catechin equivalent, (mg/g of dry weight of extract).
Sl
Retention time (min)
Compounds
Area (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
3.34 3.41 3.60 4.45 10.80 10.90 11.17 11.64 12.81 13.93 15.65 15.71 26.57 27.72 28.31 29.12 28.84 30.77
Isobenzofuran Ethylbenzene o-Xylene 1,2,4-trimethylbenzene, Card-20(22)-enolide Methyl 4-O-methyl-D-arabinopyranoside n-Decanoic acid 4-O-Methylmannose Hexadecanal Hexadecanoic acid 9,12-Octadecadienoic acid 8,11,14-Docosatrienoic acid Stigmasterol β-Asarone 24-Noroleana-3,12-diene Lupeol Urs-12-en-3-ol Lup-20(29)-en-3-ol Total area =
3.80 0.32 1.02 0.68 2.72 1.86 0.25 10.6 1.20 12.06 1.53 14.20 1.04 0.79 4.91 17.10 0.58 4.89 79.25
2.5. ABTS%+ scavenging capacity ABTS%+ assay was carried out according to the method described previously (Cai et al., 2004). The ABTS%+ solution was prepared by mixing 7 mM ABTS (Sigma Aldrich, Germany) and 2.45 mM potassium persulfate (Carl Roth, Germany) and incubating in the dark at room temperature for 12 h. The ABTS%+ solution was then diluted with water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. ABTS%+ solution (3 ml) was added to 0.1 ml of the test sample with various concentrations (0.625–3.75 μg/ml) and mixed vigorously. The absorbance was measured at 734 nm after standing for 6 min. The ABTS%+ radical scavenging activity of the samples was expressed as
2.4. DPPH% scavenging activity
ABTS%+ scavenging effect (%) = [1 − (Asample/Acont)] × 100
Free radical scavenging activity was determined by DPPH (Sigma Aldrich, Germany) radical scavenging assay as previously described method (Mohsen and Ammar, 2009) with some modification. A solution of 0.1 mM DPPH in methanol was prepared and 3 ml of this solution was mixed with 1 ml of extractives in methanol at different concentrations (6.25–37.5 μg/ml). The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as reference standard. Percentage of DPPH radical scavenging was calculated as DPPH% scavenging effect (%) = [1 − (Asample/Acont)] × 100 EC50 values (μg/ml), the effective amount of the sample needed to scavenge DPPH% by 50%, were determined from the plotted graphs of scavenging activity against the concentration of the extracts.
where Acont is the absorbance of the blank control (ABTS%+ solution without test sample) and Asample is the absorbance of the test sample.
2.6. GC-analysis The chemical composition of MELC was established by gas chromatography mass spectrometry/Quadropole detector analyses using GCMS-QP2010S (Shimadzu Kyoto, Japan) spectrometer. They equipped with a flame ionization detector and capillary column with HP-5MS (30 m × 0.25 mm × 0.25 μm). The temperature program for the column was from 120 °C (1 min) to 230 °C at a rate of 6 °C/min and then held at 200 °C for 35 min. Helium was used as a carrier gas at a flow of 14 psi (Split 1:10), and the injection volume of each sample was 1 μl. 2
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Table 2 Antioxidant properties of MELC. Sample
DPPH% scavenging activity
ABTS% + scavenging capacity
Concentration (μg/ml)
Scavenging effect (%)
EC50 (μg/ml)
Concentration (μg/ml)
Scavenging effect (%)
EC50 (μg/ml)
MELC
6.25 7.5 10 12.5 25 37.5
27.1 43.0 49.0 64.4 87.4 86.9
10.5 ± 1.2
0.625 1.25 2.0 2.5 3.0 3.75
27.1 35.8 57.0 63.2 76.5 88.5
1.75 ± 0.3
Vitamin-C
0.25 0.50 1.00 2.00 4.00 8.00
7.76 18.3 30.0 47.6 68.2 88.1
2.16 ± 0.1
0.160 0.315 0.625 1.25 1.875 2.5
10.8 27.5 41.0 77.0 74.4 97.4
0.78 ± 0.02
Fig. 2. GC–MS chromatogram of MELC. Table 3 Effect of MELC on viable EAC count, survival time and body weight gain of EAC cell bearing mice. Treatment
Viable EAC cells on day 6 after inoculation (x107 cells/mL)
MST (in days)
%ILS
Body weight gain (g) after 15 days
Untreated control EAC + MELC (25 mg/kg) EAC + Bleomycin (0.3 mg/kg)
4.21 ± 0.87 1.36 ± 0.24t
21.5 ± 2.97 30.2 ± 2.86t
– 41.8 ± 4.19
13.7 ± 3.0 8.75 ± 2.1t
0.35 ± 0.07t
39.0 ± 1.85t
81.4 ± 3.97
5.8 ± 0.57
t
Data are expressed as the mean ± S.E.M (n = 8); Significantly different from untreated group: tP < 0.05; MST: Mean survival time; %ILS: Percentage (%) increase in life span.
The database used for the identification of chemical compounds and measurements of peak areas obtained is that of NIST/EPA/NIH MS LIBRARY (NIST 05) and also AMDIS version 2.0 d.
2.7. In vitro cytotoxicity assay against SW480 and SW-48 SW-480 (ATCC CCL228; Duke’s class B/stage II, colonic adenocarcinoma) and SW-48 (ATCC CCL-231; Duke’s class C/stage III colonic adenocarcinoma) were maintained according to ATCC guidelines. 3
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Fig. 3. In vitro cytotoxic effect of MELC against SW-480 and SW-48 cell lines. Table 4 Effect of MELC on hematological parameters of EAC cell bearing mice. Parameters
Normal
Only EAC (Untreated control)
EAC + MELC (25 mg/kg)
EAC + Bleomycin (0.3 mg/kg)
Hgb (g/dL) RBC(×109 cells/mL) WBC(×106 cells/mL)
14.48 ± 1.47 6.67 ± 0.52 8.75 ± 1.12
9.86 ± 0.83* 1.57 ± 0.11* 46.2 ± 4.96*
12.28 ± 1.16t 3.33 ± 0.41t 26.6 ± 4.03t
14.57 ± 0.85t 4.80 ± 0.08t 9.27 ± 0.69t
Data are expressed as mean ± S.E.M for eight animals in each group. * P < 0.05: against normal group. t P < 0.05: against untreated control group.
perature 25 ± 2 °C; humidity 55 ± 5%) with 12 dark/light cycle. They were allowed free access to standard dry pellet diet (collected from ICDDR,B) and water ad libitum. The newly collected mice were given a week to get themselves adapted with the new environment of the laboratory before being employed in any experiment.
Colon cancer cell lines (SW-480 and SW-48) were maintained in Leibovitz’s L-15 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a CO2 incubator. Cytotoxicity assay was performed against SW-480 and SW-48 using a cell counting kit-8 (CCK-8; Sigma–Aldrich) (Amirghofran et al., 2011). Both SW-480 and SW-48 cells were first seeded in flat-bottom 96-well plates at 1 × 104 cells/well and the plate was pre-incubated for 24 h in a humidified incubator (e.g., at 37 °C, 5% CO2). Then cells were incubated with various concentrations (1, 100, 200 and 400 μg/mL) of MELC. Following 24 and 48 h of incubation, 10 μl of CCK-8 solution was added into each well and further incubated for another four hours. The absorbance was measured at 450 nm using a microplate reader. Here untreated cells were used as negative controls and Cisplatin was used as a positive reference standard. The percentage of inhibition was measured as [1 − (optical density of test/optical density of negative control)] × 100. The IC50 value (the concentration of 50% cell inhibition) was calculated from the graph of inhibition percentage against different extract concentrations.
2.9. Tumor cells EAC cells were obtained through the courtesy of Indian Institute for Chemical Biology (IICB), Kolkata, India and were maintained by weekly intraperitoneal (ip.) inoculation of 105 cells/mouse in the laboratory. 2.10. Acute toxicity study The acute toxicity study as previously described (Lorke, 1983) was conducted to determine the LD50 value of MELC in mice. In this method, a single intraperitoneal injection was applied on thirty six animals (6 in each group) at different doses (100, 200, 400, 800, 1600 and 3200 mg/kg body weight) and LD50 was evaluated by recording mortality after 24 h.
2.8. Animals
2.11. Study on in vivo EAC cell growth
This study was carried out using Swiss albino mice of either sex, 3–4 weeks of age, weighing between 20 and 25 gm. They were collected from the Animal Research Branch of the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B). The mice were grouped and housed in plastic cages with not more than eight animals per cage and maintained under standard laboratory conditions (tem-
In order to determine the effect of MELC on EAC cell growth, 24 mice were randomly divided into three groups (8 animals in each group) and 1.5 × 105 cells were inoculated (i.p.) into each mouse of each group on day 0. The group 1 was treated with vehicle (2% 4
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Fig. 4. Fluorescence microscopic view of EAC cells collected from the mice of untreated control (A) and MELC treated (B) groups. Arrows indicating apoptotic features (condensed chromatin and nuclear fragmentation).
2.12. Study on morphological appearance and p53 gene expression
Dimethylsulfoxide; DMSO) and it was considered as untreated control. Mice in group 2 and 3 received (i.p.) MELC (25 mg/kg b.w./day) and bleomycin (0.3 mg/kg b.w./day). Treatment was started and continued for 5 days. The mice were sacrificed on the 6th day after transplantation of EAC cells and cells were collected by repeated intraperitoneal wash with normal saline (0.98% NaCl). Viable EAC cells were counted with a haemocytometer using trypan blue (Sigma–Aldrich) and total number of viable cells per mouse of the treated group was compared with those of control (Osman et al., 2011). During studies on cell growth inhibition, EAC cells were also collected from mice of untreated and MELC treated group after sacrificing for observation of morphological changes of cell by 4,6diamidino-2-phenylindole (DAPI) (Carl Roth) staining and for studying the p53 gene expression by RT-PCR.
Collected EAC cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and then visual images were taken using fluorescent microscopy (Senthilkumar et al., 2008). Fragmented or condensed nuclei were defined as apoptotic cells. PCR specificity was determined using both a melting curve analysis and gel electrophoresis. To analyze the expression of p53 gene by RTPCR (Real time-PCR), TRIzol method was applied to extract total RNA from EAC cells of MELC-treated and untreated groups. Then 3 μg RNA was reversed transcribed into cDNA in a final volume of 20 μl containing100 pmol random hexamer, and 50 U of MuLV reverse transcriptase (New England Biolab) according to the manufacturer’s guideline. Expression of p53 gene was studied using this cDNA as template for PCR. β-actin was used as the housekeeping gene. A 25-μl reaction volume containing 25 pmol each of forwarded and reverse primer, 2.5 mM of each dNTP, and 0.25 U of platinum Taq polymerase 5
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3. Results 3.1. Total phenolic and flavonoid content The amounts of total phenolic and flavonoid content found in MELC are shown in Fig. 1. The total phenolic and flavonoid content of MELC were 137.2 ± 20.7 and 6.55 ± 0.47 mg/g of dry weight of extract, respectively. The results showed that MELC has higher total polyphenolic content. 3.2. Chemical composition of MELC The chemical composition of MELC was characterized by 18 constituents and these constituents were accounted for 79.25% of the total extract. The identified compounds and the chemical compositions of MELC are presented in Table 1 and Fig. 2. The other major components were lupeol (17.10%), 8,11,14-Docosatrienoic acid (14.2%), hexadecanoic acid (12.06%), 4-O-Methylmannose (10.6%), 24-Noroleana-3,12-diene (4.91%) and Lup-20(29)-en-3-ol (4.89%). In addition, MELC also contains card-20(22)-enolide (2.72%), methyl 4-Omethyl-D-arabinopyranoside (1.86%), isobenzofuran (3.80%) and βAsarone (0.79%). 3.3. Antioxidant properties The antioxidant activity of MELC was determined by the DPPH and ABTS test system. As demonstrated in Table 3, the MELC exhibited moderate DPPH% and ABTS%+ radical scavenging ability when compared with vitamin-C and the percentage scavenging effect of MELC was increased with the increasing of its concentration. In DPPH experiment, the EC50 values of MELC and vitamin-C was found to be 10.5 ± 1.2 and 2.16 ± 0.1, respectively, whereas it was 1.75 ± 0.3 and 0.78 ± 0.02 for MELC and vitamin-C in ABTS%+ experiment.
Fig. 5. Effect of MELC on p53 expression of EAC cells. Total RNA extracted from treated and untreated EAC cells were reverse transcribed using random hexamer and PCR reaction was carried out using primers specific for p53 and control gene, β-Actin. (A) Real-time PCR relative quantification of p53 levels in untreated control and MELC-treated EAC cells. (B) PCR reaction products separated on 1% agarose gel stainedwith ethidium bromide. L represents 1 kb DNA ladder; U represents RNA from EAC cells of untreated control; T represents RNA from EAC cells of MELC-treated mice.
(Tiangen, China), was prepared. A BioRad (USA) gradient thermal cycler was used for amplification. The cycling condition was initial PCR activation step of 5 min at 95 °C, followed by 35 cycles of 95 °C/min, 55 °C/min, 72 °C/min, and final 72 °C/10 min (elongation). All the PCR reactions were analyzed in 1.0% agarose gel and GeneRular 1 kb DNA ladder (Fermentus, USA) was used as marker. Moreover, we also measured relative quantities (RQ) of p53 tumour suppressor mRNA of control and treated EAC cells by quantitative real time PCR using a melting curve analysis.
3.4. In vitro cytotoxic activity In vitro cytotoxic assay, MELC had a noticeable activity against SW480 and SW-48 cell lines. Against SW-480, 140.6 ± 5.2 and 108.4 ± 8.3 μg/ml were resulted as IC50 values for MELC after 24 and 48 h treatment whereas 376.6 ± 15.0 and 290.0 ± 11.6 μg/ml were found against SW-48 cell line after 24 and 48 h treatment (Fig. 3.). However, Cisplatin showed the high inhibitory effect against these cell lines with low IC50 values as shown in Fig. 3.
2.13. Study on survival time and heamatological parameters
3.5. In vivo antineoplastic activity against EAC
Mice in three groups (8 animals per group) were inoculated with 1.5 × 105 cells/mouse on the day 0. After 24 h of inoculation, mice in group 1, 2 and 3 were treated (i.p) with 2% v/v DMSO, MELC (25 mg/ kg b.w./day) and bleomycin (0.3 mg/kg), respectively and continued for 10 days. On 15th day after tumour inoculation, hematological parameters (Hemoglobin, RBC, and WBC) were measured from tail vein blood of each mice of each group (Mukherjee, 1988). Then the average body weight changes and mean survival time of each group were noted. The mean survival time (MST) of the treated groups was then compared with that of the untreated control group and percentage increase in life span (%ILS) was calculated according to the previously described formula (Habib et al., 2010).
In acute toxicity study, intraperitoneal administration of graded doses of MELC to Swiss albino mice produced a LD50 of 2860.7 mg/kg body weight. Antineoplastic activity of MELC against EAC cell bearing mice was assessed by the parameters such as viable EAC cell count, mean survival time (MST), percentage (%) increase of life span (%ILS) and body weight gain. The average number of viable tumour cells per mouse of untreated group was found to be (4.21 ± 0.87) × 107 cells/mL. Treatment with MELC (25 mg/kg) and bleomycin (0.3 mg/kg) decreased the viable cells significantly (P < 0.05). The effect of MELC on the survival of EAC bearing mice is shown in Table 3. The MST of the untreated group was 21.5 ± 2.97 days, whereas it was 30.2 ± 2.86 and 39.0 ± 1.85 for the group treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg), respectively. The increase in the life span of EAC cell bearing mice treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg) was found to be 41.8% and 81.4%, respectively (table. 3). On 15th day of tumour cell inoculation, the average weight gain of only EAC cell bearing mice was 13.7 ± 3.0 g whereas it was 8.75 ± 2.1 and 5.8 ± 0.57 for the groups treated with MELC (25 mg/kg) and bleomycin (0.3 mg/kg),
2.14. Statistical analysis All values were expressed as mean ± S.E.M (Standard Error of Mean). Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test using SPSS statistical software of 16 version. P < 0.05 were considered to be statistically significant. 6
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Voller et al., 2010). MELC also contained lupeol and lup-20(29)-en-3-ol in rich amount. Lupeol and its derivatives have already been recognized as dietary chemopreventive compounds for cancer (Chaturvedi et al., 2008; Siddique and Saleem, 2011). So the observed activities of MELC are thought to be a result of the synergistic action of these compounds with antioxidative property.
respectively. In addition, when compared to normal mice, significant changes had been observed in hematological parameters of EAC cell bearing mice on the 15th day after EAC cell inoculation (Table 4). The total WBC count was found to increase with a reduction in the hemoglobin content and total RBC count. At the same time interval, treatment of MELC (25 mg/kg) could change these parameters near to normal. Fluorescence microscopic view of DAPI stained EAC cells demonstrated that MELC induced the alterations (i.e., nuclear condensation, fragmentation, membrane blabbing and apoptosis of cell) in the morphology of EAC cells (Fig. 4A and B). Moreover, the quantitative analysis of p53 mRNA level by RT-PCR showed that p53 gene expression was quite absent in untreated EAC control but it was increased in MELC treated EAC cells (Fig. 5A). The products of RTPCR for MELC treated EAC cells had also given clear band on agarose gel stained with ethidium bromide (Fig. 5B).
Results of this study conclude that the MELC was effective in inhibiting the growth of cancer cells in vitro and vivo conditions. The phytochemical and antioxidants studies were also supported its cytotoxic and antineoplastic properties. Therefore further studies are required to isolate and characterize the active principles of Carissa carandas Linn. leaves which can offer enhanced anti-cancer activity and to establish their mechanism of action.
4. Discussion
Funding
In this study, MELC showed in vitro cytotoxic effect against colonic adenocarcinoma (SW-480 and SW-48) cell lines and in vivo antineoplastic activity against EAC. Generally, prolongation of lifespan of the treated animal and decrease in WBC count of blood are considered as the reliable criteria for evaluating an anticancer drug (Jules Hirsch, 2006). Our results showed an increase in lifespan accompanied by a reduction in WBC count in MECL treated mice. Treatment with MELC produced a significant effect in increasing the life span of ascities tumour bearing animals and also reduced the viable EAC cells in animal models. In addition, MELC restored the hemoglobin content, RBC and WBC cell count to normal values and thereby improving the condition myelosuppression and anaemia which are major complications of cancer chemotherapy (Hogland, 1982). Moreover, fluorescence microscopic view of DAPI stained EAC cells of MELC treated mice demonstrated the apoptosis of EAC cells when compared with untreated EAC control (Fig. 4). Apoptosis (programmed cell death) plays a critical role in normal physiological functions, but there is a dysregulation of apoptosis in cancer. P53 which is one of the most important proapoptotic mediators, is lost or functionally inactivated in more than 50% of human cancers (Hanahan and Weinberg, 2000). Preneoplastic and malignant cells represent a protective mechanism against induction of P53 thereby promoting their proliferation (Mukhtar, 2012). In this study, we had also found that treatment of MELC induced the expression of p53 gene in respect to untreated control (Fig. 5). So this finding indicates that apoptosis induced in MELC treated EAC cell was probably mediated by p53-dependent pathway. However, this study also demonstrated the potent free radical scavenging activity using DPPH% and ABTS%+ experiments (Table 2). The broad range of effects of free radicals has drawn the attention of many scientists in the past decade. It has been proved that free radicals play an important role in the pathogenesis of certain diseases including cancer. (Formica and Regelson, 1995). Plant constituents like polyphenol compounds have shown free radical scavenging or antioxidant activity. Polyphenols are the major plant compounds with antioxidant activity. This activity is believed to be mainly due to their redox properties (Nitin et al., 2010) which play an important role in adsorbing and neutralizing free radicals. Moreover, several studies have showed the biological activities (Cytotoxic, anticancer, antitermitic, antibacterial, nematicidal and insecticidal) of polyphenol rich extracts from various plant species (Kaur and Arora, 2009; Mayaud et al., 2008; Pandey et al., 2009; Park et al., 2007; Seo et al., 2009). In this study, we found that the polyphenolic content of MELC was high in respect flavonoid (Fig. 1). In addition, the GC–MS profile of the MELC confirmed that it contained isobenzofuran, card-20(22)-enolide, methyl 4-O-methyl-D-arabinopyranoside, β-asarone and 24-noroleana-3,12diene all of which showed anticancer and cytotoxic activities in previous studies (Bayer et al., 2005; Wen et al., 2016; Li et al., 2015;
This study was financially supported by the Ministry of Science and Technology, Bangladesh (39.009.006.01.00.049.2013–2014/BS-105/ 105 and 39.009.006.01.00.049.2013–2014/BS-105/535).
5. Conclusions
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