Keywords
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| Aerva sanguinolenta, EAC cell line, Intraperitoneal fluid volume, Vinblastine, Survival time |
Introduction
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| The plant Aerva sanguinolenta family- Amaranthaceae, is a perennial herb. The plant is available in tropical countries of Asia such as Bhutan, India, Nepal, Pakistan and also in China, Malaysia and Indo-China regions. In China it is known as Bai-hua-mi, in Maharashtra (India) as Burval, in Uttarakhand (India) as Sufedphulia and in Assam (India) as Soru-araksan. |
| In folklore medication leaf and flower of the plant were used as wound healing and anti inflammatory for injuries from falls, rheumatic arthritis and pain in muscles [1], the whole plant was used as diuretic and demulcent [2], tender shoot of the plant used as decoction form for galactogue to nursing mother [3] and decoction of whole plant was taken twice a day to expel intestinal worms [4]. |
| Leaves and root of the plant have been used traditionally for body pain and the paste of leaf and root is applied to affected area [5].The plant extract showed significant wound healing property [6]. Other plants of this family have shown antimicrobial [7] and pharmacological properties such as cytotoxic [7], hepatoprotective [8], nephroprotective [9][10] and hypoglycemic [11]. The aim of the present study is to investigate the anticancer property of the plant extracts. Both ethanolic and aqueous extracts were used to evaluate in vivo anticancer activity and ethanolic extract produced profound anticancer activity. Cancer is a disease in which there is uncontrolled proliferation and spread within the body of abnormal forms of the body’s own cell [12]. |
| In developed countries at least one in five of the population can expect to die of cancer. Few categories of medications are commonly used with a narrow therapeutic index and have a greater potential of causing severe side effects. As the synthetic antineoplastic drugs posses comparatively more adverse effects, so herbal drugs are being evaluated as these are comparatively safe or non toxic to the host cell. |
Material and Methods
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| Collection, identification and extraction of the plant material |
| Aerial part of Aerva sanguinolenta was collected in the month of September 2009 from different part of Midnapur district, West Bengal, India and botanically identified and authenticated by the Botanical survey of India, Botanical garden, Howrah, West Bengal, India (Vide Ref. no.-CNH/II/ 44/2009/Tech.II/124).Voucher specimen has been kept in our laboratory for future reference. |
| Thereafter the whole aerial parts was dried under shade and pulverized and extracted chronologically with menstruums in an order from low polarity to high polarity i.e. Petroleum spirit, Chloroform, Ethyl acetate, Ethanol and Water. Fatty or waxy materials and pigments present in the leaves are generally removed by Petroleum spirit [13]. Except aqueous extract, other extracts were dried under reduced pressure at temperature below 40°C through Eyela Rotary Evaporator (Japan) [14] and the dried extract were kept properly. The aqueous extract was evaporated by using Rotating vacuum evaporator. All solvents (Analytical grade) were purchased from Merck (Mumbai). |
| Chemicals |
| The chemicals used were sodium chloride, propylene glycol, trypan blue, methyl violet, sodium sulphate, methylene blue (MERCK Limited, Mumbai, India) and Cytoblastine (Vinblastine Sulphate) from Cipla Limited, Goa, India. All other chemicals and reagents used were of highest analytical grade. |
| Experimental animals |
| Male Swiss albino mice (20-25g) were grouped and housed in poly acrylic cages (38x23x10cm) 6animal/cage under standard laboratory condition (temperature 22-25°C, humidity 40-60% and dark/light cycle of 12 hours). The animals had free access to food (standard dry pellet diet, Hindustan Lever, Kolkata, India) and water ad libitum. The mice were allowed to acclimatize to laboratory condition for 7days before commencement of the experiment. The animals were housed and used accordance with the guidelines of the Institutional Animal Ethical Committee. Vide Reg. No. {(0367/01/C/CPCSEA) India}. |
| Acute toxicity test |
| Male Swiss albino mice were divided into 10 groups of 10 animals each. The control group received 1ml/100g body weight of 0.9 % NaCl I.P. The other groups received 500,750, 1000, 1250, 1500, 1750, 2000, 2250 and finally 2500mg/kg body weight I.P. of ethanolic extract of Aerva sanguinolenta. At a dose of 2500mg/kg body weight all the animals were died within 48hrs but at the dose of 2250mg/kg body weight only half of animals group were died, so this dosage is determined as LD50 of ethanolic extract [15]. |
| Anticancer activity |
| The EAC cell line was maintained in the ascetic form by the sequential passages in Swiss mice, by means of every 10 days i.p transplantations of 2x106 tumor cells. The animals were divided into seven groups of 20 animals in each and labeled accordingly. All the groups except the group-? (normal saline control) were inoculated with 2x106 tumor cells suspended in 0.2 ml of phosphate buffered saline (PBS) solution (pH 7.2-7.4) i.p. The ascitic fluid was collected using a syringe and the tumor cell count was performed in a Neubauer Hemocytometer (MARIE field, Germany), using the Trypan Blue Dye exclusion method [16] [17]. This was considered as day ‘0’. Group ?? represent EAC control. From day ‘1’ to day ‘9’standard drug Vinblastine (1mg/kg body weight), ethanolic extract 200 and 400mg/kg body weight and aqueous extract 200 and 400 mg/kg body weight were administered I.P. to standard group and test groups respectively for nine consecutive days. Half of the mice in each group were weighed and sacrificed on the 10th day after 24hrs of last dose administration and 18hrs fasting by cervical dislocation [18]. Blood was collected directly by heart puncturing. Viable cell, non viable cell, i.p. fluid volume and packed cell volume were determined by I.P fluid collection. Haemoglobin, RBC and WBC counting were performed from blood [19] [20]. Mean survival time and increase in life span was determined by keeping half of animal groups with free access to food and water ad libitum [21] [8]. |
| Tumor volume determination |
| The mice were dissected and ascitic fluid was collected from peritoneal cavity. With the help of graduated centrifuge tubes, I.P. fluid volume, packed cell volume was measured by centrifuging at 1000 rpm for 5min [19][20]. |
| Tumor cell count |
| The ascitic fluid was diluted 100 times, 0.4% trypan blue stain was added, waited for 10 minutes and one drop was placed in Neubauer Hemocytometer (MARIE field. Germany) and the number of viable and non viable cells were counted. The non viable cells (shrinked and crenated) accept the stain and viable cells appear as colorless droplets [19][20]. Here, cell count = (Number of cells × dilution factor) / (Area × thickness of liquid film). |
| Percentage increase in life span |
| Mortality time was noted in standard, test (Ethanolic & aqueous extract) and EAC control groups. Percentage of increase in life span was calculated as follows [22] [23]. ILS (%) = [(Mean survival time of treated group/Mean survival time of control group) -1] ×100 Mean survival time* = (first death + last death)/2 *time denoted by days. |
| Composition of phosphate buffer-saline (PBS) |
| Sodium chloride – 8.00 gm/L Potassium chloride – 0.2 gm/L |
| Disodium hydrogen phosphate −1.44 gm/L Potassium dihydrogen phosphate – 0.24 gm/L |
| Hemoglobin estimation |
| 20μl of heparinized blood was transferred in Sahli’s Hemoglobinometer, diluted and mixed with 0.1 (N) HCL till the color match with the standard color [24]. |
| RBC count |
| Blood sample was diluted 1:100 with RBC diluting fluid, using Thoma pipette. It was mixed properly and a drop of the mixture was dropped gently under the cover slip of the Neubauer Hemocytometer and after the settlement of red corpuscles for three minutes, the number of RBC cells was counted in 5×16 small squares under binocular microscope, Magnus-ICON [19][20]. |
| Composition of RBC diluting fluid |
| for 100ml distilled water) Sodium chloride – 0.5 gm (maintain isotonicity) Sodium sulfate – 2.5 gm (fix the corpuscles) Mercuric per chloride – 0.25 gm (preservative) |
| WBC count |
| Blood sample was diluted 1:20 with WBC diluting fluid, using Thoma pipette. It was mixed properly and a drop of the mixture was added gently under the cover slip of the Neubauer Hemocytometer and after the settlement of white corpuscles for three minutes, the number of WBC cells was counted in 4×16 large squares under binocular microscope [19][20]. |
| Composition of WBC diluting fluid (for 100ml distilled water) Glacial acetic acid – 2cc. (hemolysing RBC) Trypan blue – 0.025 gm (staining the corpuscles) |
| Statistical analysis |
| All data are expressed as mean±S.E.M. (n = 10 mice per groups). Statistical significance (p) calculated by one-way ANOVA between the treated groups and the EAC control followed by Dunnett’s Post Hoc Test of significance where p < 0.05 and p < 0.01 considered to be significant and highly significant, respectively. |
| Result |
| After treating of the Ehrlich ascites tumor bearing mice with ethanolic and aqueous extract of Aerva sanguinolenta at a dose of 200 and 400mg/kg body weight i.p, the effect of ethanolic extract was more prominent. Ascitic cell volume and packed cell volume are reduced. Viable cell count was reduced and non viable cell count was increased compared to that of control group. Life span was also increased comparable to that of the standard drug (Vinblastine) (Table-I). |
| ** (p) < 0.01 while treated group compared to EAC control |
| The ethanolic extract of Aerva sanguinolenta also restored the hematological parameters towards the saline control. The number of RBC count and hemoglobin content also increased (Fig.1 and 2) but the WBC count was decreased as compared to that of EAC control (Fig. 3). |
| Illustration of figures: |
| Figure(1) represent the effect of ethanolic extract of the plant Aerva sanguinolenta on RBC count (million/μl) of each group which highly reduced on EAC cell bearing group and comparatively increased on treated groups with ethanolic extract. |
| Figure(2) represent the effect of ethanolic extract of the plant Aerva sanguinolenta on hemoglobin parameter (g/dl) of each group which in extract treated groups were highly increased compared to that of EAC control group. |
| Figure(3) represent the effect of ethanolic extract of the plant Aerva sanguinolenta on WBC count (thousand/μl) of each group and it shows significant reduction in the number of WBC on extract treated groups compare to that of EAC control group. |
| Discussion: |
| The reliable criteria for judging the value of any anticancer drug are prolongation of life span and decrease the WBC [25]. Usually in cancer chemotherapy the major problem is anemia due to reduction in RBC. Present study indicated that ethanolic extract of the plant has significantly enhanced the erythrocyte count and hemoglobin level when compared to that of EAC control. Viable cell count was decreased and non viable cell count was increased in intraperitoneal fluid by ethanolic extract in a dose dependent manner. There is a complex relationship between the components normally present in the plant extracts. The active components of A.sanguinolenta are likely to be present in the ethanolic extract. [26]. |
| Intraperitoneal fluid volume and packed cell volume was decreased. This indicated a toxic effect on these cells that resulted in cell death. It signifies that the drug was absorbed directly by the EAC cells in the peritoneal cavity and this herbal drug causes the lysis of the EAC cell by direct cytotoxic mechanism [27]. |
| Ethanolic extract normalized hematological parameters like RBC count, WBC count, and hemoglobin level. Thus ethanolic extract possesses protective role in hematological parameters. I.P. fluid accumulation is comparatively less than that of the EAC control group. Preliminary phytochemical study indicated the presence of flavonoid, alkaloids and tannins in ethanolic extract of Aerva sanguimolenta. Flavonoids have been shown to possess antimutagenic and antimalignant effect [28]. Furthermore, flavonoids have a chemopreventive role in cancer through their effect on signal transduction in cell proliferation and angiogenesis [29]. |
| Conclusion: |
| It was found that the ethanolic extract of the plant has potent dose dependent antitumor activity and that is comparable to that of vinblastine. The cytotoxicity and anticancer activity of ethanolic extract of Aerva sanguinolenta is probably due to presence of flavonoids. All of the above data and findings supporting anticancer activity of the plant and increase life span of the tumor bearing object. |
Acknowledgement
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| We express thankful appreciation to “Botanical Survey of India, Shibpur, West Bengal, India” for botanical identification and authentication of the plant (Vide Ref. no.-CNH/II/ 44/2009/Tech.II/124, dated 04th November 2009). |
Tables at a glance
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Figures at a glance
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