In vitro Antioxidant and Anticancer activity of methanolic extract of Alangium salvifolium subsp. hexapetalum (Wangerin)

 

H. Seena1*, N. Kannappan2, P. Manoj Kumar1

1Department of Pharmaceutical Chemistry, The Dale View College of Pharmacy and Research Centre, Trivandrum, 695575, Kerala.

2Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram 608002, Tamil Nadu.

*Corresponding Author E-mail: seenakannan02@gmail.com

 

ABSTRACT:

The present study was implemented for revealing the antioxidant and anticancer effect of the aerial parts of methanolic extract of Alangium salvifolium subspecie hexapetalum (Wangerin). Preliminary screening of the methanolic extract was carried out to determine the presence of plant secondary metabolites such as alkaloids, glycosides, flavonoids, terpenoids, steroids, and sterols. Free radical scavenging activity was evaluated using DPPH radical scavenging assay, ABTS radical scavenging assay and reducing power assay. Invert phase contrast microscope and MTT assay method was used to analyse the number of viable cells using HT29 cell lines treated with different concentrations of the aerial parts of methanolic extract of Alangium salvifolium subspecie hexapetalum (Wangerin). The preliminary phytochemical analysis of the methanolic extract of Alangium salvifolium subspecie hexapetalum (Wangerin) unveil the presence of alkaloids, glycosides, flavonoids, tannins, phenolic compounds, carbohydrates and proteins as secondary metabolites. The free radical scavenging activity also revealed the presence of good antioxidant activity in the extract. A dose dependent growth inhibition of HT29 cell lines were seen when treated with methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum (Wangerin). As the concentration of extract increases the percentage of viable cells decreases. The IC50 value for radical scavenging activity of methanolic extract was found to be 175µg/ml (DPPH radical scavenging assay) and 40µg/ml (ABTS scavenging assay). The IC50 value for anticancer activity was found to be 30.22µg/ml. The investigational outcome proves that the methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum (Wangerin) showed promising antioxidant and cytotoxic activities.

 

KEYWORDS: Alangium salvifolium subspecie hexapetalum (Wangerin), DPPH assay, ABTS assay, Reducing power assay, HT29 cells, MTT assay.

 

 


INTRODUCTION:

The use of medicinal plants is increasing day by day in the present scenario. Naturally synthesised drugs are considered to be safer than synthetic drugs. Phytochemicals and secondary metabolites present in plants are used for treating many human ailments. Cancer is a disease condition which leads to proliferation of abnormal cells. Chemotherapy is recently the standard method of treatment but still the need for new anticancer agents which are natural products is increasing.

 

There is a continuing demand to develop new, efficacious, and inexpensive anticancer drugs. Natural products have gained high recognition over the past 30 years for their prospective as cancer preventive and therapeutic agents. There are significant evidence for the plant-derived compounds as inhibitors of various stages of oncogenesis thus emphasizing the importance of these products in cancer prevention and therapy1.

 

Antioxidants inhibit the process of oxidation, at relatively small concentration and thus have multiple physiological roles in the body. Plant antioxidants acts as radical scavengers, and helps in converting the radicals to less reactive species. Numerous free radical scavenging antioxidants are found in dietary sources2. Antioxidants play key role in preserving the cellular integrity and thus are critical in maintaining the homeostasis of the host immune system. For decades, there have been scientific discussions on the use of antioxidants for the treatment of cancers3. Alangium salvifolium of family Alangiaceae which is commonly known as ‘Irinjil’ is traditionally used to treat many diseases like laxative, antiepileptic, jaundice, antiulcer agent, agent to alleviate spasms, anthelmintic, emetic, antiprotozoal agent and hypoglycemic agent. Its sub specie Alangium salvifolium subsp. hexapetalum (wangerin) is also known for a variety of traditional use like haemorrhoid, rheumatism and antidote for snake bite. The main objective of the current research is to determine the antioxidant and anticancer property of aerial parts of Alangium salvifolium subsp.hexapetalum (Wangerin).

 

MATERIALS AND METHODS:

Plant Collection and Verification:

The aerial parts of Alangium salvifolium subsp. hexapetalum (Wangerin) was collected from Tirunelveli district, Tamil Nadu. Mr. Chelladurai, the Research officer- Botany, in Central Council for Research in Ayurveda and Siddha, Govt. of India recognized and verified the plant (Reference No. FTN/183/2018).

 

Preparation of extract:

The plant’s aerial parts were desiccated in air shade and ground to coarse powder through a mixer. Using soxhlet extraction method, the powdered drug was processed with solvents of increasing polarity. The extract of petroleum ether, chloroform, ethyl acetate and methanol was subjected to preliminary phytochemical study.

 

Phytochemical screening:

The various extracts were subjected to preliminary phytochemical screening for the detection of alkaloids, glycosides, steroids and triterpenoids, flavonoids, proteins, carbohydrates, tannins, phenolic compounds and saponins present in them, by using standard procedures4,5,6.

 

Antioxidant Activity:

DPPH scavenging assay:

Radical scavenging activity of plant extracts against stable 2, 2diphenyl 2- picryl hydrazyl radical (DPPH) was determined. Five grams of extract was taken and treated with 25ml methanol. From the above sample 1ml was taken and mixed with an equal volume of methanol to which 1ml of 200μM DPPH was added. Control, blank and sample solutions were incubated in the dark at 37°C for 30 min7. DPPH reacts with an antioxidant compound, which can donate hydrogen, and reduce DPPH. The change in colour, from deep violet to light yellow was measured at 515nm using UV visible spectrophotometer. Standard used was ascorbic acid. Radical scavenging activity was calculated by the following formula.

 

% Inhibition = (C - T)/C1 x 100,

 

Where C= absorption of control sample (t= 0 min), C1= absorption of control (t=15 min), T=absorption of test solution.

 

ABTS radical scavenging assay:

Free radical scavenging activity of the extract was studied by ABTS radical cation decolourization assay. ABTS+ cation radical was produced by the reaction between 7mM ABTS in water and 2.45mM potassium per sulfate (1:1), stored at room temperature for 12-16 h before use in dark. ABTS+ solution was finally diluted with methanol to obtain an absorbance at 734nm. After the addition of 5μl of plant extract to diluted ABTS+ solution, the absorbance was measured within 30 min after the initial mixing. An appropriate solvent blank was run. Standard used was ascorbic acid. Percentage inhibition of absorbance at 734nm was calculated using the formula,

 

ABTS+ scavenging effect (%) = (Ac–At)/ Ac ×100,

 

where Ac- is absorbance of ABTS radical + methanol; At- is absorbance of ABTS radical + sample extract/standard 8.

 

Reducing power assay:

The ferric reducing capacity of extract was studied by using potassium ferricyanide-ferric chloride method. 0.2mL of each extract at different concentrations, 2.5mL of phosphate buffer (0.2 M, pH 6.6), and 2.5mL of potassium ferricyanide K3Fe(CN)6 (1%) was mixed and incubated for 20 min at 50˚C, to reduce ferricyanide into ferrocyanide. Add 2.5mL of 10% (w/v) trichloroacetic acid to stop the reaction followed by centrifugation at 1000rpm for 10 min. Finally, 2.5mL of the upper layer was mixed with 2.5mL of distilled water and 0.5 mL of FeCl3 (0.1%) and the absorbance was measured at 700 nm.8,9.

 

In vitro evaluation of anticancer activity:

Cell culture:

From National Centre for Cell Sciences (NCCS), Pune, India, the HT29 cell line was procured, and preserved in Dulbecos modified Eagles medium (Himedia). The cell line was cultivated in a tissue culture flask (25cm2) through DMEM augmented as sodium bicarbonate, L-glutamine, 10% FBS, and antibiotic solution having: Amphoteracin B (2.5µg/ml), Streptomycin (100µg/ml), and Penicillin (100U/ml). Cultivated cell lines were maintained at 37şC and moisturized in 5% CO2 incubator (NBS Eppendorf, US). The feasibility of the cells was determined through the explicit examination of cells using Inverted phase contrast microscope with MTT assay method.

 

Cells seeding in ninety six well plate:

The merging single-layered cells, which were two days matured, were trypsinized and were put off in 10% of progress, 100µl cell suspension (5x104cells/well) seeded in ninety six well tissue culture plates. Moreover, the tissues were kept warm at 37şC and moisturized in 5% CO2 incubator.

 

Preparation of plant extracts and compound stock:

DMEM (one ml) was mixed with the plant extract (one mg) which was totally dissipated in cyclo-mixer. Then, 0.22µm Millipore syringe filter was employed to filter the derived solution in order to assure the sterility.

 

Antiproliferative effect estimation:

One day later, the progress attained was eliminated. Furthermore, 5% DMEM of newly produced trails were thoroughly diluted using two fold dilution (50µg, 25µg, 12.5µg, 6.25µg in 100µl of 5% DMEM). All 100µl concentration were mixed in triplicates to its corresponding wells and kept warm at 37şC and moisturized in 5% CO2 incubator.

 

Anti-proliferative effect using Direct Microscopic examination:

The inverted phase contrast tissue culture microscope (Labomed TCM 400 inverted microscope) was employed to examine the overall plates with the interval of one to three days. Furthermore, the observed microscopic readings were stored as pictures. All remarkable modifications on the cell morphology like vacuolization and granulation on cell cytoplasm, and shrinking/rounding of cells were specified as cytotoxicity indicators10.

 

Cytotoxicity using MTT Process:

MTT (15mg) (Hi media) was recomposed with PBS (3 ml) till it totally dissipated, as well as cleaned using filter sterilization. One day later the incubation, the trail model in wells were separated as well as the recomposed MTT solution (30µl) was mixed with each test and cell control wells. Moreover, the plate was mildly shaken and kept warm at 37şC and moisturized in 5% CO2 incubator for a period of four hours. After incubation, the supernatants were separated as well as MTT solubilisation solution (DMSO) (100µl) was added. Besides, the formazan crystals were solubilised by the up and down pipetting of the wells. Micro plate reader was used to calculate the absorbance values at 570mm.11

 

Using the equation, the growth inhibition percentage (cells viability) was evaluated, where means OD of samples, and is the means OD of control groups.

 

 

Statistical Analysis:

Student’s t-test was used. Each test was carried out in triplicate. Data were expressed as mean±standard deviation (S.D.). The IC50 value, half maximal inhibitory concentration was also determined.

 

RESULTS AND DISCUSSION:

The therapeutic effect or the medicinal value of a plant extract depends on the nature and quantity of secondary metabolites present in it. Preliminary phytochemical study of the methanolic extract signifies the presence of major secondary metabolites such as alkaloids, glycosides, flavonoids, tannins, glycosides, saponins, phenolic compounds carbohydrates and proteins which is detailed in Table-1.

 

Table-1: Preliminary Phytochemical Screening of Various Extracts of Alangium salvifolium subsp. hexapetalum (Wangerin).

Sl. No

Phytoconstituents

PEE

CE

EAE

ME

1.

2.

3.

4.

5.

6.

7.

Alkaloids

Carbohydrates

Protein

Flavonoids

Saponin

Glycoside

Phenolic

-

+

+

-

+

+

-

-

+

+

+

+

-

+

+

+

-

+

+

-

+

+

+

+

+

+

+

+

PEE-Pet.ether extract, CE- chloroform extract, EAE- Ethyl acetate extract, ME- Methanolic extract. ‘+’ Presence, ‘-‘ Absence.

 

Based on the observation of preliminary phytochemical studies the methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum was used for antioxidant study.

 

DPPH radical scavenging activity was measured in terms of percentage inhibition of radical scavenging activity of various extracts of aerial parts of Alangium salvifolium subspecie hexapetalum compared with ascorbic acid is depicted in (Fig.-1). The IC50 value of standard ascorbic acid and the extracts were compared. The IC50 value of methanolic extract was found to be 175µg/ml which was lesser when compared to chloroform extract (200µg/ml) and ethyl acetate extract (320µg/ml). The lower the value the higher will be the radical scavenging activity. Similarly the ABTS radical scavenging activity was measured and the IC50 values were calculated and compared with standard ascorbic acid. Here it was observed that the IC50 value of chloroform extract was 48µg/ml and that of ethyl acetate extract was 44µg/ml which was greater than methanolic extract having IC50 value 40µg/ml. The ABTS radical scavenging activity was also measured in terms of percentage inhibition which is shown in (Fig.-2). In both the cases petroleum ether extract had the highest IC50 value.

 

Fig.- 1: DPPH radical scavenging activity

As.Acid- Ascorbic acid, CE- Chloroform Extract, EA- Ethyl acetate extract ,ME- Methanolic extract

 

Fig.-2: ABTS radical scavenging activity

As.Acid- Ascorbic acid, CE- Chloroform Extract, EA- Ethyl acetate extract ,ME- Methanolic extract

 

Reducing power (or antioxidant capacity) of the methanol, chloroform, ethyl acetate and petroleum ether extract of aerial parts of Alangium salvifolium subspecie hexapetalum, was determined and the results are summarised in (Fig. -3).

 

Fig.-3: Antioxidant activity using reducing power assay

CE- Chloroform Extract, EA- Ethyl acetate extract, ME- Methanolic extract

 

The antioxidant activity is exhibited by the presence of reductants which helps in breaking the free radical chain through donating a hydrogen atom. Fe3+/Fe2+ conversion was investigated in the presence of sample for the measurements of the reduced activity. The reducing capacity is estimated by the chelation of Fe2+ ions by the Decker and Welch method in which ferrozine quantitatively forms complexes with Fe2+. In the presence of chelating agents, the formation of this complex is disrupted there by formation of red color imparted by the complex. The absorbance was measured at 700nm on a UV /Visible spectrophotometer12.

 

The methanolic extract proved the highest antioxidant property, while the chloroform and ethyl acetate extracts showed almost good potency. All the extracts showed an increasing trend in reducing power with the increase in concentration. This may be due to the presence of phytoconstituents such as flavonoids and phenolics which is known to posses’ highest antioxidant property 13.

 

The difference in the antioxidant potency in different extracts may be due to the amount of phytoconstituents present in various extracts.

 

Cancer is one of the major causes of death worldwide. Adenosine triphosphate (ATP) is produced when cells generate energy by using oxygen. During this cellular redox reaction, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced as the by-products. ROS and RNS are useful for cellular functions and immune responses if they are in balanced level but the unbalance concentration of these will lead to oxidative stress and can also become mutagenic or carcinogenic leading to the cancer development14.

 

Based on the antioxidant study and preliminary phytochemical analysis the methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum (Wangerin) was used for evaluation of anticancer activity in HT29 cell lines using MTT assay method. The principle involved in MTT assay is the reduction of yellow 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells15. The intensity of colour developed by the reaction is directly proportional to the percentage of viable cancer cells. Percentage of cell viability refers to the number of living cells. As the concentration of extract increases (μg/ml), the absorbance decreases. The percentage of viable cell decreases from 90.79% to 30.43% as the concentration increases from 6.25µg/ml to 50µg/ml which is shown in Table-2.

 

Table -2: Effect of Methanolic Extract of Aerial Parts of Alangium salvifolium subsp.hexapetalum (Wangerin) on HT29 Cell Lines Using MTT Assay

Conc.(µg/ml)

Absorbance

% of viability

6.25

12.5

25

50

0.39122±0.0114

0.32501±0.00541

0.24311±0.00413

0.13112±0.00117

90.79

75.42

56.42

30.43

The values are mean ± S.D. (triplicates)

 

The half maximal inhibitory concentration (IC50) is a measure of the efficacy of a substance in interfering a specific biological or biochemical function. This quantitative measure specifies how much of sample is required to inhibit a given biological process. In the present study, the methanolic extract of Alangium salvifolium subspecie hexapetalum (Wangerin) showed IC50 of 30.22μg/ml. Thus, it can be concluded that 30.22µg/ml is the effective concentration of the sample to arrest the cancer cells which reveals that the methanolic extract has a very potent anticancer activity. The change in cell viability using methanolic extract was also examined using invert phase microscope and the pictures are depicted in (Fig.-4).

 

Fig.-4: Effect of methanolic extract on the presence of viable cells through formation of formazan crystals after MTT treatment

 

CONCLUSION:

The present study provides the fact that methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum (Wangerin) has a promising cytotoxic activity against HT29 human colon cancer cell lines. Preliminary phytochemical analysis of the extract showed the presence of potent secondary metabolites, which is responsible for various therapeutic effects. Further antioxidant study proved the presence of high free radical scavenging activity in the methanolic extract. The outcome of this study clearly indicates that the methanolic extract of aerial parts of Alangium salvifolium subspecie hexapetalum (Wangerin) has boundless biological activities mainly as a good source of antioxidant and antitumour agent.

 

CONFLICT OF INTEREST:

Authors report that there is no conflict of interest.

 

REFERENCE:

1.      Elisha Solowey.et.al. Evaluating Medicinal Plants for Anticancer Activity. The Scientific World Journal 2014.

2.      Sulekha Mandal, Satish Yadav, Sunita Yadav and Rajesh Kumar Nema. Antioxidants: A Review. Journal of Chemical and Pharmaceutical Research. 2009; 1(1) :102-104.

3.      Anita Thyagarajan and Ravi P. Sahu. Potential Contributions of Antioxidants to Cancer Therapy: Immunomodulation and Radiosensitization. Integrative cancer therapies. 2018; 17(2) : 210-216.

4.      Trease G E and Evans M C. Text book of Pharmacognosy, London: Bailier Tindall, 12th ed. 2002, 343-382.

5.      Harborne J B. Phytochemical methods, A guide to modern techniques of plant analysis, Springer India, 3rd ed. 1998,49-120.

6.      Nishandhini Marimuthu, Viswanathan T, Mahendran Radha, Rathisre. P.R, Jeyabaskar Suganya. Preliminary analysis of Phyto-constituents from the Leaf Extracts of Ballota nigra Linn. Research J. Pharm. and Tech. 2017; 10(1): 161-165.

7.      Mukesh Kumar DJ , Sonia K, Madhan R , Selvakumar K and Kalaichelvan PT. Antiyeast, Antioxidant and Anticancer Activity of Tribulus terrestris Linn and Bougainvillea spectabilis Linn. Research J. Pharm. and Tech. 2011; 4(9): 1483-1489

8.      Salah Eddine Marref, Naima Benkiki and Mohamed Akram Melakhessou. In vitro Antioxidant Activity, Total Phenolics and Flavonoids Contents of Gladiolus segetum Extracts. Research J. Pharm. and Tech 2018; 11(11): 5017-5023.

9.      Suneetha B et. al. Evaluation of In-vitro anti-oxidant activity of various extracts of Actinodaphne madraspatana leaves. Research Journal of Pharmacognosy and Phytochemistry 2014; 6 (1): 01-04

10.    Radhika Mahadev.et.al. Cytotoxic activity of methanolic extracts of Solanum Erianthum D. Don . Int J Pharm Pharm Sci. 2015; 7(2) : 106-108.

11.    Dhakshanamoorthy S, Murali Krishnan M and Arumugham MN. Synthesis, Characterisation, DNA binding/cleavage, anticancer and antimicrobial activity of Ternary Copper (II) Complexes. Asian Journal of Research in Chemistry 2017; 10(3):312-318.

12.    Dildar Ahmed , Muhammad Mehboob Khan and Ramsha Saeed. Comparative Analysis of Phenolics, Flavonoids, and Antioxidant and Antibacterial Potential of Methanolic, Hexanic and Aqueous Extracts from Adiantum caudatum Leaves. Antioxidants. 2015; 4(2) : 394-409.

13.    Duangjai Tungmunnithum, Areeya Thongboonyou, Apinan Pholboon and Aujana Yangsabai. Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines (Basel). 2018; 5(3) : 93.

14.    Neeta and Harish Dureja. Reverse Phase High-Performance Liquid Chromatographic Estimation and In vitro Cytotoxicity of Boswellic Acids on A-375 Melanoma Cancer Cell lines. Asian Journal of Pharmaceutical Analysis 2018; 8 (1): 13-19.

15.    Akila B and Manikandaselvi S. In Vitro Antioxidant and Anticancer efficacies of ethanolic fruit extract of Ziziphus jujube Mill .Asian J Pharm Clin Res. 2019; 12(6) :160-165.

16.    Merin Babu, Subin Varghese Thomas, Sruthi T P, Jipnomon Joseph. Evaluation of Cytotoxic Activity of Annona muricata Fruits and Leaves. Research J. Pharm. and Tech 2019; 12(8):3802-3806.

17.    Shelar MK, Patil MJ and Bhujbal SS. Phytochemical and Pharmacognostical Evaluation of Milky Mangrove Excoecaria agallocha Linn. Research J. Pharm. and Tech. 2019; 12(3): 1289-1293.

18.    NasimunIslam N1., Jemimah Naine S., Shaik Jameel, S.S. Khora, Venkat Kumar, Mohanasrinivasan V., C. Subathra Devi, Panneerselvam A. Cytotoxic Property of Cocos nucifera shell Extracts on HeLa Cells. Research J. Pharm. and Tech. 2014; 7(5): 521-525.

19.    Dwira S., Fadhillah M. R., Fadilah F., Azizah N. N, Putrianingsih R., Kusmardi K.. Cytotoxic Activity of Ethanol and Ethyl Acetate Extract of Kenikir (Cosmos caudatus) against Cervical Cancer Cell Line (HELA). Research J. Pharm. and Tech. 2019; 12(3): 1225-1229.

20.    Angel Steffy J, Henna Parveen M, Durga V and Manibalan S.Extraction Purification of Phlorotannins from Different Species of Marine Algae and Evaluation of their Anti-Oxidant Potential. Research Journal of Engineering and Technology 2013; 4 (4) :163-168.

21.    Datir SB et.al. Evaluation of Antioxidant Activity of the Aerial Parts of the Abutilon indicum (Linn) Sweet (Malvaceae). Research Journal of Pharmacology and Pharmacodynamics 2010; 2 (5): 324-327.

 

 

 

Received on 05.10.2019           Modified on 09.12.2019

Accepted on 11.02.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(8):3715-3719.

DOI: 10.5958/0974-360X.2020.00657.5