TWO ALGAE FROM OMAN SEA: THE IN VITRO BIOLOGICAL EFFECTS

TWO ALGAE FROM OMAN SEA: THE IN VITRO BIOLOGICAL EFFECTS

H. Hajimehdipoor ¹, B. Gharanjik ², H. Soleimani ³, Z. Mousavi ³ and M. Hamzeloo-Moghadam^*1

Traditional Medicine and Materia Medica Research Center and Department of Traditional Pharmacy ¹, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Offshore Waters Research Center ², Chabahar, Iran.

Pharmaceutical Sciences Branch ³, Islamic Azad University, Tehran, Iran.

ABSTRACT: Algae are extensively used in cosmetics, supplements and pharmaceutical industries. Researches support their hypotensive, cytotoxic, anti-inflammatory and many other biologic effects. In the present study, the cytotoxic, antioxidant and acetylcholinesterase inhibitory activities of two brown algae, Padina tetrastromatica and P. australis, have been evaluated. Total extract was prepared using methanol 80%. The extracts were evaluated for antioxidant and acetylcholinesterase inhibitory activity and cytotoxic properties against A-549, HT-29, HepG-2 and MCF-7 cell lines through DPPH, Ellman and MTT assays, respectively. The results revealed that P. australis with the IC₅₀ value of 163.9μg/mL demonstrated stronger antioxidant activity compared to P. tetrastromatica (IC₅₀ 259.1μg/mL) in DPPH assay. Neither of the total extracts showed cytotoxic activity up to the maximum amount examined (100μg/mL). The total extracts of P. australis and P. tetrastromatica in concentration of 300μg/mL presented 15.6% and 23.7% enzyme inhibition in Ellman assay, respectively. It was concluded that the two algae have shown weak antioxidant and acetylcholinesterase inhibitory activity and their further use in prevention and treatment of diseases needs more investigations especially in the field of isolation of active compounds.

Antioxidant, DPPH, Ellman assay, MTT assay, Padina tetrastromatica, Padina australis

INTRODUCTION: There are a large number of creatures living in the oceans of which only 200,000 invertebrate species have been described ¹. Many compounds with biological activities have been isolated from marine species which have shown to be promising in the primary steps of hard to manage diseases; thus more attention has been focused to research for pharmaceuticals with marine origin. Algae comprise various species; from the bacteria-sized coccoid taxa to the world’s greatest protists, including diatoms, giant kelps, red, green, and brown algae.

The brown alga possesses chlorophyll C and kind of special xanthophyll due to which they are called brown algae ^{2, 3}. They are found in rocky coasts around the world and prefer cold areas ³. Padina australias Hauck and P. tetrastramonia Hauck (Dictyotaceae family) are two known brown algae. Due to the rising interest for finding new sources of medications with natural origin and because of the increasing focus to the products with marine origin, the above two brown algae which are found at the coasts of the Oman Sea have been investigated for some biological effects in the present study.

MATERIALS AND METHODS:

Plant Material: Padina australias Hauck and P. tetrastramonia Hauck were collected from Oman Sea in 2009. Their scientific names were verified by Dr. Bairam Gharanjik, Offshore Waters Research Center, Chabahar, Iran.

Voucher specimens TMRC-3513 and TMRC-3510 were assigned to the herbarium samples, respectively and they were kept at the Herbarium of Traditional Medicine and Materia Medica Research Center, Tehran, Iran for future reference.

Chemicals and Reagents: Acetylthiocholine iodide (ATCI), acetyl-cholinesterase enzyme (AChE) from bovine erythrocytes, 5, 5′-dithiobis [2-nitrobenzoic acid] (DTNB), [3-(4, 5-dimethyl thiazol-2-yl)-2, 4-diphenyl tetrazolium bromide] (MTT) and 2, 2-diphenyl-1-picryl hydrazyl (DPPH) were provided from Sigma (Germany). All solvents were purchased from Merck (Germany). The mediums and material used in MTT assay were provided from Sigma.

Cell Lines: The cancerous cell lines to be used in the cytotoxicity evaluation assay were provided from Pasture Institute, Tehran, Iran. A-549 (non-small cell lung carcinoma), HT-29 (human colorectal adenocarcinoma, HepG-2 (human heap-tocellular carcinoma) and MCF-7 (human breast adenocarcinoma) cells were cultured in suitable medium and seeded in 96-well plates, each well containing 8×103, 5×103,  15×103 and  8×103 for A-549, HT-29, HepG-2 and MCF-7 cells, respectively. A-549 and HepG-2 cells were cultured in RPMI-1640 medium containing 5% FBS; while MCF-7 and HT-29 cells were maintained in DMEM medium enriched with 5 and 10% FBS, respectively. All media contained 1% penicillin-streptomycin and the cells were kept in humidified atmosphere with 5% CO₂.

Extraction: The algae were air dried and ground. Twenty mg of each alga powder was then extracted with 80% methanol through maceration. After 24h, the filtrate was removed and the residues of the algae were macerated again with 80% methanol. The process continued thrice. The filterate were combined and concentrated.

MTT Assay: The ability of the live cells to reduce MTT, [3-(4, 5-dimethylthiazol- 2-yl)-2, 4 diphenyl-tetrazolium bromide] to its formazan salt was measured by exposing the cell lines to different concentrations of the extracts (3.125, 6.25, 12.5, 25, 50, 100μg/mL in DMSO 1% obtained by serial dilutions) for 72 hr MTT solution (final concentration 0.5mg/mL) was added to each well after removing the media containing the extract. The resulting formazan crystals were dissolved in DMSO 4 hr later. The absorbance was recorded with an ELISA reader at 570nm. The viability was measured according to the following equation:

Viability (%) = A_s / A_c × 100

Where A_s is the absorbance of wells with sample and A_c is the absorbance of wells in absence of sample. IC₅₀ was calculated according to the data plotted with Microsoft Excel ^{4 - 6}.

Ellman Assay: The acetyl cholinesterase inhibitory activity of algae extracts was assessed with Ellman assay. Briefly, 25μL of sample (concentration 3mg/mL of each extract) in methanol was added to each well of 96-well plates. 1250μL of 3mM DTNB, 25μL of 15mM ATCI and 50μL of phosphate buffer (pH 8) were also added to each well. The absorbance was recorded every 13 sec for 65 sec (405nm) with a TECAN microplate reader. Twenty five μL of 0.22U/mL AChE enzyme was added afterwards and the absorbance was measured every 13 sec for 104 sec. By plotting the absorbance vs time, enzyme activity was determined by comparing the rates for the sample to the blank (methanol). Donepezil was used as the positive control ^{7, 8}.

DPPH Assay: The antioxidant property of the algae extracts was measured using DPPH radicals. In case DPPH radicals meet a hydrogen donator substance, they will lose their colour and the absorbance (at 520nm) would decrease. The DPPH methanol solution (2mL, 100μM) was added to 2mL of different concentrations of the extracts followed by strong shaking. The mixture was then left at room temperature (30 min). The absorbance was measured at 517nm and the antioxidant capacity was calculated using the following equation. IC₅₀ was calculated accordingly.

Scavenging capacity % = 100 - [(ABS of sample-ABS of blank) ×100/ABS of control]

The blank, negative and positive controls were 2mL of methanol along with 2mL of the extract solution, 2mL DPPH solution with 2mL methanol and Vitamin C, respectively ^{9, 10}.

RESULTS AND DISCUSSION: The methanol extracts of P. australis and P. tetrastromatica did not show cytotoxic activity on A-549, HT-29, HepG-2 and MCF-7 cells upto the concentration of 100μg/mL. The antioxidant and AChEI activities have been presented in Table 1. Each experiment was done in triplicate.

TABLE 1: THE ANTIOXIDANT AND ACHEI ACTIVITY OF PADINA AUSTRALIS AND PADINA TETRASTROMATICA TOTAL EXTRACTS

As mentioned earlier, algae have recently become the center of attention in biological researches. P. australis and P. tetrastromatica were of no exception and some biological investigations from different regions of the word have been conducted on these seaweeds in recent years.

The methanol extracts of Padina australis collected from the Pramuka Island, Indonesia, has gone through antioxidant assay and has shown the IC₅₀ of 267.1 ppm in DPPH assay ¹¹; while different fraction of this alga from the east coastal regions of Penyabong, Malaysia, have demonstrated the range of 1.63 – 130.14μg/mL for IC₅₀; the value has increased according to the polarity of the solvents ¹². However, Dotulong et al., observed almost no activity from hexane, ethyl acetate and water fractions of Padina australis from North Sulawesi Nain Islandin DPPH assay ¹³. In another study from Indonesia, P. australis ethanol and aceton extracts were evaluated in DPPH assay but the IC₅₀ values were rather high compared to other mentioned studies ¹⁴.

The IC₅₀ values of 0.276 ± 0.01 and 0.649 ± 0.03mg/mL have been reported for the dichloromethane and methanol fractions of Padina australis from Malaysia, as reported by Gany et al., ¹⁵. The free radical scavenging ability of Padina australis methanol extract was found to be 163.9 ± 13.2(μg/mL) in our study. The classified informa-tion in Table 2 show that our results are somehow in accordance with most studies though others were not able to record the antioxidant activity or they recorded rather low values. It should be noted that different methods of extraction and different geographical conditions might also be the cause of these differences.

TABLE 2: RESULTS OF DPPH ASSAY OF PADINA AUSTRALIS FROM DIFFERENT GEOGRAPHICAL LOCATIONS

Cytotoxic studies on the same species collected from Malaysia, have led to the isolation of fucoxanthin which has shown to be cytotoxic to human lung cancer cells (H1299) with IC₅₀ value of 2.45mM ¹⁶. In our current study, no cytotoxic activity was observed to the maximum tested concentration (100μg/mL). As fucoxanthin from the species has shown cytotoxicity in the study of Jaswir et al., it could be assumed that higher concentrations of the extract might be able to induce toxicity in the tested cell lines of our study.

In previous studies for finding AChE enzyme inhibitory activity in natural sources, the methanol extract of Padina australis collected from Coasts of Persian Gulf has not shown much inhibition of the enzyme (IC₅₀ 6.3mg/mL) in a study conducted by Ghnannadi et al., ¹⁷; whereas the dichloromethane fraction of the alga was reported to inhibit the acetylcholinestrase enzyme with IC₅₀ value of 0.149 ± 0.046mg/mL; while this value was > 0.2 mg/mL for inhibiting butyrylcholinesterase. IC₅₀ value was > 0.2 mg/mL for the methanol fraction in inhibition of both enzymes in the same study ¹⁵. Another study reported that the n-butanol fraction of the Padina australis collected from Malaysia, showed IC₅₀ value of 1.54 ± 0.045mg/mL for inhibiting AChE enzyme12. In our present study, Padina australis methanol extract could inhibit the AChE enzyme (15.6% ± 1.4) at the concentration of 300μg/mL. Comparing the present result with previous data, it could be suggested that our result are similar to previously published data except for the study of Ghannadi et al., ¹⁷ (Table 3).

TABLE 3: RESULTS OF ACHEI EVALUATION OF PADINA AUSTRALIS FROM DIFFERENT GEOG-RAPHICAL LOCATIONS

 As for P. tetrastromatica, several studies have been carried out to define its biological effects. In a study conducted by Sarojini et al., the ethanol extract of the alga collected from coasts of India, was able to scavenge DPPH radicals with the IC₅₀ of 574μg/mL ¹⁸. In another study, fucoidan was isolated from the ethanol extract of P. tetrastromatica which demonstrated 66.34% inhibition of DPPH radicals at the concentration of 1mg/mL ¹⁹. Also, with aqueous extract of P. tetrastromatica, at the concentrations of 0.3-0.6 mg/mL, the DPPH radicals were inhibited in the range of 13.04 ± 1.14 to 55.85 ± 56% growing as the concentration increased ²⁰; while methanol extract of samples from India demonstrated IC₅₀ of 0.61 ± 0.005mg/mL in DPPH assay. The ethyl acetate fraction of P. tetrastromatica from Malaysia gave the IC₅₀ 171.67 ± 2.89μg/mL which is a bit lower compared to the above mentioned studies ^{21, 22}. The results presented in Table 4 show that P. tetrastromatica from Iran and Malaysia has shown stronger activity for scavenging DPPH radicals. Considering that all other studies were conducted on the Indian alga, it comes in to the mind that different localities might have made some changes in the chemicals responsible for the observed effects.

Padina tetrastromatica has also been evaluated for cytotoxic activity in a few studies. Its methanol derived extract has been found to be active against MCF-7 cells (IC₅₀ 125 ± 2.03μg/mL) during a study in Malaysia ²³; while in the same study that was mentioned for P. australis from Chahbahar, again no toxicity was observed. Since our study used limited extract concentrations (up to 100μg/mL), it could be proposed that higher concentrations might lead to observation of cyto-toxicity. There is not much data about the AChEI activity of P. tetrastromatica in literature; however the methanol extract showed 23.7 ± 0.6 inhibition of the enzyme in our study at the concentration of 300μg/mL.

TABLE 4: RESULTS OF DPPH ASSAY OF P. TETRASTROMATICA FROM DIFFERENT GEOG- RAPHICAL LOCATIONS

CONCLUSION: Taking all into consideration, it is concluded that both alga do not seem to be potent in presenting antioxidant, acetylcholinestrase inhibitory or cytotoxic activity. The overall results of the present study along with the previously published data show that high amounts of the extracts are needed for presenting biological activities. As an alternative, it is suggested that instead of increasing the concentrations, one should focus on isolating the compounds that might be responsible for the biological activities. By isolating and purifying the compounds, stronger activity of potent compounds might be observed; these compounds would then be promising for continuing studies for finding medications of marine origin.

ACKNOWLEDGMENTS: The authors wish to thanks the Traditional Medicine and Materia Medica Research Center (TMRC) Shahid beheshti University of Medical Sciences, Tehran, Iran for the financial support of the project. The data have been obtained from a Pharm. D student thesis (Helia Soleimani).

DECLARATION OF INTEREST: The authors declare that there is no conflict of interest.

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    How to cite this article:

    Hajimehdipoor H, Gharanjik B, Soleimani H, Mousavi Z and Hamzeloo-Moghadam M: Two algae from oman sea: the in vitro biological effects. Int J Pharm Sci Res 2017; 8(10): 4286-90.doi: 10.13040/IJPSR.0975-8232.8(10).4286-90.

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