Defensive Consequences of Hydroalcoholic Extract and Isolated Compounds of Ipomoea aquatica Forsskal against DMBA Method
Sasikala M1,2*, Swarnakumari S1, Mohan S1
1Karpagam College of Pharmacy, Coimbatore – 641032, Tamil Nadu, India.
2Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore – 641021, Tamil Nadu, India.
*Corresponding Author E-mail: sasikalampharm@gmail.com
ABSTRACT:
Introduction: Beyond the preceding twenty years progresses in investigative systems and an upsurge in effect usage routes have remarkably enhanced breast cancer endurance proportions, regardless of a cumulative number of identified cases amongst women. Certain plant extracts have been acquired to produce anti-cancer powers and to fit into chemotherapy treatments. Aim and Objective: To accomplish the anticancer activities of phytoconstituents of Ipomoea aquatica Forsskal beside DMBA induced breast cancer in Sprague dawley rats with the addition of assessment of histopathological and biochemical parameters. Methods: There were six groups of animals such G1 (Normal), G2 (tumor), G3 (standard Vinblastine), G4 (hydroalcoholic extract), G5 (Isolated compound I) and G6 (Isolated compound II) correspondingly. Results and discussion: The body weight, defense percentage and tumor volume were determined. A substantial behaviors were detected in G3 (37.85%), G4 (24%), G5 31.70% and G6 (32.65%) after 45 days therapy. The special effects of antioxidant enzymes similar to SOD, CAT, GPX and LPx were examined and had revealed statistically significant findings. Conclusion: Bearing in mind regarding the prolonged medication of patients, natural resolves (plant of interest with its isolated compounds) will control superior deprived of any side effects to cure the ailment in effectual approach.
KEYWORDS: Anti-cancer, In-vivo, Water spinach, Breast cancer, 7,12-Dimethyl benzanthracene.
1. INTRODUCTION:
The crucial pathophysiological disorders together with mutagenesis and carcinogenesis are triggered by Reactive oxygen species (ROS). The oxidative stress causes cellular DNA impairment, lipid peroxidation and membrane distraction.
The various antioxidants in human body are superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) and so on. These could stabilize the harmful act of ROS and defend as of cellular and molecular loss.2-5
The reagent 7, 12-dimethyl benz[a]anthracene (DMBA) is adherent to the polycyclic aromatic hydrocarbons (PAH). It is a pro-carcinogen desires metabolic start to convert a critical carcinogen. It is frequently hired by way of a chemical model for bringing investigational carcinogenesis. It is a cancer originator in animal models presence of cytotoxic, carcinogenic, mutagenic and immunosuppressive properties. Since DMBA is secondary cancer-causing agent, it requires metabolic stimulation by cytochrome P450 enzymes effects in the development of oxidative free radicals.6-9
Breast cancer and other tumor linked syndromes have been cured using surgery, chemotherapy and radiation treatment or a combination of these. Even though these remedial possibilities are made accessible, malignancy leftovers with high mortality. The biological inequalities brought by a number of pollutants related with free radicals are diminished using phytoconstituents. Thus, the plant products are obvious to progress herbal drugs with no side effects to treat the desired tumors. Hence, the target of current study is to examine the anticancer activity of plant extract and isolated compounds in contrast to DMBA induced breast cancer in trial rats. Ipomoea aquatica is a river herb to be plentiful in metabolites, for instance flavonoids, alkaloids, reducing sugar and amino acids. 10
2. MATERIALS AND METHODS:
2.1 Materials:
2.1.1 Plant crude extract:
Approximately 350.0g of coarse powder of drug was balanced and was taken in a 5000.0ml round bottomed flask. Petroleum ether was drenched to abolish the fatty matters associated with the particle. The solvent possessed was evaporated at room temperature after rinsing for few minutes. Then the dried defatted powder was immersed in 2000.0ml of solvents which comprises 1000.0ml of distilled water and 1000.0ml of ethanol. The content was closed with aluminium foil and kept for cold maceration at ambient temperature with occasional shaking to bring about rapid equilibrium between intra and extra cellular fluids thereby bringing fresh menstruum to the particle surface for further extraction. The contents would take 3 days for imbibitions and another 4 days for extraction. Wherefore, totally the content was kept for 7 days of maceration. The volume of menstruum retained after extraction was 1700.0ml.
After the mentioned period, the content of extraction was constrained through a muslin cloth. The marc was separated from the menstruum. The extract was divided into two portions in two separate 1000.0ml beakers and kept at 40.00C for concentration. After 4 days, the concentrated AEIA was poured into a previously weighed china dish of big size for evaporation at the same temperature. After 3 days, the completely dried extract was cooled to room temperature and weighed. The percentage yield was calculated. Then, the product was used for further studies.
2.1.2 Isolated compounds:
The method of isolation was led by the extract and the eluents similar to petroleum ether, chloroform, ethyl acetate, methanol and distilled water through column chromatographic analysis. The yields were put in storage in the sample containers for more analysis.
2.1.3 Chemicals:
DMBA were procured from Sigma Chemicals Co. (St. Louis, MO, USA). All the other Chemicals used in this study were of analytical grade obtainable commercially.
2.1.4 Practice for inducing tumour:
Presently obtainable mouse models for human breast cancer be able to regard as three core groups:
(a) Xenograft models
(b) Chemically induced, virally brought or ionizing radiation made models
(c) Genetically engineered mice (GEM) such as transgenic and hits DMBA induced Breast cancer model.11
2.1.5 Animals:
Female Sprague–Dawley rats assessing (190g) were gotten from animal experimental laboratory and used all over the study. They were stored in fit ventilated huge airy polypropylene cages and had 12±1h light and dark sequence during the investigational period. The animals usual a balanced diet of commercially existing pellet rat provender and water ad libitum. In accordance with the customary exercise, the rats were confined for 15 days earlier the origination of experiment. The protocol was permitted by the Institutional Animal Ethics Committee. 12-13
2.2 Methods:
2.2.1 Acute Oral Toxicity Analysis:
It was carried out as per the guidelines Organization of Economic Co-operation and Development (OECD) – 423 guidelines after the animal ethical clearance from Institutional Animal Ethics Committee. The procedure is done with doses such as 50, 300 and 2,000 mg.kg-1 body weight.
2.2.2 Experimental design:
Female albino rats (36 numbers) weighing 190g were set apart into 6 groups, every group encompassing of 6 animals respectively.
Group 1 served as the normal control (received vehicle).
Group 2 functioned in place of the tumor control, was preserved with a single dose of 7.5mg of DMBA dissolved in sun flower oil (0.5ml) given subcutaneously one time a week for four sequential weeks. Group 1 and 2 were received normal diet and Water.14,15
Group 3 worked as per the positive control, was set by injection Vinblastine at 0.5mg/kg body weight, intra-peritoneal just the once a week aimed at 45 days.16
Group 4 assisted by means of the treatment control, which was administered with hydroalcoholic extract of plant material (200mg/kg body weight) through oral way day-to-day used for 45 days.
Group 5 helped as treatment control and was cured with isolated compound-I (10mg/kg body weight) by oral route daily for 45 days.
Group 6 served as treatment control, was treated using isolated compound-II (10mg/kg body weight) orally every day for 45 days.
The trial rats were recurrently observed for food and water ingestion, the specious marks of toxicity, weight loss and mortality. Forty-five days afterwards DMBA administration, entirely the experimental animals were sacrificed. Toward the end of the probationary period, wholly the rats were alive, starving instantaneous anesthetized with diethyl ether and sacrificed via euthanasia method. The breast tumor was surgically cut apart. The growth volumes (mm in diameter) of both cancer controls, along with the investigational groups were measured and total body weight (g) was correspondingly verified.
Breast was separated out, cut into small pieces with a durable blade and washed with ice-cold 0.9% NaCl solution. The tissues of 100mg were homogenized in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was meant for assessment of biochemical parameters, for example SOD radical scavenging action, Inhibition of LPx formation, GPx activity, CAT analysis. The consequent factors as Body weight, Tumor Volume and Percentage reduction of growth volume [17,18] were too evaluted. Tumor size was measured weekly and calculated using the formula. (cc) = 4/3 πr3. On scheduled 45th day, rats were forfeited, tumors were detached from the animals, weighed and determined for the above said features.19
2.2.3 Evaluation of Biochemical Parameters:
The special effects of plant extract and isolated compounds on superoxide dismutase, catalase, glutathione peroxidase and lipid peroxide were predicted for treated as well as untreated rats’ breast tissues.
2.2.3.1 Assay of Superoxide dismutase radical scavenging activity:
SOD activity was assessed by the nitroblue tetrazolium (NBT) reduction scheme. Nearly, a known concentration of tissue supernatant was supplemented to a reaction mixture comprising 0.1mM EDTA, 0.12mM Riboflavin and 0.6M phosphate buffer (pH 7.8) in an ultimate volume of 3.0ml. The optical density was measured at 560nm.
2.2.3.2 Evaluation of lipid Peroxidase action:
Stimulation by Fe3+/ascorbate system: the response mixture enclosing rat breast homogenate (0.1ml, 50%, w/v) in Tris–HCl (30.0mM), ferrous ammonium sulfate (0.16mM) and ascorbic acid (0.06mM). It was incubated for 1h at 37◦C and the resulting thiobarbituric reacting substances (TBARS) were estimated. [23,24] For a moment, a 0.4ml aliquot of the mixture was preserved with sodium dodecyl sulfate (0.2ml, 8%), thiobarbituric acid (1.5ml, 0.8%) and acetic acid (1.5ml, pH 3.5), made up to a total volume of 4.0ml by adding distilled water and then kept in a water bath at 95◦C for 1h. When cooling, 1.0ml of distilled water and 5ml of n-butanol/pyridine (15:1, v/v) were added. The organic layer was parted later centrifugation. LPx activity was studied in terms of thiobarbituric acid formation. The color intensity observed spectrophotometrically at 530nm.
2.2.3.3 Testing of Glutathione peroxidase activity:
The effect of GPx was assayed in the following reaction combination. It contained 0.2ml of EDTA (0.8mM, pH 7.0), 0.4ml of phosphate buffer (10mM) and 0.2ml of tissue homogenate was nurtured with 0.1M of H2O2 and 0.2ml of glutathione for 10min. The spectrophotometric determination of enzymatic glutathione oxidation at 420nm had been done. The activity was conveyed as 1.0mol glutathione oxidized/min/mg protein.
2.2.3.4 Analysis of Catalase activity:
Breast catalase doings were monitored spectrophotometrically. The reaction mix (2.0ml) contained 1.95ml of 10mM H2O2 in 60mM phosphate buffer (pH 7.0). The response was begun by adding 0.5ml supernatant to it. The absorbance was observed for 3min at 240nm taking phosphate buffer (60mM, pH 7.0) as a standard. The extinction coefficient of 0.04mM−1 cm−1 was considered to study the detailed activity of CAT. The data has been quantified as mol H2O2 consumed/ (min (mg protein)).
2.2.4 Histopathological Consideration:
Mammary tissue fragments were secured in 10% buffered formalin. The paraffin block of 5µm section was stained through haematoxylin and eosin intended for histologic analysis. The image of successive paraffin segments were caught using light microscopy.
2.2.4 Statistical exploration:
The comparisons between control and treatment mean values of two parameters were scrutinized using the Student’s t-test. Whereas, the manifold evaluations were completed using ANOVA with Newman Keul’s multiple range test. The modifications were statistically substantial at P < 0.01; P < 0.05.
3. RESULTS AND DISCUSSION:
3.1 Acute oral toxicity results:
The toxic symptoms were observed for 72hr including behavioral changes, locomotion, convulsions and mortality. The results are tabulated in the table 1.
Table 1. Acute toxicity study of hydroalcoholic extract on experimental mice
|
Treatment |
Dose (mg.kg-1) |
Sign of Toxicity (ST.NB-1) |
Mortality (D.S-1) |
|
Group I |
0 |
0/3 |
0/3 |
|
Group II |
300 |
0/3 |
0/3 |
|
Group III |
2000 |
0/3 |
3/3 |
3.2Anti-tumor activity:
The body mass was considerably reduced in tumor-induced animals set against control. Growth promotional phase is a revocable stage in the multistage carcinogenesis which is more suitable anti-carcinogenic agent to prevent, reverse, or slow down the process. This was reached with the group 2 animals after 45 days. Tumor incidence and multiplicity linked to the normal control rats had been augmented at the end of the experiment in non-treated rats. A substantial decrease in Tumor capacity was detected in G3 (37.85%), G4 (24%), G5 31.70% and G6 (32.65%) after 45 days treatment. The body weight, protection percentage and tumor volume with DMBA brought Breast cancer in rats are explained in the figure 1 and table 2.
Figure 1. The Effect of samples on Antitumour Activity in Experimental Breast Tissue Cancer in Rats
Table 2. The Effect of samples on Antitumour Activity
|
Group |
Body Weight (g) |
Tumour Volume [TV = L X W(2) / 2] (mm) |
Percentage Tumour Growth Inhibition [%TGI = (MTV of Control – MTV of Treated / MTV of Control) X 100](%) |
|
G1 |
214.6±4.52 |
- |
- |
|
G2 |
158±3.80**a |
46.80±1.90**a |
- |
|
G3 |
185.30±3.62**b |
35.53±0.85**b |
37.91**b |
|
G4 |
171.6±3.55**b |
29.06±1.55**b |
24.08**b |
|
G5 |
190.10±2.72**b |
31.94±0.92**b |
31.75**b |
|
G6 |
190.10±2.72**b |
32.80±0.99**b |
29.91**b |
Note:
· Values are expressed as mean ± S.E.M.
· Values are found out by using one way ANOVA followed by Newman Keul’s multiple range tests.
· **a values are significantly different from normal control at p<0.001.
· **b values are significantly different from pathogenic control at p<0.001.
· TV: Tumour Volume, MTV: Median Tumour Volume; L: Length; W: Width
Antioxidant effect
The free radical scavengers were revealed a major fall in the actions of antioxidant enzymes like SOD, CAT, GPX and LPx in G2, while the values were pointedly raised then preserve with Vinblastine, plant extract and isolated compounds. The results are enumerated in the figure 2 and table 3. SOD and CAT turn as conjointly helpful antioxidative enzymes, which offer protective defense beside reactive oxygen species. GPx is in the same way vital antioxidant, which counters by H2O2, thus preventing intracellular damage. Augmented lipid peroxidation modifies membrane fluidity, potential leading to injury of cellular function and cell death
Figure 2. The Effect of Plant Extract and Isolated Compounds on Enzymatic Antioxidants in the Breast Tissue of Control and Experimental Animals
Table 3. The Effect of AEIA and Isolated Compounds on Enzymatic Antioxidants
|
GROUP |
SOD (units/mg protein) |
CAT (μmol H2O2 consumed/ [min(mg protein)]) |
GPx (μ gm GSH utilized/[min(mg protein)]) |
LPO (n mol MDA found/[min/(mg protein)]) |
|
G1 |
3.80±0.09 |
46.90±0.49 |
3.85±0.12 |
0.76±0.03 |
|
G2 |
1.56±0.05**a |
14.54±1.10**a |
2.12±0.04**a |
2.17±0.09**a |
|
G3 |
3.24±0.08**b |
36.90±0.82**b |
3.56±0.08**b |
1.13±0.06**b |
|
G4 |
2.86±0.06**b |
27.65±0.68**b |
3.10±0.05**b |
1.48±0.08**b |
|
G5 |
3.17±0.07**b |
33.12±0.74**b |
3.25±0.06**b |
1.34±0.07**b |
|
G6 |
3.19±0.08**b |
33.20±0.76**b |
3.28±0.07**b |
1.32±0.06**b |
Note:
· Values are expressed as mean ± S.E.M.
· Values are found out by using one way ANOVA followed by Newman Keul’s multiple range tests.
· **a values are significantly different from normal control at p<0.001.
· **b values are significantly different from pathogenic control at p<0.001.
3.2 Histopathological inspection:
The untreated groups were typically rising carcinomas exposed a matching nuclear outline per major epithelial cell and fibrous tissue adjacent to the mammary ducts, a mixed structural arrangement with attack of adjoining tissues and extreme stromal desmoplastic response. The in-vivo treated groups exhibited tubules development, lessening of intra-tumor vascularization, reduced proliferation and addition of regular ductile and alveolar structure of mammary tissue. The microscopical images are illustrated in the figures 3, 4, 5, 6 and 7.
Figure 3. Section shows Breast Parenchyma with Ductules, Ducts and Fibrous Stroma of G1, G2 and G3 respectively
Figure 4. Section shows Breast Parenchyma with Ductules, Ducts and Fibrous Stroma of G1, G2 and G4 respectively
Figure 5. Section shows Breast Parenchyma with Ductules, Ducts and Fibrous Stroma of G1, G2 and G5 respectively
Figure 6. Section shows Breast Parenchyma with Ductules, Ducts and Fibrous Stroma of G1, G2 and G6 respectively
Figure 7. Section shows Breast Parenchyma with Ductules, Ducts and Fibrous Stroma of G3, G4, G5 and G6 respectively
4. CONCLUSION:
Since the outcomes concluded that the antioxidant activity of extract and isolated compounds confidently controlled by extinguishing and detoxifying the free radicals persuaded by DMBA. Histopathological investigation also maintenances the studies. The diminution of induced oxidative stress by means of the plant extract could be recognized to the antioxidants of flavonoids, terpenoids, phenolic compounds and alkaloids. The two isolated compounds of the plant has noteworthy anticancer activity on the designated animal models. These will optimistically encounter the necessity of herbal drug progress in the region of female breast cancer studies and as a therapy to the widespread and life threatening syndrome in the sphere.
5. ACKNOWLEDGEMENT:
This research was supported by the Management and Principal of Karpagam Educational Institutions, Coimbatore. We express thankfulness to our associates from Karpagam College of Pharmacy and Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore who provided perception and proficiency that exceptionally help out the study.
6. CONFLICT OF INTEREST:
It is declared none.
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Received on 22.06.2020 Modified on 31.07.2020
Accepted on 08.09.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(7):3863-3868.
DOI: 10.52711/0974-360X.2021.00670