In vitro Biological Evaluation of Acanthophora spicifera

 

Shabna Roupal Morais, R. Ram Narayanan*, K. P. Sushmitha, N. Suchithra, Shruti Shankar, M. Sugumar

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Porur, Chennai - 600116.

 

ABSTRACT:

Over the years, the pursuit for new antioxidant compounds from natural products has mounted, due to health concerns regarding the potential toxic and side effects spawn from synthetic antioxidants. In this study the antioxidant potential and the cytotoxic activity was studied using hydroxyl radical scavenging assay and MTT assay using MCF 7 breast cancer cell lines. The hydroxyl radical scavenging potential of various solvent extracts of A.spicifera showed that hydroxyl radical scavenging capacity was increased with decreasing concentration of sample extracts. In the present investigation, out of the four extracts (hexane, ethyl acetate, chloroform, methanol) of   A. spicifera methanol showed potential hydroxyl radical scavenging activity than vitamin E in the range of 94.84-99.81% at the concentration range of 50μg/ml-1000μg/ml where the percentage inhibition increased with decrease in concentration. While scavenging hydroxyl radical, the ability of methanol extract (99.81%) to scavenge the hydroxyl radical was the highest among other extracts and Vitamin E. The methanolic extract of A.spicifera was evaluated for in-vitro anti-proliferative assay against MCF-7 cell line, where the cells were exposed to different concentrations of the extract. It is inferred from the results that the appearance of cytotoxic changes to the extract was in a concentration dependant manner. The results also indicate that the IC₅₀ value of the extract was found to be 47.58µg/ml.

 

 

 

INTRODUCTION:

Antioxidant compounds play an important role in various fields such as medical fields, cosmetics and food industries. Many commercialized synthetic antioxidants such as butylated hydroxyanisole (BHA) are used under strict regulation in certain countries because of their potential health hazards. Thus, the hunt for various antioxidants from natural merchandise has raised and among them, aquatic plants have gained the focus. Sea weeds have been as rich sources of various natural antioxidants. Acanthophora spicifera is an erect edible marine plant which belongs to the family of rhodophyta, Rhodomelaceae².They possess significant antimicrobial,³ hypolipidemic⁴, antibacterial⁵ and antiviral⁶ activity. The current study is to inspect the antioxidant activity and cytotoxicity of Acanthophora spicifera.

 

Figure 1: Acanthophora spicifera.

 

Plan of work:

1.       Collection of Seaweeds

2.       Preparation of Extract

3.       Phytochemical Screening

4.       Estimation of Tannic Acid

5.       Hydroxyl Ion Scavenging Assay

6.        MTT Assay

 

Collection of seaweeds:

Acanthophora spicefera was collected in the coastal area of Rameswaram, India during the month of November 2016. The collected seaweed material was recognized and authenticated by Mr. M. Rajendrakumar, R. K. Algae Project Centre, Mandapam, Ramanathapuram District. The whole seaweed material was shade dried and coarsely powdered.

 

Figure 2: Acanthophora spicefera

 

Preparation of extract:

The seaweed of Acanthophora spicifera was dried under shade and powdered. The powdered material was extracted successively with solvents of increasing polarity like Hexane, Chloroform, Ethyl acetate and Methanol. The extracts were concentrated and stored in a dessicator.

 

Figure 3. Hexane Extract

 

Figure 4. Chloroform Extract

 

Figure 5. Ethyl acetate Extract

 

Figure 6. Methanol Extract

 

Preliminary phytochemical screening:

Test for Metals:

Prepare ash of drug material. Add 50% v/v HCl or 50% v/v HNO₃ to ash. Keep for 1 hr or longer. Filter. With filtrate perform the following tests:

 

Test for Calcium:

1.     To 10ml filtrate, add 1 drop dil.NH₄OH and saturated ammonium oxalate solution. White precipitate of calcium oxalate forms. Precipitate is soluble in HCl but insoluble in acetic acid.

2.     With solution of ammonium carbonate it gives white precipitate which is insoluble in ammonium chloride solution.

 

Test for Magnesium:

1.     Filter and separate the white calcium oxalate precipitate obtained above. Heat and cool the filtrate with which a solution of sodium phosphate in dilute ammonia gives white crystalline precipitate.

2.     Gives white precipitate with ammonium carbonate solution but not with ammonium chloride solution.

 

Test for Sodium:

1.     To 2ml solution, add little uranyl magnesium acetate reagent. Shake well and keep it for few minutes. Yellow crystalline precipitate of sodium magnesium uranyl acetate is observed.

2.     Flame test: Prepare thick paste of ash of drug with conc. HCl. Take paste on platinum wire loop, introduce in Bunsen flame. Golden yellow flame is observed.

3.     10ml ash extract + 2ml of potassium pyroanthllollate gives white precipitate.

 

Test for Potassium:

1.     To 2-3ml test solution add few drops of sodium cobalt nitrate solution. Yellow precipitate of potassium cobalt nitrite observed.

2.     Flame test: Gives violet colour to flame.

 

Test for Iron:

1.     To 5ml test solution, add few drops 2% potassium ferrocyanide solution. Dark blue colouration is observed.

2.     To 5ml test solution, add few drops 5% ammonium thiocyanate (or 5% potassium thiocynate) solution. Solution turns blood red.

 

Test for Sulphate:

1.     To 5ml of test solution add few drops 5% BaCl₂ solution. White crystalline BaSO₄ precipitate appears which is insoluble in HCl.

2.     With lead acetate reagent it gives white precipitate soluble in NaOH.

 

Test for Phosphate:

To 5ml test solution prepared in HNO_3, add few drops ammoniummolybdate solution. Heat for 10min. Cool. Yellow crystalline precipitate of ammonium phosphomolybdate is observed.

 

Test for Chloride:

1.     To 3ml test solution prepared in HNO_3, add few drops of 10% AgNO₃ solution. White precipitate of AgCl₂ is observed. Precipitate is soluble in dil. Ammonia solution.

2.     To about 5ml to 7ml filtrate, add 3ml to 5ml of lead acetate solution. White precipitate soluble in hot water is observed.

3.     On heating with MnO₂ and H₂SO_{4 ,}chlorine is liberated which gives blue colour to starch solution.

 

Test for Carbonate:

1.     With dilute acid carbon dioxide is liberated.

2.     With mercuric chloride solution a brownish-red precipitate is obtained.

3.     With solution of magnesium sulphate, white precipitate is formed.

 

Test for Nitrates:

1.     Red fumes are liberated when warmed with sulphuric acid and copper.

2.     With solution of ferrous sulphate no brown colour can be seen but if sulphuric acid is added, a brown coloured ring is produced at the junction of two liquids.

 

Test for secondary metabolites:

Test for Terpenoids- Nollers Test:

The extract is warmed with tin and thionyl chloride. Pink colouration indicates the presence of terpenoids.

 

Test for Flavones:

Shinoda’s Test:

1.     To the extract in alcohol, few magnesium turnings and few drops of concentrated hydrochloric acid are added and boiled for 5mins. Red colouration shows the presence of flavones.

2.     To the extract in alcohol, 100% caustic soda solution or ammonia is added. Dark yellow color indicates the presence of flavones.

 

Test for Steroids - Lieberemann- Burchard test:

1mg of the test extract is dissolved in a few drops of chloroform, 3ml of acetic anhydride and 3ml of glacial acetic acid are added, warmed and cooled under water and few drops of concentrated sulphuric acid are added on the edges of the tube. appearance of blue green color shows the presence of steroids.

 

Test for Anthraquinones:

Borntrager’s test:

1.     The extract is macerated with ether. after filtration, aqueous ammonia or caustic acid is added. Pink, red or violet colour in the aqueous layer after shaking indicates the presence of anthraquinones.

2.     If present as a glycoside then the test should be changed by hydrolyzing with hydrochloric acid, as the initiative.

 

Test for Glycosides:

The extract is mixed with a little anthrone in a watch glass. One drop of sulphuric acid is added and is made into a paste and warm gently over a water bath. Dark green coloration is the indication of the presence of glycosides.

 

Test for Sugars:

The extract is mixed with Fehling’s solution A and B  and heated. Formation of red coloration is the indication of the presence of sugars.

 

Test for Alkaloids:

1.     To the extract a few drops of acetic acid are added, followed by Dragendroff”s reagent and jolted well. Formation of orange red precipitate indicates the presence of alkaloids.

2.     The extract is mixed with very little quantity of dil. hydrochloric acid and Mayer’s reagent. Formation of white precipitate indicates the presence of alkaloids.

 

Test for Quinones:

To the test extract, caustic soda is added. Blue, green or red color indicates the presence of quinine.

 

Test for Phenol:

To the extract, a few drops of alcohol and ferric chloride are added. blue green or red color indicates the presence of phenol.

 

Test for Tannins:

The extract is mixed with basic lead acetate solution. Formation of white precipitate indicates the presence of tannins.

 

Test for Saponins:

The extract is shaken with water. Copious lather formation indicates the presence of saponins.

 

Estimation of tannic acid:

Reagents required:

·       Folin’s phenol reagent (1:2)

·       35% w/v Sodium carbonate.

·       Stock tannin solution: Weigh precisely 100mg of tannic acid. Dissolve it in distilled water and make up to 100ml in standard flask. (conc. 1mg/ml)

·       Working standard: Dilute 1ml of the stock solution to 25ml with distilled water in a standard flask. 1ml of this solution contains 40µg of tannins.

·       Preparation of extract: 1mg of tannin positive extract (Chloroform, Ethyl acetate, Methanol) was weighed and dissolved in 10ml of 1:1 DMSO and Methanol-Water system (70:30). From this 1ml was used for estimation.

 

Procedure:

·       0.2ml (8µg), 0.4ml (16µg), 0.6ml (24µg), 0.8ml (32µg) and 1.0ml (40µg) of the working standard were pipetted out into a sequences of test tubes.

·       1ml of the extract was pipetted out in duplicates.

·       The volume was made up to 1ml with distilled water. 1ml of water aids as the blank.

·       To this, 0.5ml of Folin’s phenol reagent (1:2) trailed by 5ml of 35%w/v sodium carbonate were added and kept at room temperature for 5 min.

·       The intensity of the blue colour formed was measured at 640nm and standard graph was plotted.

 

In vitro antioxidant activity:

Determination of Hydroxyl Ion Scavenging Activity:

Materials required:

·       2-deoxy-D-ribose (2.8mM)

·       Potassium dihydrogen phosphate (20mM)

·       Ethylenediaminetetraacetate (EDTA – 1.04mM)

·       Ferric chloride (200µM)

·       Hydrogen peroxide (1mM)

·       Vitamin E (1mM - 50, 100, 200, 400, 600, 800, 1000 µg/ml)

·       Hydrogen peroxide (1mM)

·       Thiobarbituric acid (TBA -1%)

·       Trichloroacetic acid (TCA – 2.8%)

·       Extract (50, 100, 200, 400, 600, 800, 1000µg/ml)

 

Principle:

The hydroxyl radical can induce oxidative damage to DNA, lipids and proteins. The hydroxyl radical reacts with 2-deoxy-D-ribose to undergo an oxidative degradation which leads to formation of a complex. This complex on treatment with thiobarbituric acid will enhance the chromogen formation (pink colour). The hydroxyl radical scavenging ability of the extracts was determined by its ability to compete with 2-deoxy-D-ribose and diminish chromogen formation (pink).

 

Procedure:

·       The reaction mixture contained 100µl of 2- deoxy -D- ribose (2.8mM), 500µl of extract dissolved in (0.1 mM) phosphate buffer (pH 7.4), 200µl of a mixture of ferric chloride and EDTA (1:1v/v), 100µl of hydrogen peroxide (1mM).

·       The mixture was incubated for 1 hour at 37^OC.

·       After the incubation, 1ml of TBA (1% w/v) and 1ml of TCA (2.8 % w/v) were added.

·       The mixture was then heated at 90^oC for 20min. to develop the colour.

·       The mixture was cooled and centrifuged at 3000rpm.

·       The absorbance of the supernatant was measured at 532 nm against an appropriate blank solution.

 

In- vitro antiproliferative activity against mcf-7 breast cancer cell lines:

MTT Assay:

Reagents required:

·       0.5% DMSO

·       MTT(3-[4,5-dimethylthiozole-2-yl]-2,5-diphenyltetrazolium bromide)

·       RPMI-1640 medium

·       20% FBS

·       2mM L-glutamine

·       Phosphate buffered saline (pH 7.4)

 

Principle:

The MTT assay⁷ is a colorimetric assay for measuring the activity of enzymes that reduce MTT insoluble to formazan dyes, which gives purple colour. A main application allows to access the viability (Cell counting) and the proliferation of cells (Cell Culture assays). It can also be used to determine the cytotoxicity of potential medical agents and toxic materials, since those agents would stimulate MTT. 3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, (a yellow tetrazole), is reduced to purple formazan in living cells. A solubalization solution (Usually either dimethyl sulfoxide and acidified ethanol solution, or a solution of the detergent sodium dodecyl sulphate in dilute hydrochloric acid) is added to dissolve the insoluble purple formazan product into a coloured solution. The absorbance of this coloured solution can be quantified by measuring at a certain wavelength (Usually between 500 and 600nm) by spectrophotometer. The absorption maximum is dependent on the solvent employed.

 

Cell lines and cultural conditions:

Human cancer cell lines were cultured in RPMI-1640 medium with 20% FBS, 2mM L-glutamine, 1% penicillin or streptomycin under a fully humidified atmosphere 5% CO₂ at 37^oC.

 

Procedure:

·       The effect of plant extract on the viability of human cancer cell lines were determined by MTT (3-[4,5-dimethylthiozole-2-yl]-2,5-diphenyltetrazolium bromide) assay.

·       100μl of cell suspensions in growth medium were plated in 96 well microtitre plates at concentrations of 1x10⁴cells/well and incubated for 48hours at 37^oC in a humidified incubator.

·       After 48 hours of incubation, the cell reaches confluence.

·       Then, cells were incubated in the presence of various concentrations of the samples in 0.1% DMSO for 72 hours at 37^oC.

·       After removal of the sample solution and washing with phosphate buffered saline (pH 7.4), 20μL of MTT (5mg/ml) was added to each well of the plate.

·       The plate was incubated for 4hours at 37^oC.

·       The solution in each well along with MTT was aspirated and 100μL of buffered DMSO was added to dissolve formazan.

·       The plates were quivered for 5min.

·       Optical density was measured on a microplate ELISA reader at 540nm with DMSO as control.

·       The cytotoxicity was attained by relating the absorbance between the samples and control.

 

                      Abs (control) – Abs (Test)

% Inhibition = --------------------------------- x100

                                      Abs (control)

IC₅₀was premeditated from dose-response curve.

 

RESULTS:

Table 1: Preliminary phytochemical screening

Sl. No	Tests	Hexane Extract	Chloroform Extract	Ethyl acetate Extract	Methanol Extract
1.	Test for Calcium	-	-	-	-
2.	Test for Magnesium	-	-	-	-
3.	Test for Sodium	-	-	-	-
4.	Test for Potassium	-	-	-	-
5.	Test for Iron	-	-	-	-
6.	Test for Sulphate	-	-	-	-
7.	Test for Phosphate	-	-	-	-
8.	Test for Chloride	-	-	-	-
9.	Test for Carbonates	-	-	-	-
10.	Test for Nitrates	+	+	+	+

 

Table 1: shows the results for presence of metals in Acanthophora spicefera. The preliminary phytochemical screening test for A.spicifera such as calcium, magnesium, sodium, potassium, iron, sulphate, phosphate, nitrates etc was performed for all the four extracts. Nitrates were present in all the four extracts.

 

Table 2: Test for secondary metabolites

Sl. No	Tests	Hexane Extract	Chloroform Extract	Ethyl acetate Extract	Methanol Extract
1.	Test for Terpenoids [Noller’s Test]	-	-	-	-
2.	Test for Flavones [Shinoda Test]	-	-	-	-
3.	Test for Steroids [Libermann Burchard Test]	-	-	-	-
4.	Test for Anthraquinone Glycoside [Borntrager’s Test]	-	-	-	-
5.	Test for Glycoside	-	+	+	+
6.	Test for Sugars	-	+	+	+
7.	Test for Alkaloids	-	-	-	-
8.	Test for Quinones	-	-	-	-
9.	Test for Phenol	-	-	-	-
10.	Test for Tannins	-	+	+	+
11.	Test for Saponins	-	+	+	+

 

Table 2: shows the results for presence of secondary metabolites in Acanthophora spicifera. The secondary metabolities test for terpenoids, quinones, phenol, tannins, sugars and saponins was performed⁸ for all the four extracts. Glycoside, sugars, tannins and saponins were present in the Chloroform, Ethyl acetate and Methanol extracts.

 

Estimation of tannins:

Standard curve:

Table 3: Estimation of Tannins (Standard)

Concentration (µg/ml)	Absorbance (at 640nm)
4	1.388
8	1.875
16	2.396
24	2.679
32	2.919
40	3.121

 

Figure 7: Estimation of tannins (Standard curve)

Estimation of tannins:

Table 4: Tannic Acid Equivalent

Extracts	Tannic acid equivalent (mg/g)
Chloroform	3.225
Ethyl Acetate	3.273
Methanol	4.273

 

The table 3 shows the absorbance values for different concentrations of the standard. Table 4 shows the tannin content in terms of tannic acid equivalent for different extracts of A.spicifera The graph showing a plot of the standard curve was shown in the Figure 7.From the results, it was interpreted that the various extracts contained tannic acid in a solvent dependent manner. In Acanthophora spicifera, the tannic acid content in the Methanolic extract was found to be the highest with a tannic acid⁹ equivalent of 4.273mg/g.

 

Hydroxyl Radical Scavenging Assay:

Table 5: Hydroxyl Radical Scavenging Assay of Acanthophora spicifera

Concentration (µg/ml)	Percentage Inhibition
	Vitamin-E	Hexane	Chloroform	Ethyl acetate	Methanol
50	93.12	97.06	98.01	98.65	99.81
100	92.8	96.58	97.96	97.49	98.91
200	92.65	93.69	94.28	96.98	98.02
400	92.65	90.01	84.81	96.01	97.6
600	92.03	89.9	83.12	75.15	96.84
800	91.09	88.43	77.5	62.18	95.62
1000	88.28	76.09	60.62	53.43	94.84

 

 

Figure 8: Hydroxyl Radical Scavenging Assay of Acanthophora spicifera

 

The hydroxyl radical scavenging potential of various solvent extracts of A. spicifera exhibited that hydroxyl radical scavenging capacity was increased with decreasing concentration of sample extracts. In the present investigation, out of the four extracts (hexane, ethyl acetate, chloroform, methanol) of A. spicifera methanol showed potential hydroxyl radical scavenging activity than vitamin E in the range of 94.84-99.81% at the concentration range of 50μg/ml-1000μg/ml where the percentage inhibition increased with decrease in concentration. While scavenging hydroxyl radical, the ability of methanol extract¹⁰ (99.81%) to scavenge the hydroxyl radical was the highest among other extracts and Vitamin E.

MTT Assay:

 

Figure 9: Direct microscopic observations

 

The representative snapshots of the treated cells at various concentrations displays the presence of cell blebbing, chromatin condensation and apoptotic fragments at high concentration.

 

MTT Assay:

Table 6. MTT Assay of Acanthophora spicifera

Drug Conc. (µg/ml)	MCF7			Average	Average-Blank	%viability	%IC
0.1	1.019	.019	1.018	1.01867	1.016666667	85.219335	14.75593
1	0.95	0.96	0.96	0.95667	0.954666667	80.0223526	19.94421
10	0.78	0.781	0.78	0.78033	0.778333333	65.2416876	34.70014
100	0.428	0.428	0.428	0.428	0.426	35.7082984	64.1841
300	0.103	0.103	0.103	0.103	0.101	8.46605197	91.38075
Untreated	1.196	0.196	1.193	1.195	1.193	100	0
Blank	0.002	0.002	0.002	0.002	0	0	100
IC50=47.58µg/ml

 

Fig.10: MTT Assay of Acanthophora spicifera

 

The methanolic extract of A. spicifera was appraised for in-vitro antiproliferative assay against MCF-7 cell line, where the cells were exposed to diverse concentrations of the extract. It is inferred from the fig.9 that the appearance of cytotoxic changes to the extract was in a concentration dependant manner. The results also specify that the IC₅₀ value of the extract was found to be 47.58µg/ml.

 

CONCLUSION:

The objective of the current study was to scrutinize and appraise the biological potential of Acanthophora spicifera. It was vetted mainly for their antioxidant activity using hydroxyl radical scavenging assay and the latter was found to possess effective in vitro antiproliferative activity against Human breast cancer Cell lines- MCF 7. The various extracts of Acanthophora spicifera was vetted for its antioxidant potential using hydroxyl radical scavenging assay. The extracts showed a prominent antioxidant activity in a dose dependant manner with a good scavenging capability at low concentration and contrariwise. Therefore, plants rich in tannins are considered as potential antioxidant as they neutralize the free radicals via donation of hydrogen atom, quenching of oxygen and by chelation of metals thus, minimizing oxidative stress¹¹. Tannins have the ability to chelate metal ions such as Fe(Ⅱ) and interfere with one of steps in the fenton reaction and thereby retard oxidation. The in vitro anti-proliferative study was performed for the methanolic extract of Acanthophora spicifera and it showed a dose dependant cytotoxic effect against MCF-7 human breast cancer cell lines with the IC₅₀ value of 47.58µg/ml.

 

ACKNOWLEDGEMENT:

We would like to thank the management of Sri Ramachandra Institute of Higher Education and Research (DU), Porur, Chennai-600116 for their continuous support during the research work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      "The Significance of Seaweed | KAUST Discovery". Discovery.kaust.edu.sa. N.P., 2016. Web. 19 Dec. 2016.

2.      "Main Characteristics of Rhodophyceae (Red Algae)". Plantscience4u.com. N.P., 2016. Web. 21 Dec. 2016.

3.      Nurul ZA, Darah I, Shaida SF, Nor SA. Screening for antimicrobial activity of various extracts of Acanthophora spicifera (Rhodomelaceae, Ceramiales) from Malaysian waters. Res J Biol Sci. 2010;5:368-75.

4.      Hetta M, Hassan S, Abdel-Tawab S, Bastawy M, Mahmoud B. Hypolipidaemic effect of Acanthophora spicifera (red alga) and Cystoseiratrinode (Brown alga) on albino rats. Iranian Journal of Science and Technology (Sciences). 2009 Dec 12;33(4):287-97.

5.      Pandian P, Selvamuthukumar S, Manavalan R, Parthasarthy V. Screening of antibacterial and antifungal activities of red marine algae Acanthaphora spicifera (Rhodophyceae). J. Biomed. Sci. Res.2011;3(3):444-8.

6.      Nogueira CC, de Palmer Paixão IC, Cirne-Santos CC, Stephens PR, Villaça RC, de Souza Pereira H, Teixeira VE. Anti-HIV-1 activity in human primary cells and Anti-HIV-1 RT inhibitory activity of extracts from the red seaweed Acanthophora spicifera Journal of Medicinal Plants Research. 2016 Sep 17;10(35):621-5.

7.      Van Meerloo J, et al. "Cell Sensitivity Assays: The MTT Assay. - Pubmed - NCBI". Ncbi.nlm.nih.gov. N.p., 2016. Web. 19 Dec. 2016.

8.      Abirami RG, Kowsalya S. Phytochemical screening, microbial load and antimicrobial activity of underexploited seaweeds. International Research Journal of Microbiology. 2012;3(10):328-32.

9.      Lavakumar V, Ahamed KF, Ravichandiran V. Anticancer and antioxidant effect of Acanthophora spicifera against EAC induced carcinoma in mice. J Pharm Res. 2012 Mar;5:1503-7.

10.   Zakaria NA, Ibrahim D, Sulaiman SF, Supardy A. Assessment of antioxidant activity, total phenolic content and in vitro toxicity of Malaysian red seaweed, Acanthophora spicifera. J Chem Pharm Res. 2011;3(3):182-91.

11.   Patel DK, Kumar R, Laloo D, Sairam K, Hemalatha S. Evaluation of phytochemical and antioxidant activities of the different fractions of Hybanthus enneaspermus (Linn.) F. Muell. (Violaceae). Asian Pac J Trop Med 2011;4:391-396.

 

 

 

Received on 13.12.2019           Modified on 28.01.2020

Accepted on 14.03.2020         © RJPT All right reserved