Salla Pujitha1*, A. Jaswanth2, A. Ganesh3, Irfana Begum4, Noor Jahan5
1Assistant Professor, Department of Pharmacology, Jyothishmathi College of Pharmacy, JNTUH.
2Principal, Department of Pharmacology, Surabhi Dayakar Rao College of Pharmacy, JNTUH.
3Associate Professor, Department of Chemistry, Surabhi Dayakar Rao College of Pharmacy, JNTUH.
4Assistant Professor, Dept. of Pharmacology, Surabhi Dayakar Rao College of Pharmacy, JNTUH.
5Assistant Professor, Department of Quality Assurance, Jyothishmathi College of Pharmacy, JNTUH.
*Corresponding Author E-mail: sallapujitha@gmail.com
ABSTRACT:
PURPOSE-The objective of present study is to explore the anticancer activity of the Ethanolic Extract of the Maytenus emarginata (EEME) in Swiss albino mice against Ehrlich ascites Carcinoma (EAC) cell-line. METHOD-The dose of 100 and 200 mg/kg body weight EEME was administeredintra-peritoneally for nine consecutive days. Twenty-four hours of last dose and 18hrs of fasting, the mice were sacrificed and antitumor effect of EEME was assessed by evaluating tumor volume, viable and non-viable tumor cell count, tumor weight and hematological parameters of EAC bearing host. RESULTS-EEME showed significant decrease in (p<0.01) tumor volume, viable cell count, tumor weight and elevated the life span of EAC tumor bearing mice. Hematological profile such as RBC, hemoglobin, WBC and lymphocyte count reverted to normal level in EEME treated mice. CONCLUSION-From the result it was showed that the extract has potent dose dependent anticancer activity and that is comparable to that of 5-fluorouracil.
KEYWORDS: Maytenus emarginata, Ehrlich Ascites Carcinoma (EAC), tumor volume, viable cell count, tumor weight.
INTRODUCTION:
Botanical Classification:
Kingdom: Plantae
Phylum: Magnoliophyta
Class: Magnoliopsida
Order: Celastrales
Family: Celastraceae
Genus: Maytenus
Botanical Description:
The members of Celastraceae family generally are trees and shrubscomprising about 55 genera and 850 species that are sometimesclimbing or vining. Maytenus emarginata is a small, compact tree, 3‐5 meter high; young branches purple, often spiny, with leaves andflowers on the spine.
Chemotherapy is a kind of treatment that uses drugs to attack cancer cells. It is called a "systemic treatment" since the drug entering through the blood stream, travels throughout the body and kills cancer cells at their sites. The drugs may rarely be intended to have a local effect, but in most cases, the intention is to destroy cancer cells wherever they may exist in the body. Chemotherapeutic drugs are chemically designed to target cells that are dividing and growing rapidly(11,13). Once they reach the cancer cells, they act to retard their growth, eventually resulting in their destruction. Chemotherapy works by stopping or slowing the growth of cancer cells, which grow and divide quickly. The era of cancer chemotherapy began in the 1940s with the first use of nitrogen mustards and folic acid antagonist drugs. Cancer drug development has exploded since then into a multi‐billion dollar industry(4,7,9,10,14).
Plants of this family generally grow as small trees, bushes or lianas and have resinous stems and leaves. They have been valued since antiquity because their extracts have useful medicinal properties.
The crude plant extracts of the Celastraceae in traditional medicine and agriculture is astonishing, and includes stimulant, restorative,male contraceptive, anti‐tumor, anti‐leukemic, anti‐bacterial,insecticidal and insect repellent activities(1,2,12).
It is an evergreen tree that tolerates various types of stresses of the desert, locally known as “Kankero” in Hindi, “Thorny staff tree” in English.Traditionally species of Maytenus has been used for fever, asthma, rheumatism and gastrointestinal disorders worldwide. Recently some biomolecules from Maytenus species has been reported to be active against HIV‐Protease.
(a)
(b)
Figure A. Maytenus emarginata (Willd.) a. Plant b. Leaf
Various parts of this plant contain immense medicinal properties which are mentioned under:
Root- Used in gastrointestinal troubles, especially dysentery.
Stem- Tender shoots of the plant help for mouth ulcer. The bark is ground to a paste and applied with mustard oil to kill lice in the hair.
Leaf‐ Pulverized leaves of Maytenus emarginata are given in milk to children as a vermifuge. A decoction of the leafy twigs is used as a mouthwash to relieve toothache. Ash of leaves used to heal up sores and wound gives cooling effect. The leaves are burnt and mixed with ghee to form an ointment used to healsores.The tender leaves are chewed raw in the treatment of jaundice.
Fruit‐The fruits are used in medicines to purify blood.
ECOLOGY:
Grows at elevations from near sea level and locally abundant on the coast on sand, at the edge of mangrove forest or secondary forest, at forest margins, in thickets on branches and hillsides and on sea cliffs, often on limestone. Long, hot summers are needed for production of flowers and fruits.
PROPAGATION:
Maytenus emarginata is an out breeding tree therefore it shows great variability. Sow seed under glass in season of autumn. Remove suckers, which may appear at some distance from the parent plant in spring season. Root semi‐ripe cuttings with bottom heat in summer.
GROWTH PATTERNS:
Maytenus emarginata (Willd.) grow in moderately fertile, moist but well‐drained soil in full sun with midday shade. Fruit appears in January to February and fruit ripens start from March to April. Flowers appears in October to January. Develop new leaves from June to August. The best season for collecting of this plant for medicinal uses is monsoon.
MATERIALS AND METHOD:
CHEMICALS:
The chemicals used were sodium chloride, propylene glycol, trypan blue, methyl violet, sodium sulphate, methylene blue, 5-flurouracil.All the chemicals and reagents used were of highest analytical grade.
ANIMALS:
Male Swiss albino mice weighing 20-22g were taken. The mice were grouped and housed in poly acrylic cages (38cm x 23cm x 10cm) with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25 +2oc and dark/light cycle 14/10h).They were allowed free access to standard dry pellet diet and water ad libitum. The mice were acclimatized to laboratory conditions for 7days before commencement of the experiment. All procedures described were reviewed and approved by the institutional Animal Ethical Committee(6,8).
TRANSPLANTATION OF TUMOR:
EAC cells were obtained from Amala Cancer Research Institute, Thirussur, India. The EAC cells were maintained in vivoin Swiss albino mice by intra-peritoneal transplantation of 2 x 106 cells per mouse after every 10 days. Ascitic fluid was drawn out from EAC tumor bearing mouse at the log phase (days 7-8 of tumor bearing) of the tumor cells. Each animal received 0.1 ml of tumor cells suspension containing 2 x 106 tumor cells intraperitoneally.
TREATMENT SCHEDULE:
100 Swiss albino mice were divided into five groups (n = 20) and given food and water ad libitum. All the animals in each groups except Group – I received EAC cells (2 x 106 cells/mouse i.p.)This was taken as day 0 .Group-I served as normal saline control (5 ml/kg i.p.) and Group-II served as EAC control. 24-h after EAC transplantation, Group-III and Group-IV received ethanol extract of Maytenus emarginata (EEME) at a dose of 100 and 200 mg/kg i.p. for nine consecutive days, respectively. Group-V received reference drug 5-FU (20 mg/kg i.p) for nine consecutive days. Twenty-four hours of last dose and 18h of fasting, 10 animals of each group were sacrificed by cervical dislocation to measure antitumor and hematological parameters and the rest were kept with food and water and libitum to check percentage increase in life span of the tumor host. The antitumor activity of the ethanol extracts of Maytenus emarginata was measured in EAC animals with respect to the following parameters.
TUMOR VOLUME:
The ascitic fluid was collected from the peritoneal cavity. The volume was measured by taking it in a graduated centrifuge tube.
TUMOR WEIGHT:
The tumor weight was measured by taking the weight of the mice before and after the collection of the ascitic fluid from peritoneal activity.
PERCENTAGE INCREASE IN LIFE SPAN:
The effect of EEME on percentage increases in life span was calculated on the basis of morality of the experimental mice.
Mean survival time of the treated group
ILS (%) = [------------------------------------------------ -1 ] X 100
Mean survival time of the control group
First Death + Last Death
Mean survival time =-----------------------------------------
2
TUMOR CELL COUNT:
The ascitic fluid was taken in a WBC pipette and diluted 100 times.Then a drop of the diluted cell suspension was placed on the Neubauer’s counting chamber and the numbers of cells in the 64 small squares were counted(15).
VIABLE/NON-VIABLE TUMOR CELL COUNT:
The viability and non-viability of the cell were checked by trypan blue assay. The cells were stained with trypan blue (0.4% in normal saline) dye.The cells that did not take up the dye were viable and those that took the dye were non-viable. These viable and non-viable cells were counted.
Number of Cells X Dilution Factor
Cell Count = -------------------------------------------
Area X Tickness of Liquid Film
HEMATOLOGICAL PARAMETERS:
At the end of the experimental period, the next day after an overnight fasting blood was collected from freely flowing tail vein and used for the estimation of hemoglobin (Hb) content, red blood cell (RBC) count, white blood cell (WBC) count and lymphocyte count by standard procedures(8,16,3,5).
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 byDunnet’s post hoc test of significance where p<0.05 and p<0.01 considered to be significant and highly significant, respectively.
RESULTS:
Table 1: Effect of EEME plant on tumor volume, tumor weight, mean survival time (MST), percentage increase life span (%ILS), viable and nonviable tumor cell count in EAC bearing mice.
|
Parameters |
EAC control |
100 mg/kg |
200 mg/kg |
5-FU |
|
Tumor volume (ml) |
3.01 ± 0.19 |
1.45 ± 0.72 |
0.99±0.25 |
0.54 ± 0.014 |
|
Tumor weight (g) |
3.40 ± 0.24 |
1.63 ±0.57 |
0.96 ± 0.32 |
0.49 ± 0.15 |
|
MST (days) |
19.5 ±1.2 |
27.5±1.3 |
34.3 ±1.6 |
43.0 ±3.1 |
|
%ILS |
00.00 |
41.02 |
75.89 |
120.5 |
|
Viable cell |
7.9x107 ±0.05 |
3.6x107±0.07 |
1.4x107 ±0.04 |
0.91x107± 0.05 |
|
Nonviable cell |
0.4x107± 0.03 |
1.1x107± 0.03 |
2.7x107± 0.04 |
3.6x107±0.05 |
|
Total cell % |
8.3x107 |
4.7x107 |
4.1x107 |
4.51x107 |
|
viable % |
95.1 |
76.59 |
34.14 |
20.1 |
|
Nonviable % |
4.81 |
23.40 |
65.85 |
79.82 |
Each point represents the mean + S.E.M. (n=10 mice per groups).
*p<0.01,when treated is compared with control.
**p<0.01, when treated is compared with control.
Table 2: Effect of the EEME plant on hematological parameters of EAC treated mice.
|
S.No. |
Parameters |
No.of RBC cells (cellsx106/µl) |
No.of WBC cells (cellsx103/µl) |
Hemoglobin (g/dl) |
Lymphocytes (%) |
|
1 |
Normal |
4.23 |
3.8 |
11.1 |
71.1 |
|
2 |
EAC control |
3.01 |
5.4 |
4.56 |
80.1 |
|
3 |
100 mg/dl |
3.23 |
4.3 |
6.9 |
74.2 |
|
4 |
200 mg/dl |
3.94 |
3.7 |
9.8 |
69.1 |
|
5 |
Standard (5FU) |
4.13 |
4.1 |
10.9 |
70.5 |
DISCUSSION:
Intra-peritoneal administration of the EEME at the dose of 100 and 200 mg/kg body weight increased the life span (ILS) and nonviable cell count, decreased tumor volume, tumor weight and viable cell count of the tumor bearing mice when compared to that of EAC control mice (Table 1). The EEME also restored the hematological parameters towards the saline control. The number of RBC count and hemoglobin content also increased but the WBC and lymphocyte count was decreased as compared to that of EAC control.
Statistical significance (p) calculated by one-way ANOVA between the treated groups and EAC control followed by Dunnet’s post hoc test of significance. Each point represents the mean + S.E.M. (n = 10 mice per groups).*p< 0.01 and **p< 0.05.
CONCLUSION:
The present study showed that EEME significantly increased the life span than that of the EAC control. The reliable criteria for judging the value of any anticancer drug are prolongation of life span and decrease the WBC. Further the reduced volume of EAC and increased survival time of mice suggests the delaying impact of EEME on cell division. Usually in cancer chemotherapy the major problem is anemia due to reduction in RBC. Present study indicates that EEME have significantly enhanced the erythrocyte count and hemoglobin level when compared to that of EAC control. The WBC level decreased when compared with the EAC control.
Reduction in viablecell count and increased non-viable cell count towards normal in host suggest antitumour effect against EAC cell in mice. These suggested that EEME have direct relationship with tumor cells as these tumour cells are observed the anticancer drug by direct absorption in peritoneal cavity and this anticancer agent lysis the cells by direct cytotoxic mechanism.
Preliminary phytochemical study indicated thepresenceof sugar, flavonoids, steroids, fats and oils. Flavonoids have a chemopreventive role in cancer through their effect on signal transduction in cell proliferation and angiogenesis. The cytotoxic and anticancer activity of EEME are probably due to presence of flavonoids.
Thus our present study suggests that the EEME possess potent anticancer activity, increase life span of the tumor bearing host.
ACKNOWLEDGEMENT:
The Authors acknowledge the management of Surabi Dayakar Rao College of Pharmacy, JNTUH, Pregnapur to provide facilities to perform this research work.
CONFLICT OF INTEREST:
No Conflict of Interest by the authors.
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Received on 04.06.2018 Modified on 13.07.2018
Accepted on 02.08.2018 © A&V Publications All right reserved
Asian J. Res. Pharm. Sci. 2018; 8(3):133-136.
DOI: 10.5958/2231-5659.2018.00023.1