Chemicals and reagents
Reagents and chemicals used for this experiment are all of reagent grade. Trypan blue dye, DAPI, PBS: phosphate-buffered saline, methanol, and ethanol (Sigma, USA); DMSO (Merck, Germany); Solo™ 0.9% (NaCl) IV Infusion (Square Pharmaceuticals Ltd, Bangladesh); Vacutainer® EDTA Tubes (Thomas Scientific).
Collection and identification of plant material
Fresh, young, and mature C. album leaves were collected from Jashore, Bangladesh, in July 2019. The Naimur Rahman (taxonomist and senior scientific officer at National Herbarium, Dhaka, Bangladesh, identified and authenticated the leaves). The accession number for the plant is 46989.
Preparation of plant extract
To remove dirt, collected fresh leaves of the C. album were washed with cold and then hot water to remove any kind of organism. The leaves were dried in a shaded area for 15 days (at room temperature, 25 ± 2 °C) to prevent photo-oxidation of bioactive components and then ground into a fine powder. The leaves fine powder (500 g) extracted by using methanol (99.8%) (3 L) at room temperature (25 ± 2 °C) for 15 days with agitation three times in a day. Finally, filtration performed by using cotton and then no.1 Whatman filter paper. The filtrate is then concentrated by using a rotary evaporator and finally air dried. The extract yield was 6.78% (w/w) with methanol.
Animals
Adult male Swiss albino mice (8-9 weeks old), weighing 28–30 g, were taken for the experiment. We collected mice from the department of pharmacy, Jahangirnagar University, Dhaka, Bangladesh. The animals were kept in standard environmental conditions including relative humidity (RH) 55 ± 5% and temperature (22 ± 2) °C in the Pharmacology lab. The 24 h were divided into 12 h light-dark cycles for 1 week in the animal house for adaptation to a new climate before the experiment. The animals took tap water ad libitum and standard laboratory food. After the completion of the experiment, all the remaining living mice that were used under this laboratory experiment euthanized and anesthetized for their normal death.
The study protocol was approved by the institutional animal ethical committee of Jashore University of Science and Technology, Jashore, Bangladesh. This research work was approved by the Ethical Review Committee of Research cell of Jashore University of Science and Technology, Jashore-7408, Bangladesh (Ref: ERC/FBS/JUST/2019-16).
Tumor cells
Ehrlich ascites carcinoma (EAC) cells were inoculated by Professor Dr. Abu Reza, Protein Science Lab, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh, and EAC cells were maintained by weekly intraperitoneal (i.p.) inoculation of 1 × 105cells/mouse in the laboratory.
Determination of median lethal dose (LD50)
The median lethal dose (LD50) value was determined by the following conventional methods with minor modifications [8]. The C. album leaf extract was dissolved in 0.1% (v/v) dimethylsulfoxide (DMSO) with 0.9% normal saline for performing the experiments and injected intraperitoneally to six groups of mice (n = 6) at various doses (100, 200, 400, 800, 1600, 3200 mg/kg). The median lethal dose (LD50) was determined by taking a record of mortality at the end of the 24 h experiment.
Experimental design
Total mice were subdivided into four groups, in where group I, group II, group III, and group IV were considered and treated with negative control, positive standard, 200 (mg/kg), and 400 (mg/kg) respectively. Group I received only 0.1% (v/v) DMSO (dimethylsulfoxide) and 0.9% normal saline and treated as a control group, group II received standard drug vincristine sulfate 0.3 mg/kg and treated as a standard group, group III and IV received 200 and 400 (mg/kg) C. album leaf extract to identify the potentiality of plant extract as an anticancer drug at lower concentration and treated as experiment group. Treatment of mice was initiated at the end of the 24 h of tumor cells inoculation through intraperitoneally (i.p.) by Professor Dr. Abu Reza (Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh).
Measurement of cell growth inhibition
Cell growth inhibition was measured through conventional methods with minor modifications [2]. Mice of each group (n = 6) were inoculated as 1 × 105 EAC cells in 1 mL concentration (10 μL) with a thin (5 mL) syringe. Treatment was started at the end of the 24 h of EAC cell introduction and carried out 6 days. On the 7th day, the animals were anesthetized using chloroform and from the intraperitoneal cavity of mice, cancer cells were collected, and cells were then diluted with 0.9% NaCl saline water. By using trypan blue stain, different slides were prepared to count viable cells. Finally, viable cells were counted under the microscope using hemocytometer through the following equation:
$$ \%\kern0.5em \mathrm{Cell}\kern0.5em \mathrm{Growth}\kern0.5em \mathrm{Inhibition}=\left(1\hbox{-} \mathrm{Tw}/\mathrm{Cw}\right)\times 100 $$
where,
Tw = mean number of EAC cells in the treated mice
Cw = mean number of EAC cells in the control mice
Apoptosis determination by DAPI staining
Apoptosis determination by DAPI staining of EAC cell was conducted by the conventional methods with minor modifications [2]. On the 7th day, 1 mL EAC cells were collected from each group of mice, and centrifugation was performed for 2 min at 705×g and the plate was washed with PBS (phosphate buffer solution) each time. The obtained cells were then incubated in dark for 10 min with 5 μL DAPI staining solution. The phosphate buffer solution (PBS) was then added to the DAPI containing pellets. The mixer then centrifuged for 2 min at 705×g. The microscopic slide was loaded with 10 μL of the supernatant and 200 μL phosphate buffer solution with the obtained pellet. The morphological change was observed through a fluorescence microscope (Optika, Italy) of EAC cancerous cells.
Assessment of average tumor weight and mean survival time
Assessment of average tumor weight and mean survival time of EAC cell bearing Swiss albino mice was conducted by the conventional methods with minor modifications [9, 10]. At the end of the 24 h of tumor cells, introduction and treatment were continued for 15 days. The tumor weight (g) was assessed through a daily basis weight change record. The survival time of mice was noted and stated as MST (mean survival time) in days and % increase of life span was calculated through the following equation:
$$ \mathrm{Mean}\kern0.5em \mathrm{survival}\kern0.5em \mathrm{time}:\sum \frac{\mathrm{Survival}\kern0.5em \mathrm{time}\kern0.5em \left(\mathrm{days}\right)\mathrm{of}\kern0.5em \mathrm{each}\kern0.5em \mathrm{group}\kern0.5em \mathrm{mouse}}{\mathrm{Total}\kern0.5em \mathrm{number}\kern0.5em \mathrm{of}\kern0.5em \mathrm{mice}}\kern0.5em $$
$$ \%\kern0.5em \mathrm{in}\mathrm{crease}\kern0.5em \mathrm{in}\kern0.5em \mathrm{life}\kern0.5em \mathrm{span}\kern0.5em =\left(\frac{\mathrm{MST}\kern0.5em \mathrm{of}\kern0.5em \mathrm{Treated}\kern0.5em \mathrm{Group}}{\mathrm{MST}\kern0.5em \mathrm{of}\kern0.5em \mathrm{Control}\kern0.5em \mathrm{Group}}\hbox{-} 1\right)\times 100 $$
$$ \mathrm{Average}\kern0.5em \mathrm{Tumor}\kern0.5em \mathrm{Weight}\kern0.5em \left(\%\right)=\left(\frac{\mathrm{Tumour}\kern0.5em \mathrm{weight}\kern0.5em \mathrm{in}\kern0.5em \mathrm{mg}\kern0.5em \mathrm{of}\kern0.5em \mathrm{each}\kern0.5em \mathrm{mouse}\kern0.5em \mathrm{group}}{\mathrm{Total}\kern0.5em \mathrm{Number}}\kern0.5em \right)\kern0.5em \times 100 $$
where Tumor weight in mg = weight after treatment and weight before treatment of mouse
Monitoring of the hematological parameters
The experiment was performed to conduct the hematological profile of EAC cell bearing mice by conventional methods with minor modifications [10]. To assess the anticancer effects of C. album leaf extract for the hematological profile including RBC, WBC, and hemoglobin content. Treatment of mice was initiated at the end of the 24 h of EAC cells inoculation and carried out 6 days. On the 7th day, blood was collected from six mice of each group by tail puncturing and preserved in EDTA tubes. Through the centrifugation, serum was separated from plasma at 2352×g for 10 min and blood was analyzed using Bioanalyzer (Microlab 200) with the help of commercial kits (Atlas Medica, UK).
Brine shrimp lethality bioassay
Brine shrimp lethality bioassay of Chenopodium album leaf extract was conducted by the conventional method with minor modification [11]. Brine shrimps (Scientific name: Artemia salina leach) nauplii considered as a test organism for this study. For this experiment, 16 mg of the test sample (extract) was dissolved in 200 μL of pure dimethylsulfoxide (DMSO) and finally, the volume was made to 20 mL with seawater and obtained stock solution with concentration 800 μg/mL. Then the solutions of various concentrations (400, 200, 100, 50, 25, 12.5, 6.25 μg/mL) were obtained through serial dilution. A total of 2.5 mL of seawater containing pre-marked vials and there 2.5 mL of plant extract solution was added to adjust 5 mL final volume. Each premarked vial contains 10 live brine shrimp nauplii. The experiment was performed twice. At the end of the 24 h, a magnifying glass was used for the counting and recording and the numbered of survived nauplii in each vial.
Statistical analysis
The obtained results were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA followed by Bonferroni test where *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the control group using SPSS software (16, New York, USA). The graph was prepared by using the Graph Pad Prism 8.0.1 version software.