Abstract
Context: In India, Dregea volubilis (L.f.) Benth. ex Hook.f. (Asclepediaceae), a large twining shrub with a woody vine, is used to treat tumors traditionally.
Objective: This study evaluated the in vitro and in vivo antitumor activity of the methanol extract of Dregea volubilis leaves (MEDV) and elucidated its possible mechanism of action.
Materials and methods: In vitro antitumor activity of MEDV was evaluated against Ehrlich ascites carcinoma (EAC) cell-line. In vivo antitumor and antioxidant activity of MEDV at three dose levels (50, 100, and 200 mg/kg) were determined against EAC tumor-bearing mice. After 24 h of EAC inoculation, the extract was administered for 9 consecutive days. After the administration of the last dose on the 9th day followed by 18 h fasting, mice from all groups were sacrificed to determine antitumor activity and hematological profiles along with liver related biochemical parameters like lipid peroxidation, antioxidant enzymatic activity, etc.
Results: For in vitro antitumor activity, IC50 value of MEDV for EAC tumor cells was 85.51 ± 4.07 µg/ml. The MEDV showed a decrease in tumor volume, packed cell volume and viable cell count and an increase in the non-viable cell count of the EAC tumor-bearing mice (p < 0.001). Hematological profile reverted near to normal level in extract treated mice. MEDV decreased the hepatic lipid peroxidation level and enhanced superoxide dismutase and catalase level in tumor-bearing mice (p < 0.001).
Discussion and conclusion: MEDV exhibited in vitro and in vivo antitumor activity in EAC tumor-bearing mice mediated through augmenting antioxidant defense system.
Introduction
India has a rich tradition of using medicinal plants to treat different diseases. Plant derived secondary metabolites such as alkaloids, flavonoids, terpenoids, saponins, tannins, etc, are responsible for different pharmacological activities such as antidiabetic, analgesic, anti-inflammatory, antioxidant, anticancer, antidiarrheal, antiviral, etc. (CitationGupta et al., 2004a). It is true that several plant metabolites (paclitaxel, vincristine, podophyllotoxin, and camptothecin) are now available as drugs of choice for the treatment of cancer. In spite of this, there is a lack of truly effective drugs for various forms of cancer, and it is still a fatal disease rating in the top three causes of death (CitationKumarappan & Mandal, 2007). Many of the chemotherapeutic agents sold for the treatment of cancer are highly expensive, mutagenic, carcinogenic, teratogenic, and bone marrow depressants, which limit their application. Therefore, active research for the discovery of effective anticancer drugs is still needed. Thus, efforts are being made to identify naturally occurring anticarcinogens, which would, either prevent, slow down, or otherwise reverse the cancer development process (CitationKumarappan & Mandal, 2007).
In Ayurveda, Dregea volubilis (L.f.) Benth. ex Hook.f. (Asclepediaceae) [Synonym: Wattakaka volubilis (L.f.) Stapf.; Asclepias volubilis L.f.] is extensively used to treat inflammation, piles, leucoderma, asthma, tumors, urinary discharges, etc. (CitationKirtikar & Basu, 1935). It is a large twining shrub with a woody vine, widely distributed in India, Sri Lanka, Myanmar, Indonesia, Thailand, and China. The leaves and flowers are used as vegetables. Drevogenin D and kaempferol have been isolated from the leaves, whereas dregeosides, hyperoside, drevogenin A and P, drebbysogenin were isolated from its seed, stem, and root (CitationAnon, 1976; CitationYoshimura et al., 1983, Citation1985). CitationSahu et al. (2002) isolated volubilioside A, B, C and drevogenin D and P from its flowers. Dregeoside Ap1 and dregeoside A01, isolated from stem of the plant, showed antitumor activity against melanoma B-16 (CitationYoshimura et al., 1983). Taraxerone isolated from chloroform extract of the fruit of the plant show in vitro antitumor and antileishmanial activity (CitationMoulisha et al., 2009, Citation2010). Aqueous and ethanol extracts of its leaves, exhibited good and sustainable in vitro rancid inhibition ability (CitationTangkanakul et al., 2005). It was reported earlier by our group that the methanol extract of Dregea volubilis leaves was non-toxic up to an oral dose of 2 g/kg for mice (CitationHossain et al., 2010).
Based on a literature search, no report on antitumor activity of the leaves of Dregea volubilis was found. The purpose of the present work was to investigate in vitro and in vivo antitumor activity of methanol extract of Dregea volubilis leaves (MEDV) and to establish its possible mechanism of action.
Material and methods
Plant material
The fresh leaves of Dregea volubilis were collected from Ramchandrakhali, District of South 24 Parganas, West Bengal, India, on 11th November, 2008 by Emdad Hossain (First author) and its botanical identification was done by Dr H. J. Chowdhery, a taxonomist and Joint Director of the Central National Herbarium (CNH), Botanical Survey of India, Howrah, India (Voucher specimen no. CNH/1-I(54)/2004-Tech II/886). The voucher specimen was deposited in the herbarium of the Department of Pharmaceutical Technology of Jadavpur University, Kolkata, India, for future reference. The leaves were dried in shade, powdered, and stored in an air-tight container for further use.
Preparation of extracts
The powdered leaves of Dregea volubilis were extracted with methanol in a Soxhlet apparatus. The solvent was removed in a rotary vacuum evaporator first, and then subsequently in a vacuum oven at 45°C to get the crude extract (MEDV, 27%, w/w), which was kept in a desiccator prior to use for the in vitro and in vivo studies.
Test animals
Swiss albino mice of either sex weighing 18–22 g were used. They were acclimatized for one week before the experiment and maintained under controlled conditions (temperature, 25 ± 2°C along with a light and dark cycle of 12 h each) and were fed a standard pellet diet along with water ad libitum. Approval from the Institutional Animal Ethical Committee, Pharmacy College, Azamgarh, India (937/c/06/CPCSEA) was achieved prior to this study.
Cell lines
Ehrlich ascites carcinoma (EAC) cell line was obtained from the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. The EAC cells were maintained by intra-peritoneal inoculation of 1 × 106 viable cells/mouse.
Chemicals
All the chemicals were of analytical grade. Ethylene diamine tetraacetic acid (EDTA), pyrogallol, hydrogen peroxide, sodium di-hydrogen orthophosphate, potassium dihydrogen orthophosphate, ferric chloride, hematoxylin, trichloroacetic acid and thiobarbituric acid were obtained from SD Fine Chem (Mumbai, India). Methanol was obtained from CDH (India).
In vitro cytotoxicity
The in vitro cytotoxicity study was done with MEDV using EAC cell line. For this, 0.1 ml of 1 × 106 viable EAC cells were suspended in 0.1 ml of phosphate buffered saline (PBS, 0.02 M, pH 7.4) and mixed with various concentrations of extracts (0–1000 µg/ml) in phosphate buffer to make final volume to 1 ml and incubated for 3 h at 37°C. After incubation, cell viability was determined using trypan blue method. Percent in vitro cell viability was calculated in comparison to the treated control (CitationAjith & Janardhanan 2003). Similar method was followed for the 5-flurouracil (FU, standard) (CitationGupta et al., 2004a).
In vivo antitumor study
Male Swiss albino mice were divided into 6 groups (n = 6). Antitumor activity of the extract was measured by slightly modifying the method described by CitationGupta et al. (2004b). Briefly, all the groups were injected with EAC cells (0.2 ml of 2 × 106 cells/mouse) intra-peritoneally (i.p.) except the normal group. This was considered as day zero. From the first day 5% v/v aqueous propylene glycol (5 ml/kg/mouse/day) were injected to normal and EAC control groups respectively for 9 days intra-peritoneally. Similarly, MEDV at different doses (50, 100, and 200 mg/kg/mouse/day in 5% v/v aqueous propylene glycol) and standard drug 5-flurouracil (20 mg/kg/mouse/day) were administered in groups 3, 4, 5, and 6, respectively. After the administration of the last dose followed by 18 h fasting, mice from all groups were sacrificed to determine the antitumor activity of the drug and also to evaluate its effect on hematological and liver related biochemical parameters.
The antitumor effect of MEDV was assessed by observation of changes with respect to body weight, ascites tumor volume, packed cell volume along with viable and nonviable tumor cell counts. Hemoglobin content, red blood cell (RBC) and white blood cell (WBC) counts were measured from freely flowing tail vein blood (CitationGupta et al., 2004b).
After the collection of blood samples, the mice were sacrificed and their livers were excised, rinsed in ice-cold normal saline followed by cold 0.15 mol/L Tris-HCl buffer (pH 7.4), blotted dry, and weighed. A 10% w/v homogenate was prepared in 0.15 mol/L Tris-HCl buffer (pH 7.4) and a portion utilized for the estimation of lipid peroxidation. The remaining homogenate was centrifuged at 1500 rpm for 15 min at 4°C. The supernatant thus obtained was used for the estimation of superoxide dismutase and catalase (CitationGupta et al., 2004b).
Determination of superoxide dismutase and catalase activities
For the biochemical analyses of these enzymes, livers of EAC-bearing mice were homogenized in phosphate buffer (pH 6.5). Superoxide dismutase (SOD) activity was measured in both tissues by the ability of this enzyme to inhibit pyrogallol auto-oxidation using a spectrophotometer at 420 nm (CitationHodgson & Fridovich, 1975). The amount of enzyme that inhibited the reaction by 50% (IC50) was defined as one unit of SOD, and the enzyme activity was expressed in units of SOD per mg of protein. Catalase (CAT) activity was measured according to CitationTakahara et al. (1960). The reaction was monitored at 240 nm in spectrophotometer for liver samples to detect H2O2 degradation per minute.
Determination of lipid peroxidation rate in liver
Lipid peroxidation (LPO) rate was measured according to the method of CitationJordan and Schenkman, (1982). Liver samples were homogenized in 0.1 M phosphate buffer saline and then centrifuged at 5000 × g at 4°C for 5 min. To 100 µl separated microsomes in 0.1 M phosphate buffer saline, 1 ml of 28% aqueous trichloroacetic acid was added, and centrifuged at 2000 × g at 4°C and for 20 min. Supernatant (1 ml) was separated and 900 µl of 1% aqueous thiobarbituric acid was added and the volume was adjusted to 3 ml by using phosphate buffer (pH 7.0). It was heated on a water bath for 60 min and then cooled in ice bath. The absorbance was measured at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction co-efficient (1.56 × 105) of malondialdehyde.
Statistical analysis
All results are expressed as the mean ± S.E.M. The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey test using computerized Graph Pad InStat version 3.06, Graph Pad Software, USA. Here, p < 0.05 was considered as statistically significant.
Results
In vitro cytotoxicity assay
The methanol extract of Dregea volubilis leaves (MEDV) showed marked cytotoxic activity. The concentration of MEDV required for 50% death of the EAC cell lines (IC50) was found to be 85.51 ± 4.07 µg/ml; values were expressed as mean ± SEM (n = 3). Cell viability was decreased with increase of MEDV concentration in dose dependent manner (p < 0.001) (). The standard drug, 5-fluorouracil, showed significant cytotoxic activity (IC50 = 37.96 ± 3.73 µg/ml; p < 0.001) ().
Table 1. In vitro effect of methanol extract of Dregea volubilis leaves (MEDV) on EAC-cell line.
Effect of MEDV on tumor growth
MEDV at the doses of 50, 100, and 200 mg/kg body weight reduced the four parameters, namely, body weight, tumor volume, packed cell volume, and viable tumor cell count in a dose-dependent manner as compared to that of EAC control group (p < 0.001). Furthermore, non-viable tumor cell counts at different doses of MEDV were also found to decrease when compared with the EAC control (p < 0.001) ().
Table 2. Effect of methanol extract of Dregea volubilis leaves (MEDV) on body weight, tumor volume, packed cell volume, viable and non-viable tumor cell count of EAC-bearing mice.
Effect of MEDV on hematological parameters
Hemoglobin content and RBC count in the EAC control group were decreased significantly (p < 0.001) as compared to normal group. Treatment with MEDV at the dose of 50, 100, and 200 mg/kg increased the hemoglobin content and RBC count to near normal level. The total WBC count was increased in EAC control group when compared with normal group. Administration of MEDV at doses of 50, 100, and 200 mg/kg in EAC-bearing mice reduced WBC count as compared with EAC control (p < 0.001). Treatment with MEDV at different doses changed these altered parameters to near normal ().
Table 3. Effect of the methanol extract of Dregea volubilis leaves (MEDV) on hematological parameters of EAC-bearing mice.
Effect on antioxidant enzymes (SOD and catalase)
shows that the superoxide dismutase level in the liver of EAC-bearing mice was significantly decreased by 46.5% in comparison to the normal group (p < 0.001). Administration of MEDV at doses of 50, 100, and 200 mg/kg increased the level of SOD by 46.4, 75.7, and 79.7%, respectively, as compared to that of the EAC control group (p < 0.001), whereas 5-fluouracil (standard) at the dose of 20 mg/kg increased the SOD level to 80.9% (p < 0.001). The liver catalase level in the EAC control group was significantly decreased by 45.6% in comparison to the normal group (p < 0.001). Treatment with MEDV at doses of 50, 100, and 200 mg/kg increased catalase levels by 32.8% (p < 0.05), 45.7% (p < 0.01), and 54.3% (p < 0.001), respectively, when compared to that of the EAC control; the standard increased catalase levels by 58.6% (p < 0.001) ().
Table 4. Effect of the methanol extract of Dregea volubilis leaves (MEDV) on liver enzymes of EAC-bearing mice.
Effect of MEDV on lipid peroxidation
Experimental results revealed that the level of lipid peroxidation in liver tissue was increased by 36.2% in EAC control group as compared to the normal group (p < 0.001). After treatment of the EAC-bearing mice with MEDV at different dose levels (50, 100, and 200 mg/kg), the lipid peroxidation values were reduced by 9.3% (p > 0.05), 15.6% (p < 0.01) and 17.5% (p < 0.01), respectively, in comparison to EAC control group ().
Discussion
The present study evaluated in vitro cytotoxicity and in vivo antitumor activity of MEDV on EAC-bearing mice along with its effect on lipid peroxidation and antioxidant status. The in vitro study clearly indicated that MEDV effectively inhibited EAC cells at a low dose (i.e., IC50 = 85.51 ± 4.07 µg/ml), which helped further in vivo studies. The in vivo study revealed that the MEDV-treated animals at the doses of 50, 100, and 200 mg/kg significantly inhibited the tumor volume, packed cell volume, tumor cell count and also modified the hematological parameters to near normal level. The extract reduced the hepatic lipid peroxidation in EAC-bearing mice. Additionally it enhanced the level of antioxidant enzymes such as SOD and catalase in tumor-bearing mice.
It was observed that the ascites tumor volume increased rapidly in EAC-bearing mice in subsequent days. Ascites fluid is the direct nutritional source for tumor cells and faster increase in ascites fluid will help for further growth of tumor cells (CitationGupta et al., 2004b). MEDV significantly reduced ascites tumor volume, packed cell volume and tumor cell count in the EAC-bearing mice. It may be expected that MEDV arrested the tumor growth by decreasing the nutritional fluid volume of EAC-bearing mice.
In cancer chemotherapy, the major problems encountered are myelo-suppression and anemia. The anemia in EAC-bearing mice may be due to reduction of hemoglobin level or RBC count (CitationPrice & Greenfield, 1958). MEDV brought back the hemoglobin content, RBC and WBC count near to normal value.
The improper balance between ROMs (Reactive Oxygen Metabolites) and antioxidant defenses results in oxidative stress. It disturbs the cellular functions leading toward various pathological conditions, including cancer (CitationBandyopadhyay et al., 1999). Overproduction of ROMs induced by different endogenous and exogenous mechanism in the body may exhaust the antioxidant system of cells and contribute to a number of destructive processes and diseases, including cancer (CitationKandasamy et al., 2005). Excessive production of free radicals also resulted in oxidative stress and leads to damage of macromolecules such as lipids, and, thus, can stimulate lipid peroxidation in vivo. Malondialdehyde (MDA), the oxidative end product of lipid peroxidation, was reported to be at a higher level in cancer tissues than in unaffected organs (CitationYagi, 1987). CitationDeWys (1982) reported that the presence of tumors in the human body or in experimental animals affected many functions of the important organs, especially the liver, even when the site of the tumor does not obstruct directly the organ function. From the present experimental results it was found that MEDV at doses of 100 and 200 mg/kg significantly reduced the elevated levels of lipid peroxidation in EAC-treated mice. It might be expected that the phytoconstituents of MEDV reduced the free radical quenching property in the tumor cells due to the antioxidant effect ().
It is known that antioxidant enzymes SOD and catalase provide defense mechanism against damaging effect of superoxide and hydrogen peroxide specifically to DNA. SOD converts superoxide to hydrogen peroxide in conjunction with two other antioxidant enzymes, CAT and peroxidase (CitationLi & Oberley, 1997). An earlier report found that a decrease in SOD activity in EAC-bearing mice may be due to loss of Mn2+-containing SOD activity in tumor cells and the loss of mitochondria, leading to a decrease in total SOD activity in the liver (CitationGupta et al., 2004b). CitationMarklund et al. (1982) reported that inhibition of SOD and catalase help tumor growth. Similar effects were found in the present investigation with EAC-bearing mice. The administration of MEDV at different doses increased the SOD and CAT levels in a dose-dependent manner.
Plant derived extracts containing antioxidant principles showed cytotoxicity toward tumor cells and antitumor activity in experimental animals (CitationGupta et al., 2004b). We propose that the additive and synergistic antioxidant activity of phytochemicals, such as saponins, triterpenoids, steroids, etc., found in MEDV in our earlier study (CitationHossain et al., 2010), may be responsible for its potent antitumor activity.
Conclusion
The results obtained demonstrate significant antitumor activity of methanol extract of Dregea volubilis leaves. It reduced body weight, tumor volume, and viable cell count in EAC-bearing mice. MEDV modified hemoglobin, RBC and WBC levels near to normal. The increase in hepatic SOD and catalase levels with lowering of lipid peroxidation in EAC-bearing mice after MEDV treatment indicated the potential activity of MEDV as an inhibitor of intracellular oxidative stress. Further investigations are in progress in our laboratory to identity the active principles involved in this antitumor and antioxidant activity.
Acknowledgments
We are thankful to Mr. Debobrata Devbhuti and Ashish Bala, Jadavpur University, Kolkata, India for their technical help.
Declaration of interest
There is no conflict of interest to report.
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