2.1 Chemicals and reagents

Hoechst 33342, RPMI 1640-medium and MTT were obtained from Sigma–Aldrich, USA. Penicillin-streptomycin and fetal calf serum were purchased from Invitrogen (USA). Methanol was obtained from Labscan, Thailand. Trypan blue and all other chemicals/reagents were of analytical grade obtained commercially.

2.2 Collection of plant materials and authentication

Rhizome of Kaempferia galanga L. was collected from the hilly areas of Chittagong, Bangladesh. Authentication of the plant material was identified by a taxonomist at the Department of Botany, University of Rajshahi. The voucher sample (No. 06) of this collection was deposited for further reference.

2.3 Preparation of extract

The dried and powdered rhizome of Kaempferia galanga L. was used for the extract preparation with methanol (200 g powder in 500 ml methanol) at room temperature and after filtration, filtrates were evaporated under reduced pressure at 40 °C using a rotary evaporator to have methanol extract (about 2% yield) and it was designated as MEKGR (Methanol extract of Kaempferia galanga rhizome).

2.4 Estimation of total phenolic and flavonoid content

Previously described methods were used to estimate the total phenolic (TPC) and flavonoid (TFC) content of MEKGR (Singleton and Rossi, 1965; Dewanto et al., 2002). TPC of MEKGR was calculated as gallic acid (used as reference standard) equivalents (y = 0.117x + 0.051, R² = 0.998) per gram of dry weight whereas TFC was calculated from the standard curve of catechin (y = 0.005x + 0.047, R² = 0.998) as catechin equivalents per gram of dry weight.

2.5 Determination of total antioxidant and ferrous reducing capacity

The total antioxidant and ferrous reducing potential of the extract/catechin as standard was assessed by the method of Prieto et al. (1999) and Oyaizu (1986).

2.6 DPPH, ABTS and nitric oxide radical scavenging assay

The DPPH free radical scavenging capacity of MEKGR was measured by the method Choi et al. (2000) with some modification whereas the ABTS method was used according to Cai et al. (2004). Nitric oxide scavenging activity of MEKGR extract was carried out as reported by Marcocci et al. (1994) with some modification. The absorbance was taken at 517 nm for DPPH assay, 734 nm for ABTS assay and 546 nm for nitric oxide assay. Catechin was used as reference in all cases. Percentage of DPPH/ABTS/nitric oxide radical scavenging was calculated using the following formula: $$\text{Percentage}(\%)\text{scavenging effect}=[1-({\text{A}}_{\text{sample}}/{\text{A}}_{\text{cont}})]\times 100$$

where A_sample = Absorbance of sample and A_cont = Absorbance of control.

IC₅₀ values (µg/ml) were determined from the plotted graphs of scavenging activity against the concentration of the extract.

2.7 Animals and ethical clearance

Swiss-albino mice of both sexes, average weighing 28–32 g 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 polypropylene cages (maximum six mice per cage). Sterile paddy husk was used as bedding material and the mice were fed with standard mouse food-pellets (collected from ICDDR’B) and water ad libitum. The room was maintained at a temperature of 25 ± 2 °C, humidity 55 ± 5% with 12:12 h light–dark cycle. Protocol used in this study for the use of mice as a 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, (225/320-IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh.

2.8 In vitro cell viability test by MTT colorimetric assay

MTT assay was performed to assess the viability of EAC cells as reported by Kabir et al. (2016). In shortly, the dose-dependent effects of extract on the viability of EAC cells were investigated by MTT assay. The cells (2 × 10⁶) were plated in 200 µl RPMI 1640 media in the presence of various concentrations (3.125–100 µg/ml) of extracts and allowed to stand for 24 h at 37 °C in CO₂ incubator. Following incubation, aliquot were removed and 180 µl of PBS and 20 µl of MTT were added to each well and again incubated at 37 °C for 8 h. After clearing away the supernatant, 200 µl of acidic isopropanol was put into each well of the culture plate and finally incubation was carried out at 37 °C for 1 h. Titer plate reader was used to measure the absorbance at 570 nm.

2.9 Determination of cell growth inhibition (in vivo)

To explore the in vivo cell growth inhibition properties of the extract, three groups of Swiss albino mice (n = 6) weighing 26 ± 3 gm were used (Islam et al., 2016). For this purpose, 1.6 × 10⁶ EAC mice cells were inoculated into each group of mice on the first day. After 24 h of tumor injection treatments were commenced and continued for five days. In brief, groups 1 and 2 were received MEKGR at the doses of 5 and 10 mg/kg/per day, respectively, via intraperitoneal injection. Group 3 was used as untreated control mice. On day 6, mice were sacrificed and viable tumor cells of MEKGR-treated groups were compared with those of control group.

2.10 Survival time and tumor weight

Studies on survival time and tumor weight of extract were carried out using the method of Khanam et al. (2010). In summary, Swiss albino mice were divided into three groups (six in each group) and each group of mice received 1.6 × 10⁶ EAC cells. Group 1 and group 2 mice were administered with MEKGR at 5 and 10 mg/kg/mouse/day, respectively for 10 days whereas group 3 was used as untreated control. Then tumor weight gain and mean survival time of each group were recorded.

2.11 Observation of EAC cells morphological change

The nuclear morphological changes of EAC cells in MEKGR-treated (10 mg/kg/mouse/day) and untreated control mice were investigated using a fluorescence microscope (Olympus iX71, Korea). Briefly, the EAC cells from both groups of mice were collected and stained with Hoechst 33342 at 37 °C for 10 min in the dark. The cells were then washed with PBS. Finally, the morphological changes were studied using a fluorescence microscope.

2.12 Studies on hematological parameters

Studies on hematological profiles of extract were carried out using the method of Islam et al. (2014a). Experimental mice were divided into four groups (n = 6) and each mice were injected with EAC cells (1.6 × 10⁶ cells/mouse) intraperitoneally except the normal group (Group 1). Group 2 was used as control. Group 3 and group 4 mice were administered with MEKGR at 5 and 10 mg/kg/mouse/day, respectively for 10 days. On the 12th day, after tumor transplantation, tail vein blood was collected which was used to measure hematological profile.

2.13 Brine shrimp lethality bioassay

Brine shrimp lethality bioassay was carried out according to published method for the determination of cytotoxic property of the sample extracts (Kabir et al., 2011). Briefly, 3 mg of the extract was prepared in 0.6 ml of distilled water to get a concentration of 5 μg/μl and solution of different concentrations such as 10, 20, 30, 40 and 50 μg/ml were obtained. After 24 h. of incubation the percent of lethality of the brine shrimp nauplii was calculated for each concentration.

2.14 GC–MS analysis of MEKGR

Separation and identification of the components of ethanol extract were performed by GC–MS agilent 6890 N gas chromatography hooked to agilent 5973 N mass selective detector. They equipped with a flame ionization detector and capillary column with HP-5MS (30 m × 0.25 mm × 0.25 μm). In GC settings: the initial oven temperature was set at 60 °C for 1 min and ramped at 10 °C min⁻¹to 180 °C for 1 min and then ramped at 20 °C min⁻¹ to 280 °C for 15 min. The temperature of the injector was controlled at 270 °C. The samples (1 μl) were injected neat, with a split ratio of 1:10. Helium was used as the carrier gas at a flow rate of 1.0 ml min⁻¹. Spectra were scanned from 20 to 550 m/z at 2 scans s⁻¹. Identification of most constituents by gas chromatography was done by comparing their retention indices with those reported in the literature or with those of authentic components available in database.

2.15 Statistical analysis

All results were represented as mean ± standard deviation (SD). SPSS software (version 16) was used to perform one way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test and IC₅₀ was calculated using GraphPad Prism 6. P < 0.05 were considered to be statistically significant.

3.1 Total phenolic and flavonoid contents

The TPC and TFC of MEKGR which were 15.40 ± 0.35 mg of gallic acid equivalent/g of extract and 37.72 ± 0.50 mg of catechin equivalent/g of extract, respectively (Table 1).

3.2 Total antioxidant and ferrous reducing capacity

As shown in Table 1, The MEKGR was found to increase the total antioxidant and ferrous reducing capacities with the increasing concentration of the extract.

3.3 DPPH, ABTS and nitric oxide radical scavenging activity

Table 1 shows DPPH, ABTS and nitric oxide radical scavenging activities of MEKGR and catechin (CA). In each radical scavenging assay, the activity of MEKGR was found to be increased with increasing its concentration. In case of DPPH, ABTS and nitric oxide scavenging assay, the IC₅₀ values of MEKGR were 16.58, 8.24 and 38.16 µg/ml, respectively whereas for catechin these were 2.67, 4.53 and 3.18 µg/ml, respectively.

3.4 Inhibition of cancer cell growth with MEKGR (in vitro)

In MTT assay, MEKGR induced EAC cell death in a dose dependent manner (Fig. 1A). A reduced cell growth was observed with MEKGR at a concentration as low as 3.125 µg/ml which markedly increased with increasing concentration of MEKGR as compared to control. A strong inhibition (76.56%) of EAC cell growth was observed at concentration 50 μg/ml which is further increased (89.37%) at concentration 100 μg/ml of MEKGR. The IC₅₀ value of the MEKGR was determined as 17.10 µg/ml against EAC cell (Fig. 1B).

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Fig. 1

(A) Growth inhibition of EAC cells by MEKGR when EAC cells were treated with various doses of MEKGR for 5 days. The inhibition ratios were measured by the MTT assay (n = 3, Mean ± SD). (B) IC₅₀ value of MEKGR was calculated from the dose–response curve.

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3.5 Cell growth inhibition (in vivo)

Table 2 showed the cell growth inhibition of MEKGR. Maximum cell growth inhibition was observed with the treatment of MEKGR at the dose of 10 mg/kg (i.p.) (70.58% inhibition).

3.6 Average tumor weight and survival time

The effect of MEKGR on survival time at different doses had been summarized in Fig. 2A. Administration of methanol extract at doses 5 and 10 mg/kg in EAC induced mice resulted in increase of life span markedly by 12.95% and 38.39%, respectively, compared with the control (p < 0.05) (Fig. 2B). MEKGR also reduced the tumor weight, compared with control group (p < 0.05) (Fig. 2C).

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Fig. 2

(A) Mean survival time of experimental mice. (B) Percentage of life span increase with the treatment and (C) reduction of body weight gain due to tumor burden at different doses. Data are expressed as mean ± standard deviation (n = 6). Level of significance ^*p < 0.05 when compared with that of control group.

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3.7 Effect of MEKGR on cell morphological change

Hoechst 33342 staining was performed to confirm the methanol extract induced apoptosis of EAC cells and the results were presented in Fig. 3. In this morphological examination, the control cells were round, regular, and homogeneously stained with Hoechst 33342 (Fig. 3A) while treated EAC cells exhibited membrane and nuclear fragmentation a hall mark of apoptosis (Fig. 3B). These results indicate that the extract could induce apoptosis of EAC cells.

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Fig. 3

MEKGR induced apoptosis is in Ehrlich ascites carcinoma (EAC) cell. EAC cells were treated for 24 h then cells were collected from the treated and non-treated EAC-bearing mice and stained with Hoechst 33342 and observed by fluorescence microscopy. Left panel (A) indicates control and right panel (B) indicates MEKGR (10 mg/kg/day) noted that apoptotic characteristics e.g. nuclear condensation and fragmentation are seen in figure B.

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3.8 Hematological studies

Effects of the methanol extract on the hematological profile of the EAC cell bearing mice were examined and the results were presented in table 3. Administration of EAC cells resulted in the reduction of Hb content and RBC counts, whereas an increase was found in WBC counts. Interestingly, the EAC-bearing mice restored their hematological profiles moderately after administration of the extract.

3.9 Brine shrimp lethality bioassay

The brine shrimp lethality bioassay was carried out to assess the in vitro cytotoxic effect of MEKGR. Percent of mortality of nauplii were increased with the increasing concentration of MEKGR and the medium lethal concentration (LC₅₀) was found to be 11.37 µg/ml.

3.10 Chemical profile of MEKGR analyzed by GC-MS

The chemical profile of MEKGR extract that was identified by GC–MS spectrum, are summarized in table 4. A total of eight components, 2-propenoic acid (10.18%), phthalic acid (3.37%), palmitic acid (35.17%), sandaracopimaradiene (8.20%), oleic acid (22.15%), octadecanoic acid (10.10%), 2-[2-(4-nonylphenoxy)ethoxy]ethanol (3.57%) and glycidyl stearate (7.27%) were identified in the methanol extract and the presence of these compounds had a significant correlation with the inhibition of EAC cell growth (R² = 0.078, p < 0.05) in vitro.