In- vitro Antioxidant and Cardio-protective effect of Delonix elata (L.) Leaf extract against Doxorubicin induced toxicity in H9c2 Cardio-myocyte cell line

Archana V*, Indumathy R

Institute of Pharmacology, Madras Medical College, Chennai.

*Corresponding Author E-mail: indumathy.s2007@gmail.com

ABSTRACT:

Objective: The aim of this study is to evaluate the protective effect of Delonix elata (L.) leaf extract against doxorubicin-induced cardiotoxicity in H9c2 cells. Methods: Doxorubicin has been used to treat cancer, but its clinical uses are limited because of its dose-dependent cardiotoxicity. Reactive oxygen species play an important role in the pathological process of cardiotoxicity. The various extracts (pet.ether, ethyl acetate and ethanol) of Delonix elata leaves antioxidant property was evaluated by SOD antioxidant assay and DPPH free radical scavenging assay. The cells were incubated with different concentrations of various extracts of Delonix elata leaves for 2 hr, followed by incubation with 5µM doxorubicin for 24 hr. Cell viability was determined by using MTT assay, respectively. Results: The various extracts of Delonix elata leaves exhibits antioxidant activity. The Doxorubicin significantly decreased cell viability which was accompanied by an increased ROS production. Pre-treatment with various extracts of Delonix elata leaves increased the viability ofcells and inhibit the generation of reactive oxygen species. Conclusion: In this study, findings how that Delonix elata leaf extract exhibited a protective effect against oxidative stress-induced cardiomyocyte damage. The ethanolic extract of Delonix elata leaves possesses significant antioxidant and cardioprotective activity.

KEYWORDS: Delonix elata, Doxorubicin, ROS, Cardiomyocyte damage.

INTRODUCTION:

Cardiovascular diseases represent a secondary cause of morbidity and mortality in today's world. Adriamycin (also named as doxorubicin) is an anthracycline antibiotic that has been used for more than 30 years for the treatment of a wide variety of cancers. It is highly effective in many malignant diseases like breast and esophageal carcinomas, osteosarcoma, Kaposi's sarcoma, soft tissue sarcoma, and Hodgkin's and non-Hodgkin's lymphomas. The clinical use of doxorubicin is limited because of its undesirable serious dose-dependent cardiotoxic side effects, which frequently lead to congestive heart failure.

ROS (Reactive Oxygen Species) is a key factor causing oxidative damage to a variety of cellular components. Oxidative stress causes increased toxicity in the cardio-myocytes irreversible damage of DNA and alteration of cardiac energetic levels¹. Peroxynitrites, a reactive oxidant produced by the rapid reaction of nitric oxide and superoxide, is mainly involved in the doxorubicin cardiotoxicity by understanding the free radical mechanism of anthracyclines induced cardiotoxicity. The overexpression of endogenous anti-oxidants is not yet feasible in human hearts, alternative strategies have been tested in an attempt to increase anti-oxidants defense mechanism in subjects receiving anthracyclines². Antioxidants are very promising in animal models of anthracycline-induced cardiotoxicity and there is increasing interesting in their use for the prevention of DOX-induced cardiomyopathy. Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties.

Delonix elata belongs to the family Fabaceae, subfamily Caesalpinaceae widely distributed within the Indian subcontinent. Delonix consists of two species growing in India (Delonix elata and Delonix regia). It is a deciduous tree and commonly called as White Gulmohur, White Poinciana, as it bears creamy white flowers and it has many regional names like Vathanarayana, sidhsaru, sunkesula, nirangi. The plant leaf is possessed Flavanoids, Tannin, Saponin, Quinone, Terpenoids, Steroids, Alkaloid, Phenol, Cardiac glycosides, Coumarins. The plant used for Siddha and folk medicine. The leaf extracts possess anti-inflammatory activity; a root decoction is drunk for abdominal pains^3-6. The plant is used for anti-oxidant, anti rheumatism and antibacterial activity. Based on the literature, the leaf of Delonix elata was selected to investigate the cardioprotective effect against Doxorubicin-induced cardiotoxicity.

MATERIALS AND METHODS:

Collection and preparation of plant extract:

The fresh leaves of Delonix elata (L.) Gamble was collected from Tharamangalam, Salem District, Tamilnadu, India in the month of August 2018. The plant was authenticated by Dr. K. N. Sunil Kumar, Research officer and HOD of pharmacognosy, Siddha Central Research Institute, Anna Govt. Hospital campus, Arumbakkam, Chennai. The dried material was powdered and passed through a 10-mesh sieve. The powdered plant material was then extracted with petroleum ether, ethyl acetate and ethanol by successive extraction method using Soxhlet apparatus. At the completion of the extraction procedure, the solvent was removed by means of evaporation at room temperature, yielding the extracted compound.

In vitro antioxidant study:

Estimation of Superoxide dismutase (SOD):

To estimate the SOD level by using the procedure of Das et al., 2000 (enzymatic method). The method involves generation of superoxide radical by photoreduction of riboflavin and its detection by nitrite formation from hydroxylamine hydrochloride at 543nm. The method is more sensitive than the NBT method and less sensitive than the NADH method⁷.

DPPH radical scavenging activity:

The DPPH free radical scavenging activity was evaluated by Mensor LL et al., 2001. DPPH radical is scavenged by antioxidants through the donation of a proton forming the reduced DPPH⁸. The color change from purple to yellow after reduction can be quantified by its decrease in absorbance at wavelength 517nm.

In vitro Cardioprotective study using H9c2 (Rat cardiomyocyte) cell line:

Cell culture:

Normal H9c2 (rat cardiomyocyte) cell line was obtained from National Centre for Cell Sciences (NCCS), Pune. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100U/ml) and streptomycin (100µg/ml) in a humidified atmosphere of 50µg/ml at 37⁰C in 5% CO₂and subcultured when they reached 70% confluence.

Cell viability (MTT) assay:

The cell viability was determined by using MTT assay as described procedure of Mosmann, 1983. Cells (1 × 10⁵/well) were plated in 24-well plates and incubated in 37⁰C with 5% CO₂ condition. After the cell reaches the confluence, the various concentrations of the samples were added and incubated for 2hrs. After 2 hrs, 5µM Doxorubicin was added in all the wells and incubated for 24 hrs. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) or DMEM without serum. 100µl/well (5mg/ml) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl--tetrazolium bromide (MTT) was added and incubated for 4 hours. After incubation, 1ml of DMSO was added in all the wells. The absorbance at 570nm was measured with UV- Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50% inhibition (IC50) was determined graphically⁷. The % cell viability was calculated using the following formula:

% Cell viability = A₅₇₀ of treated cells / A₅₇₀ of control cells × 100

RESULTS:

In vitro Antioxidant study:

SOD Assay:

The antioxidant effect of petroleum ether, ethyl acetate and ethanol extract of Delonix elata (L.) leaves were evaluated by using SOD assay and the results were shown in the following Table 1. The IC₅₀ value of these three extracts was calculated and it was compared with the standard (Ascorbic acid). The ethanolic extract of Delonix elata (L.) leaf showed significant IC₅₀value (209.03U/mg Protein) than petroleum ether (1528.08U/mg Protein) and ethyl acetate (613.33 U/mg Protein) extract. The IC₅₀ value of ethanolic extract of Delonix elata (L.) leaves has produced significant IC₅₀ value near to standard (AA) 166.59U/mg Protein.

Table 1. Estimation SOD activity of various extracts of Delonix elata (L.) leaves

S.No	Concentration (µg/ml)	SOD (U/mg Protein)
S.No	Concentration (µg/ml)	Standard (Ascorbic Acid)	Pet. Ether Extract	Ethyl acetate Extract	Ethanol Extract
1	200	52.89	7.84	30.95	49.71
2	400	67.83	17.23	41.46	60.08
3	600	78.31	23.39	49.86	73.24
4	800	85.9	29.41	58.82	83.89
5	1000	95.78	31.93	66.11	89.92
	IC₅₀ value	166.59	1528.08	613.33	209.03

Table 2. DPPH free radical scavenging assay of various extracts of Delonix elata (L.) leaves

S.No	Concentration (µg/ml)	DPPH (U/mg Protein)
S.No	Concentration (µg/ml)	Standard (Quercetin)	Pet Ether Extract	Ethylacetate Extract	Ethanol Extract
1	200	61.53	5.66	13.76	29.96
2	400	70.85	8.91	24.29	44.53
3	600	78.54	14.17	39.67	60.32
4	800	85.02	18.62	55.06	73.68
5	1000	91.49	24.29	60.32	84.21
	IC₅₀ value	142.14	2117.48	784.28	476.09

DISCUSSION:

Antioxidants are the substance which is used to delay or prevent the oxidation of the substrate. It may help the body to protect itself against various types of oxidative damage caused by reactive oxygen species, which are linked to a variety of diseases including cancer, diabetes, arthritis, and acceleration of the aging process¹⁰. Free radicals and other reactive oxygen species are considered as an important causative factor for various diseases like neurodegenerative diseases, cancer, and cardiovascular diseases^11,12. Antioxidants are act by decreasing oxygen concentration, intercepting singlet oxygen, preventing first chain initiation by scavenging initial radicals, binding metal ion catalysts, decomposing primary products to non-radical compounds and chain breaking to prevent continued hydrogen abstraction from substances.

The oxidative damages caused by ROS on lipids, proteins and nucleic acids may trigger various chronic diseases, such as coronary heart diseases, atherosclerosis, cancer, and aging¹³. The health-promoting effect of antioxidants from plant materials is thought to arise from their protective effects by counteracting ROS¹¹. Assessing the antioxidant capacity of foods, botanicals and other nutritional antioxidant supplements. Plants are expressed their significant amount of antioxidants to prevent the oxidative stress caused by photons and oxygen and they represent a potential source of new compounds with antioxidant activity.

Many medicinal plants possessed large amounts of antioxidants such as polyphenols (phenolic acids, flavonoids, and anthocyanin) which can play an important role in adsorbing and neutralizing free radicals^14,15. There is increasing interest in natural antioxidant products for use as medicines and food additives. Many studies have shown that antioxidants present in plants at a higher level and these biomolecules exert a potential protective effect against oxidative damage.

Superoxide dismutase (SOD) is a primary antioxidant enzyme and it was directly involved in the elimination of ROS. SOD is an enzyme that alternately catalyzes the dismutation of superoxide radical into their ordinary molecular oxygen or hydrogen peroxide and provides cellular defense against reactive oxygen species.

DPPH radical scavenging activity is a widely used method for assessing the antioxidant effect of the plant extracts. DPPH is a well-known stable radical and scavenger for other radicals. The rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction. A strong absorption band centered at 520nm. DPPH radical has a deep violet color in solution and it becomes colorless or pale yellow when it neutralized. The free radical scavenging activities of extracts depends on the ability of antioxidant compounds to loss hydrogen and structural conformation of these components. The DPPH radical's binding ability with hydrogen (H) is considered as a radical scavenging property. A solution of DPPH radicals prepared in methanol is converted into DPPH-H molecules in the presence of antioxidants.

From the results of SOD and DPPH assay showed that the ethanolic extract of Delonix elata (L.) leaves possess the potent antioxidant effect than petroleum ether and ethyl acetate extract of Delonix elata (L.) leaves.

H9c2 (rat cardiomyocyte) cell line derived from rat heart ventricle of the embryo and it was widely used as an in-vitro cellular cardiac model because they have most of the molecular and functional features of adult cardiomyocytes. Cardiac hypertrophy is a major risk factor for heart failure. H9c2 cell line has the advantage of being animal-free alternative and it can accurately mimic the hypertrophic responses^16,17. So these findings are helping to choose H9c2 cell line as a model for the study of In-vitro cardioprotective effect of Delonix elata (L.) leaf extracts.

The MTT assay is the most commonly used method for determining the viability of the cells. It is a colorimetric assay for assessing cell metabolic activity. The viable cell contains NAD (P) H - dependent oxidoreductase enzymes which reduce the MTT reagent to formazan, an insoluble crystalline product with a deep purple color. Formazan crystals are dissolved using a solubilizing solution and absorbance was measured at 500-600nm. When the cells die they lose their ability to convert MTT into formazan. If more cells are inviable they show thick color formazan formation. The color formation serves as a convenient marker of the viable cells.

Doxorubicin has several mechanisms for cardiotoxic effects, but an excessive generation of free radicals (ROS) is the major mechanism behind drug-induced toxicity¹⁸. Doxorubicin has Quinone moiety in their molecule which is converted enzymatically or non-enzymatically by cytochrome P₄₅₀ into its semiquinone moiety by the acquisition of electrons. This semiquinone form is then oxidized by molecular oxygen to yield free radicals which are leading to cardiotoxicity. Doxorubicin has reduced the half percentage of cell viability on H9c2 cell line at the concentration of 4.38µg/ml after 24hrs. So the Doxorubicin has a cardiotoxic effect in a dose-dependent manner. The pretreatment with Delonix elata leaf extracts (pet.ether, ethyl acetate and ethanol) showed significant increased in cell viability and reduced the cell death.

From the result of antioxidant study and cardioprotective cell line study (cell viability assay), the ethanolic extract of Delonix elata (L.) leaf was possessed significant antioxidant and cardioprotective activity than petroleum ether and ethyl acetate extract.

In this study concluded that ethanolic extract of Delonix elata (L.) leaves possess significant antioxidant and cardioprotective activity. Further study is needed forthe evaluation of the mechanism of action of plant extract.

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Received on 26.04.2019 Modified on 10.10.2020

Research J. Pharm. and Tech 2021; 14(11):5635-5641.

DOI: 10.52711/0974-360X.2021.00980