Cytotoxic and Antiradical Activities of Extracts of Rhizopora apiculata (L)
B. Vijayakumar1*, Banurekha J2, G. Swarnalatha3, D. Jothieswari4
1Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Koudunthirapully,
Palakkad, Kerala-678004, India.
4Department of Pharmaceutical Ananlysis, Sri Venkateswara College of Pharmacy, RVS Nagar, Chittoor, Andhra Pradesh-517127, India
*Corresponding Author E-mail: vijaykumarbvk86@gmail.com
ABSTRACT:
The petroleum ether and methanol crude extracts of Rhizopora apiculata (L) (Rhizoporaceae) were screened for cytotoxicity by MTT bioassay and antioxidant activity using three different methods. The active constituents of the plant Rhizopora apiculata (L) were extracted using petroleum ether and methanol. Both extracts were tested for cytotoxicity by MTT assay at a concentration range of 18.75, 37.5, 100, 150 and 300µg/ml. The percentage cell viability shown in Pet ether extract for human cervical adenocarcinoma cell lines (HeLa), human osteosarcoma cell lines (MG 63) and Breast adenocarcinoma (MC 67) were in the range of 102-91%, 100-84% and 101-78% respectively shows that pet ether extract does not have considerable cytotoxic activity.On the other hand, Petroleum ether and methanol crude extracts of Rhizopora apiculata (L) showed mild antioxidant activity with compared to ascorbic acid.
KEYWORDS: Rhizopora apiculata, Breast adenocarcinoma, Human osteosarcoma cell lines Ascorbic acid
INTRODUCTION:
Rhizopora apiculata is one member of Rhizoporaceae family that grows in Australia, Guam, India, Indonesia, Malaysia, Micronesia, New Caledonia, Papua New Guinea, the Philippines, Singapore, the Solomon Islands, Sri Lanka, Taiwan, the Maldives, Thailand, Vanuatu, and Vietnam [1]. Mangroves occur in 121 countries covering 15 million hectares worldwide. Asia harbors the largest mangroves in the world; India alone contributes 3% of the global mangrove habitat. Mangrove plants are widely used in medicine to treat a variety of diseases [2]. Specifically, there has yet to be a study examining these types of activities with the widely used R. apiculata. Most of the mangroves found along Mumbai coast are used as alternative medicine for the treatment of various diseases.
Rhizophora apiculata is considered having astringent, antidiarrhoea, antiemetic and haemostatic properties. Larvicidal and antiviral properties are also reported of these species [3]. only some have been assessed for biological activities; even less of these very few have been studied for potential antioxidant and cytotoxic activity Hence, to provide strength to the claims that R. apiculata has various potential health benefits; in this study we investigated the above mentioned antioxidant and cytotoxic activities (present in an extract) in an in-vitro model [4-5].
MATERIAL AND METHODS:
Plant Material:
The whole plant of Rhizopora apiculata (L) was collected from Andaman Islands, India, in the month of December, 2014. The taxonomical identification of the plant was done by Taxonomist, Dr. K. Madhava Chetty and Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati-517502, Andhra Paradesh, India. The voucher specimen bearing the number 1566 was preserved in our laboratory for future reference.
Extraction:
The plant material was dried in the shade, then the shade dried plant material was subjected to pulverization to get coarse powder and it was extracted in Soxhlet apparatus using various solvents. Powdered material was extracted successively with petroleum ether (60-80°C) and Methanol (60°C) [6-7]. Extracts solution was evaporated to obtain dried extracts and analyzed using chemical reagent for preliminary phytochemical investigation of various groups of phytoconstituents such as alkaloids, flavonoids, saponins, terpenoids, tannins, carotenoids, and steroids by using the standard procedure.
ANTIOXIDANT STUDIES (In vitro models):
Reducing Power Assay:
The reducing power was determined to the method of various concentration of sample were mixed with 1ml of 200m mol/l sodium phosphate buffer (pH6.6) and 1% potassium ferric cyanide. The mixture was incubated at 50°c for 20minites. After 1ml of 10% trichloroacetic acid was added, the mixture was centrifuged at 2000rpm for 10minites. The upper layer solution (2.5ml) was mixed with 2.5ml of deionised water and 0.3ml of fresh ferric chloride (0.1%). The absorbance was measured at 700nm.
Determination of Total Antioxidant Capacity:
The total antioxidant capacity (TAOC) was evaluated by the method of prieto et al. An aliquot of 0.1ml of sample solution (1mg/ml) was combined with 1 ml of reagent solution (600mm H2SO4, 28mm sodium phosphate and 4mm ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95°c for 90min. After the sample had cooled to room temperature. The absorbance of the aqueous solution of each was measured at 695nm against a blank. A typical blank solution contained 1ml of reagent solution and the appropriate volume of the same solvent used for the sample and it was incubated under the same concentration. The antioxidant capacity was expressed as the number of equivalents of Ascorbic acid.
Scavenging of hydrogen peroxide:
A solution of H2O2 (20mm) was prepared in phosphate buffer saline (PBS, PH 7.4). Various concentration (10µg-100µg) of standard and extracts was prepared, 1ml of the extract and standard was dissolved in methanol in a separate volumetric flask and to this solution 2ml of H2O2 solution in PBS was added, the absorbance was measured at 230nm, after 10min against blank solution [8-11].
CYTOTOXICITY STUDIES:
Cell lines:
The human cervical adenocarcinoma cell lines (HeLa), human osteosarcoma cell lines (MG 63) and Breast adenocarcinoma (MC 67), were obtained from National Centre for Cell Science (NCCS), Pune and grown in Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 370C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.
Cell treatment procedure:
The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x105 cells/ml. One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the test samples. They were initially dissolved in neat dimethylsulfoxide (DMSO) and an aliquot of the sample solution was diluted to twice the desired final maximum test concentration with serum free medium. Additional four serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100µl of these different sample dilutions were added to the appropriate wells already containing 100µl of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 370C, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.
MTT assay:
3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore,the amount of formazan produced is directly proportional to the number of viable cells.
After 48 h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570nm using micro plate reader [12-13]. The percentage cell viability was then calculated with respect to control as follows
% Cell viability = [A] Test/[A] control x 100
RESULTS AND DISCUSSION:
Extractive Yield:
The percentage yield and preliminary phytochemicals present in various extracts of Rhizopora apiculata (L).given Table 1.
Table 1. Percentage yield, colour, consistency of extracts of Rhizopora apiculata (L)
|
Extracts |
Dry wt. % w/w |
Colour |
Consistency |
|
Petroleum Ether |
2.93 |
Greenish Colour |
Slight sticky mass |
|
Methanol |
3.28 |
Blackish green |
Slight sticky mass |
Preliminary Phytochemical Screening
The result of preliminary phytochemical analysis of Rhizopora apiculata (L)extracts is shown in table 2.
Table 2. Results of Phytochemical screening of Rhizopora apiculata (L).
|
Chemical constituents |
Petroleum ether extract |
Methanol extract |
|
Alkaloids |
- |
- |
|
Glycosides |
- |
- |
|
Flavanoids |
- |
- |
|
Carbohydrates |
+ |
+ |
|
Proteins |
- |
- |
|
Steroids |
+ |
+ |
|
Tannins |
+ |
+ |
|
Triterperoids |
- |
- |
‘-’ = Absence, ‘+’ = Presence of chemical constituents
Anti Oxidant Activity (In-vitro Model):
The antioxidant activity of Pet ether and methanolic extract of Rhizopora apiculata (L)were carried out using Reducing Power assay, Phosphomolybdneum and Hydrogen Peroxide methods and the results are recorded in the following Tables: 3.
Table 3. Antioxidant activity of Pet ether and Methanolic Extracts of Rhizopora apiculata (L)
|
S. No
|
Extracts |
Concentration |
||
|
50µg/ml |
100µg/ml |
150µg/ml |
||
|
Reducing Power Assay |
||||
|
1 |
Petroleum ether extract |
0.35± 0.001 |
0.45± 0.002 |
0.46± 0.009 |
|
2 |
Methanolic extract |
0.41± 0.10 |
0.42± 0.09 |
0.64± 0.010 |
|
3 |
Standard (Ascorbic acid) |
0.73± 0.005 |
0.88± 0.010 |
0.94± 0.010 |
|
Phosphomolybdneum Method |
||||
|
1 |
Petroleum ether extract |
0.018± 0.007 |
0.022± 0.009 |
0.026± 0.01 |
|
2 |
Methanolic extract |
0.019± 0.003 |
0.024± 0.005 |
0.033± 0.018 |
|
3 |
Standard (Ascorbic acid) |
0.035± 0.004 |
0.042± 0.004 |
0.064± 0.007 |
|
H2O2 Scavenging Activity |
||||
|
1 |
Petroleum ether extract |
0.011± 0.005 |
0.019± 0.006 |
0.029± 0.005 |
|
2 |
Methanolic extract |
0.033± 0.004 |
0.059± 0.005 |
0.060± 0.004 |
|
3 |
Standard (Ascorbic acid) |
0.044± 0.002 |
0.063± 0.005 |
0.072± 0.03 |
CYTOTOXITY STUDIES:
The cytotoxicity activity of different cell lines by using Pet ether and methanolic extract of Rhizopora apiculata (L) were carried out using MTT assay method and the results are recorded in the following Tables: 4.
Table 4. Percentage Cell viability of different cell lines in Pet ether and Methanolic Extract of Rhizopora apiculata (L)
|
S. No |
Extracts |
Conc (µg/ml) |
% Cell viability |
||
|
|
|
|
Human cervical adenocarcinoma cell lines (HeLa) |
Human osteosarcoma cell lines (MG 63) |
Breast adenocarcinoma (MCF7 PE) |
|
1 |
Pet ether |
18.75 |
102 |
100 |
101 |
|
37.5 |
101 |
98 |
101 |
||
|
75 |
100 |
95 |
99 |
||
|
150 |
95 |
93 |
84 |
||
|
300 |
91 |
84 |
78 |
||
|
2 |
Methanol |
18.75 |
102 |
100 |
99 |
|
37.5 |
98 |
99 |
97 |
||
|
75 |
93 |
91 |
97 |
||
|
150 |
90 |
87 |
92 |
||
|
300 |
75 |
79 |
80 |
||
DISCUSSION:
The physicochemical evaluation using percentage yield (% w/w), color and consistency parameters is useful in the identification and authentication of crude extract. Preliminary phytochemical evaluation will express the chemical contents of each extract.All the extracts showed the presence of various phytochemical constituents like Steroid, Tannins, Carbohydrates. However the extracts showed negative results for Alkaloids, Glycosides, Flavanoids, Proteins and Triterpenoids.
In this reducing power method, the absorbance values of Pet ether and methanolic extracts showed lower activity than that of standard values. Ascorbic acid was used as standard.
In this phosphomolybdenum method the absorbance values of Pet ether and methanolic extracts showed lower activity than that of standard values. Ascorbic acid was used as standard.
In hydrogen peroxide method, H2O2 scavenging activity was determined from absorbance values of Pet ether extracts and methanolic extracts. The Pet Ether extract showed lower activity, but methanolic extract show significant when compared with standard values. Ascorbic acid was used as standard.
The active constituents of the plant Rhizopora apiculata (L) were extracted using petroleum ether and methanol. Both extracts were tested for cytotoxicity by MTT assay at a concentration range of 18.75, 37.5, 100, 150 and 300µg/ml. The percentage cell viability shown in Pet ether extract for human cervical adenocarcinoma cell lines (HeLa), human osteosarcoma cell lines (MG 63) and Breast adenocarcinoma (MC 67) were in the range of 102-91%, 100-84% and 101-78% respectively shows that pet ether extract does not have considerable cytotoxic activity. The percentage cell viability shown Methanolic extract for human cervical adenocarcinoma cell lines (HeLa), human osteosarcoma cell lines (MG 63) and Breast adenocarcinoma (MC 67) were in the range of 102-75%, 100-79% and 99-80% respectively shows that methanolic extract does not have considerable cytotoxic activity.
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Received on 27.07.2019 Modified on 17.09.2019
Accepted on 06.11.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(5):2155-2158.
DOI: 10.5958/0974-360X.2020.00387.X