Antitumour and anti-haematotoxic activity of Asparagus racemosus L total dissolved solids in co-administration with cyclophosphamide in mice

2.1 Collection of plant material

Fresh tubers were collected from mature plants growing at Kottakkal (11.0001°N and 75.9988°E), Kerala, India. The tubers were washed to remove all the foreign matter and were mopped dry and split vertically to remove the central ‘rib’ and was then dried under shade. The dried tubers were pulverised and reduced to powder straining through a mesh of size 80. The powder was stored in air tight glass vials till the experiments begun.

2.2 Determination of physico-chemical characters

The physico-chemical parameters of the drug were determined following the standard procedures as explained in the Ayurvedic Pharmacopoeia of India (The Ayurvedic Pharmacopoeia of India, 2008).

2.3 Preparation of aqueous extract

Aqueous extract of Asparagus racemosus (AEAR) was made following the conventional method of preparing a decoction as explained in Ayurvedic Formulary of India. 96 g of the drug powder was boiled in 16 times quantity of water (1536 ml) and was reduced to 1/8th (192 ml). The decoction was strained through a muslin cloth to remove the entire drug from the decoction. The strained decoction was again concentrated under reduced temperature and pressure in a rotary evaporator to obtain a sticky concentrate. The concentrate was then dried on a water bath to obtain dried total dissolved solids (TDS) of the drug. The TDS was powdered and stored in glass vials and kept freezed at −70 °C till the study started.

2.4 Preliminary phytochemical screening

The AEAR was subjected to qualitative tests to determine the major groups of phytochemicals present in them as per the standard procedures (Harborne, 1984).

2.5 Experimental animals

Male BALB/c mice weighing 25 ± 2.3 g were utilized for the study. The animals were procured from the Small Animal Breeding Station, Kerala Veterinary and Animal Sciences University, Thrissur. The animals were housed in standard mice cages with rice husk bedding and were maintained at 25 ± 3.8 °C with a relative humidity of 65 ± 6.5%- and 12-hour dark and light cycle. The animals were fed with standard mouse feed (Lipton India Pvt. Ltd.) and were provided with tap water ad libitum. All experiments were carried out at Amala Cancer Research Centre, Thrissur after obtaining permission from the Institutional Animal Ethics Committee (No: ACRC/IAEC/2016[7]).

2.6 Determination of haemo-protective effect

Twenty-five adult male BALB/c mice weighing 25 ± 5.2 g were taken and divided in to five groups of 5 animals each. Group 1 served as the Normal animals receiving regular diet and Group 2 received Cyclophosphamide (CTX) at 25 mg/kg body weight as intra-peritoneal injection. Group 3 to 5 received AEAR at doses 216, 432 and 864 mg/kg body weight orally along with CTX and they were designated as the Low dose, medium dose and high dose groups respectively. AEAR was given orally through an oral gavage at their respective doses for 15 days. All animals were euthanized after 15 days using ether anaesthesia. Necropsy was performed on the animals and thymus, spleen, liver, bladder and kidney were taken and weighed. Blood was collected from heart puncture and was used for determination of Haemoglobin, Total RBC count, Total WBC count, Total platelet count, Differential count and packed cell volume. Bone marrow cellularity and α-esterase activity was assessed after collecting the bone marrow cells from the femur.

2.7 Determination of haematological parameters

2 ml of blood was collected by heart puncture into a heparinized tube and Total WBC count, Differential count, Total RBC count, Packed cell volume and Total platelet count were assessed using an automated analyser (Sysmex India Pvt. Ltd.).

2.8 Determination of haemoglobin concentration

Haemoglobin concentration was determined spectrophotometrically using Drabkin’s method (Drabkin and Austin, 1935). 20 µl of blood was added to 5 ml of Drabkin’s solution and the resultant colour of cyanmethemoglobin developed was read at 530 nm.

2.9 Determination of bone marrow cellularity

Bone marrow cellularity was estimated after collecting the bone marrow from the animals at the end of the study. Femur of the animals were collected after euthanizing the animals and the marrow cells were collected with a jet of Phosphate buffered saline (PBS) as described by (Sredni et al., 1992). Bone marrow cells were counted using a haemocytometer after making a single cell suspension.

2.10 Determination of α-esterase positive cells

A thin smear of bone marrow was prepared on a glass slide and treated with α-naphthyl acetate and pararosaniline hydrochloride for 45 min followed by Hematoxylin counter staining as described by Bancroft and Cook, 1994).

2.11 Determination of anti-tumour activity

36 adult male BALB/c mice weighing 24 ± 5.5 g were divided into six groups of 5 animals each and were inoculated with DLA tumour cells. Group 1 DLA tumour cells inoculated mice served as control. Group 2 received CTX at 25 mg/kg body weight intra-peritoneally. Group 3 to 5 received AEAR at 216, 432 and 864 mg/kg body weight along with CTX. Group 6 received only AEAR at 864 mg/kg body weight.

2.12 Determination of percentage increase in lifespan (PILS) and mean survival time (MST)

All animals were inoculated with 10⁸ Dalton’s Lymphoma Ascites Tumour cells intra-peritoneally. CTX and AEAR were given at their respective doses to various groups of animals for a period of 10 days. Death of the animals per day was recorded and mean survival time and percentage increase in life span were calculated (Eluru et al., 2015).$$\text{Percentage}\text{Increase}\text{in}\text{Life}\text{Span}=\left(\frac{\text{Mean}\text{survival}\text{time}\text{of}\text{the}\text{treated}\text{group}}{\text{Mean}\text{survival}\text{time}\text{of}\text{the}\text{control}\text{group}}-1\right)\times 100$$$$\text{Mean}\text{Survival}\text{Time}=\frac{\text{Day}\text{of}\text{first}\text{death}+\text{Day}\text{of}\text{last}\text{death}}{2}$$

2.13 Determination of tumour volume

The capability of AEAR to act as an anti-tumour agent was determined by the reduction in volume of the tumour with drug administration (Nalini et al., 2011). The greater (a) and lesser (b) diameters of the tumour was measured using Vernier callipers and the volume was computed using the formula:$$\text{V}=\frac{\frac{4}{3}\pi {\text{a}}^2\text{b}}{2}$$

2.14 Determination of percentage increase in size of tumour

The solid tumour measurements at every fifth day starting from day 15 of tumour cell implant were taken to calculate the percentage increase in tumour size according to the formula:$$\text{PIST}=\frac{\left(\text{Present}\text{tumour}\text{size}-\text{Previous}\text{tumour}\text{size}\right)}{\text{Present}\text{tumour}\text{size}}$$

Similarly, percentage decrease in size of tumour (PDST) was determined by the formula;$$\text{PDST}=\frac{\left(\text{Tumour}\text{size}\text{in}\text{Control}-\text{Tumour}\text{size}\text{in}\text{respective}\text{group}\right)}{\text{Tumour}\text{size}\text{in}\text{Control}}$$

2.15 Determination of percentage increase in body weight

Weight of the animals was recorded every third day and percentage increase was calculated by the formula:$$\text{Percentage}\text{increase}\text{in}\text{Body}\text{Weight}=\frac{{\text{W}}_{\text{n}}-{\text{W}}_0}{{\text{t}}_0}\times 100$$where (W_n − W₀) = Difference in body weight of animal from initial weight (W₀).

2.16 Determination of relative organ weight

At the end of the study, animals were sacrificed and Spleen, thymus, kidney, liver and bladder were harvested from each group and washed with ice cold PBS and blotted dry. The weights of the organs were recorded accurately using a sensitive weighing balance.

2.17 Determination of antioxidant enzyme levels

By the end of the study, a 10% homogenate of liver was made with 10% KCl solution and total protein (Lowry et al., 1951) and level of lipid peroxidation (Ohkawa et al., 1979) was estimated. A portion of the homogenate was used for glutathione estimation (Beutler and Kelly, 1963). The remaining homogenate was used for estimation of SOD (Kakkar et al., 1998) and Catalase (Maehly and Chance, 1954).

2.18 Determination of short-term in vitro cytotoxicity

Trypan blue exclusion assay was utilized to test short term in vitro cytotoxicity (Shrivastava and Ganesh, 2010). Cells were aspirated from the tumour bearing mice from their peritoneal cavity and washed with PBS for three times and the suspension of cells were added to tubes previously added with different concentrations of AEAR and the final volume of the tubes were made up to 1 ml using PBS. A control tube was also maintained containing only the cell suspension. The tubes were incubated for 3 h at 37 °C and trypan blue was added at the end of the incubation and the non-viable cells were counted using a haemocytometer and the percentage cytotoxicity was calculated using the equation:$$\mathit{Percentage}Cytotoxicity=\left[\frac{\mathit{No}.ofdeadCells}{\mathit{No}.oflivecells+No.ofdeadcells}\right]\times 100$$

2.19 Determination of Long-term in vitro cytotoxicity

DLA cells were aspirated from tumour bearing mice and adjusted the concentration of the cells to 1 × 10⁵ cells/mL using sterile PBS. Cytotoxic effect of AEAR was assessed using MTT dye reduction assay (Ramnath et al., 2009).

2.20 Statistical analysis

All data were represented as mean ± SEM. The difference in means between different groups were tested using one-way ANOVA and multiple comparisons between various groups with the control animals were done using Dunnett’s multiple comparison tests. Percentage increase in life span and percentage decrease in tumour size were compared using Kruskal-Wallis test and multiple comparisons with Mann-Whitney U test. 5% was fixed as the level of statistical significance. All statistical tests were performed using statistical software SPSS 16.0.

3.1 Determination of physico-chemical characters

The physico-chemical parameters of the powdered tubers of Asparagus racemosus are tabulated in Table 1.

3.2 Preliminary phytochemical screening

The results of the phytochemical screening of AEAR is as shown in Table 2.

3.3 Determination of haematological parameters

On treating the BALB/c mice induced with hemo-toxicity by treating intra-peritoneally with Cyclophosphamide and subsequent treatment with AEAR at three doses showed statistically significant normalization of the hemogram with that of the control animals. Haemoglobin, haematocrit, total RBC count, MCV, MCH, MCHC, total platelet count, lymphocyte and neutrophils showed equivalent values as compared to the control animals. Only total WBC count showed slight reduction statistically significant at 5% (p < 0.05) as compared to the control animals (Table 3).

3.4 Determination of anti-tumour activity

Anti-tumour activity of AEAR against Dalton’s Lymphoma Ascites (DLA) tumour bearing BALB/c mice was performed by assessing the change in organ weight, body weight (Fig. 1), mean survival time (MST) (Fig. 2), percentage increase in life span (PILS), tumour volume, tumour size (PITS), percentage decrease in tumour size (PDTS), tumour cell growth and non-viable cells. Results are shown in Table 4, Table 5 and Table 7 respectively.

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Fig. 1

Body weight of BALB/c mice treated with Cyclophosphamide and AEAR.

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Fig. 2

Survival of BALB/c mice treated with Cyclophosphamide and AEAR.

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3.5 Determination of mean survival time (MST)

The DLA tumour bearing mice survived on an average to 19.32 ± 0.14 days. Treating with CTX had elevated the survival of the animals to 22.98 ± 0.32 days. The lower dose of AEAR treated animals survived for 22.32 ± 0.99 days while those in Medium dose treated and High dose treated survived up to 23.40 ± 0.39 and 24.33 + 0.15 days respectively. The group of animals treated alone with the high dose of AEAR survived for 23.1 ± 0.42 days. The increase in MSL was significant (p < 0.05) for Medium dose treated and high dose treated animals.

3.6 Determination of percentage increase in life span (PILS)

Considering the life span of animals in tumour control group as zero, the CTX treated animals showed an increase in life span to 18.9 ± 1.5%. Animals treated with Lower dose of AEAR showed an increase in life span of 15.7 ± 5.8% only which was found statistically insignificant. Medium dose of AEAR on co-administration with CTX showed an increase in life span of 21.1 ± 2.3% and higher dose showed an increase of 25.9 ± 0.7% which were found to be statistically significant at p < 0.001. Animals treated with AEAR at high dose alone showed an increase in life span of 13.3 ± 7.8% only which was statistically insignificant.

3.7 Determination of body weight

DLA tumour bearing animals showed significant increase in the body weight after tumour initiation. The maximum weight gain reported was 30.8 ± 1.6%. CTX administration produced a weight gain of 8.9 ± 21.7%. The lower dose of AEAR produced a maximum weight gain of 6.9 ± 1.7% and the medium dose of AEAR produced 2.45 ± 1.78% weight gain. The high dose of AEAR attained a maximum weight gain of 8.22 ± 1.4% and animals received AEAR at high dose alone showed a weight gain of 8.54 ± 1.3%. The results on the body weight of the animals are shown in Table 5 and Fig. 1.

3.8 Determination of antioxidant enzymes

AEAR on administration along with CTX has significantly enhanced the levels of antioxidant enzymes in the animal tissues. AEAR could reduce lipid peroxidation significantly in a dose dependent manner. Levels of glutathione, SOD and CAT were significantly increased by the administration of AEAR (p < 0.001). The results are shown in Table 6.

3.9 Determination of short-term in vitro cytotoxicity by trypan blue dye exclusion method

Cytotoxicity of AEAR on DLA cells was evaluated by Trypan blue dye exclusion method. The viable cells unstained by trypan blue was counted and the percentage cytotoxicity of the tumour cells at different concentrations are shown in Table 7. The co-administration of AEAR along with cyclophosphamide significantly increased the cytotoxicity AT P < 0.001.

3.10 Determination of long-term in vitro toxicity using MTT assay

Cytotoxic effect of AEAR on co-administration with CTX was investigated using MTT assay and the results are shown in Table 7. Cells were treated with different concentrations of AEAR along with CTX and the percentage of cell viability was computed. AEAR significantly inhibited the proliferation of DLA cells which was found to be statistically significant at p < 0.001.