A REVIEW ON BIOACTIVE SECONDARY METABOLITES OF SEAGRASS OF THE SOUTHERN RED

Mehari Ghebretinsae M. 2 , Awet Adhanom B. 1 , Henok Ghebremedhin G. 1 and Biniam Tsegay B 1 . 1. Qualty control laboratory, Ministry of Marine Resources, MassawaEritrea. 2. Massawa College of Marine Science and Technology, MassawaEritrea. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 06 January 2018 Final Accepted: 08 February 2019 Published: March 2019


ISSN: 2320-5407
Int. J. Adv. Res. 7(3), 306-314 307 100 million years ago from land plants that returned to the sea in a least three separate lineages or families [12]. Moreover the term seagrass was probably introduced into the scientific literature by the first researcher Ascherson (1871), as reported by C. den Hartog, 2006 [11]. Importantly, Seagrass have a long history of being used for a variety of remedial purposes such as fever, skin diseases, muscle pains, wounds, and stomach problems, against stings of different kinds of rays, tranquillizers for babies [6] and for diabetes mellitus [12]. Besides of their pharmacological importance marine seagrass can also be used as a food (nutritious seeds), fertilizer (nutrient rich biomass), livestock feed (goats and sheep), packing material to transport fragile items and to ship fresh fish from the coast to cities [10].
In the sea, 3 types of plants occur: phytoplankton, seaweeds and seagrass. Seagrass are mostly found attached to the substrate with most restricted to sand habitats [13]. They form highly productive and dynamic ecosystems [14], which rank among the most productive ecosystems of the ocean [12]. These flowering plants have true roots, rhizomes or vertical stem, leaves that are interconnected by vascular system [15]. They belong to superorder Alismatiflorae (Monocotyledonae) in the plant kingdom. Globally there are about 72 species of seagrass distributed worldwide [3]. Of which 13 species of seagrass are known from the Western Indian Ocean, while 12 of them are found extending in the Red Sea [16]. Seagrass are classified into four families based on their morphological, anatomical and reproductive structure, namely Hydrocharitaceae, Zosteraceae, Cymodoceaceae, and Posidoniaceae [11].

Seagrass in Eritrea
Eritrea is located in between 12 45'N and 18 03'N latitude, and, 37 35'E and 43 07'E longitude. It possesses a mainland coastline of more than 1350 km along the south-western coast of the Red Sea. The Eritrean coastline stretches from its northern border with Sudan at Ras Kesar to its southern border with Djibouti at Ras Dumera and is endowed with biodiversified marine biota. In addition, the Red Sea of Eritrea contains more than 350 islands that form a shoreline of more than 1,950 km. The geographical extent of the Eritrean landmass is estimated about 124,000 km 2 and its territorial water covers an area of about 55,000 km 2 [17].
According to the Israeli expeditions done by Y. Lipkin Price et al. (1988) depicts that out of the 72 seagrass species found in the world, 11 species are available in the southern and central Red Sea [19]. Unpublished data of MMR shows that 8 out of the 11 seagrass species were identified in the Eritrean Red Sea, namely Halophyla ovalis, Halophyla stipulacea, Thalassia hempricae, Halodule uninervis, Syringodium isoetifolium, Thalassodendron ciliatum, Enhalus acoroides and Cymodecia rutandata. In addition, they reported that no significant geographical variation was observed among these species.

Antimicrobial Activity of Seagrass
The marine species from the sea offers a huge number of novel compounds, and it portrays a plethora of natural molecules to be evaluated for drug and other therapeutic activity [20]. Among them the ethanol leave extract of Halodule uninervis showed the strongest activity against Pseudomonas aeruginosa, while displaying the lowest activity against Staphylococcus aureus. Every solvent extract have shown activity against one or more of the selected bacterial pathogens, except the aqueous extract had exhibited no kind of inhibition with an exception to P. aeruginosa exhibiting at 14.67 mm zone. In general the result showed that the ethanolic leave extracts of seagrass H. uninervis gave better performance than other solvent extracts [5].
R. Karthikeyan and M.Sundarapandian (2017) tested the methanolic extract of H. uninervis plant in streptozotocin induced diabetic rats. The diabetic rats were administered orally with H. uninervis methanolic extract at two different doses (150 and 250 mg/kg b.wt./day) for 18 days and have showed a significant antidiabetic activity and improvements in hepatic and renal function. At dose levels of 150 and 250 mg/kg, glucose levels were decreased by 24.8% and 29.9% at the 6 th hour, respectively. Antidiabetic effect of the extract was slightly decreased at the 8 th 308 hour, but remained statistically significant. An overall reduction of 26% was observed on the 18 th day by 50mg/kg administration (p < 0.01). 52.5% reduction of glucose level in the serum observed at 18 th day administration at dose level of 150 mg/kg (p < 0.0001). 250mg/kg extract administration is more effective from 6 th day onwards with a reduction rate of 18.9% (p < 0.01) and maximum reduction of serum glucose level by 61.9% on the 18 th day. In addition the presence of saponin glycosides, steroids, and phenolic compounds in the phytochemical study of the extracts was inferred to be a reason for the good activity of extracts [2].
Likewise endophytic fungi isolated from seagrass Cymodocea serrulata, Halophila ovalis and Thalassia hemprichii of southern Thailand were screened for their antimicrobial activity against ten human pathogenic microorganisms by a colorimetric broth microdilution test. They obtained 160 endophytic fungi from these three seagrass species, of which 69% of the isolates have exhibited antimicrobial activity against at least one test strain. Isolates like Penicillium sp. PSU-ES43, Fusarium sp. PSU-ES73, Stephanonectria sp. PSU-ES172 and an unidentified endophyte PSU-ES190 from T. hemprichii have shown to possess strong antimicrobial activity against human pathogens with minimum inhibitory concentrations (MIC), less than 10µg/m. The most active genera found in this study were Fusarium, Penicillium, Stephanonectria and Trichoderma. The overall results have established that seagrass are a good source of antimicrobial producing endophytic fungi [21].
Research on antimicrobial and antioxidant activities of the tropical Seagrass, Halophila stipulacea, from El-Bardawil Lake, Egypt, was investigated by Howayda et al. (2012). Methanolic extract showed 40% antioxidant activity and aqueous extract of H. stipulacea was active against Gram positive bacteria (Bacillus subtilis), yeast (Candida albicans) and fungi (Aspergillus niger) with inhibition zone 15, 15 and 20 mm, respectively. Whereas the extract was not active against Gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa. In comprise to their antimicrobial activity the total phenolic extract was found to be 0.523 mg tannic acid per gram, which are reported to be responsible for the antioxidants and free radical scavengers potential of the seagrass. According to McMillan et al. (1980) the presence of unidentified sulphated phenolic compounds from H. stipulaceae has been reported [23].
In other research, antibacterial potential of three seagrass species extracts, Halophila stipulacea, Cymodocea serrulata and Halodule pinifolia, was tested against seven human bacterial pathogens viz S. Bodii, V. Cholerae, K. Pneumonia, S. Dysentriae, P. Eruginosa, S. paratyphii and S. Aureus using methanol, Hexane and chloroform solvents. Methanolic and chloroform extracts of all the three seagrass were active against all the tested pathogens, of which methanol extracts were more active. Whereas the hexane extract of all seagrass was not active against Staphylococcus aureus. Moreover hexane extract of C. serrulata was also not active against V. cholerae, S. paratyphi and K. pneumoniae. The highest zone of inhibition (11 mm) was recorded in methanol extract of H.
309 stipulacea against S. dysenteriae with MIC value of 100 µg/mL followed by (8 mm) with H. pinifolia and chloroform extract of C. serrulata against K. Pneumonia. Of the three seagrass species screened for their antibacterial activity in the investigation, H. pinifolia and H. stipulacea were more superior to C. serrulata against the human pathogens in controlling the microbial growth [24].
The antibacterial, antioxidant, and anti-inflammatory activities of methanolic extracts of H. ovalis were investigated. Methanol extract inhibited the growth of Bacillus cereus at a minimum inhibitory concentration of 50µg/mL and other Gram-negative pathogens at 75µg/mL, except Vibrio vulnificus. Reducing power and total antioxidant level increased with increasing extract concentration. H. ovalis exhibited strong scavenging activity on 2,2-diphenyl-1picrylhydrazyl (DPPH) and superoxide radicals at IC50 of 0.13 and 0.65 mg/mL, respectively. Methanol extract of H. ovalis showed noticeable anti-inflammatory activity at IC50 of 78.72µg/mL. The GC-MS analysis of H. ovalis revealed the presence of triacylglycerols as major components in purified fractions. Quantitative analysis of phytochemicals revealed that seagrass H. ovalis are rich in phenols. These findings demonstrated that the methanol extract of H. ovalis exhibited appreciable antibacterial, noticeable antioxidant, and anti-inflammatory activities, and thus could be used as a potential source for natural health products [9]. It was reported that C. serrulata, H. ovalis and H. pinifolia extracts have showed antibacterial activity against some ocular pathogens, while the control disks (DMSO) showed no inhibition. The negative control antibiotic amikacin inhibited all the pathogens. The extracts obtained using chloroform, Ethyl acetate and Ethanol of the three seagrass showed maximum activity against E.coli (8.66mm, 9mm and 7.33mm; 8.33mm, 9mm and 8mm; and 7.6mm, 7.3mm 7mm) and Corynebacterium sps (7mm, 8mm; 13.66mm, 11mm, 14.33mm; and 11.66mm, 13mm 14.66mm) respectively. No zones of inhibition were recorded for the remaining six pathogens in all the extracts. Of the three seagrass studied, minimum inhibitory concentrations of Halodule pinifolia extracts were effective in controlling the growth of Corynebacterium species and Cymodocea serrulata extracts in E.coli. H. ovalis extracts were effective in controlling the growth of Corynebacterium sps [20].
Vietnam and Indian seagrass species Enhalus acoroides, Halophila ovalis and Halodule pinifolia were studied for mediating non-lethal interactions on sessile bacterial (E.coli) and fungal (Candida albicans) cultures. The focus was mostly on anti-biofilm activity of the seagrass extracts without killing cells. Seagrass extracts seem to be more effective in preventing microbial adhesion on hydrophobic surfaces. The results showed that non-lethal concentrations of E. acoroides leaf extract: reduce bacterial and fungal coverage by 60.9 and 73.9% respectively; affect bacterial biofilm maturation and promote dispersion, up to 70%, in fungal biofilm; increase luminescence in Vibrio harveyi by 25.8%. In addition it was reported that E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extracts were having 310 ant-biofilm effect on C. albicans and E.coli biofilm in the first phase of biofilm formation, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces [26].
C.rotundata and S.isoetifolium contains large amount of bioactive compounds like saponins, flavonoids and alkaloids, so they are suspected to have pharmacological role [30,31]. The methanolic extract of both seagrass was tested against Sitophilus oryzae and Sitophilus zeamais. The result showed 96.5 % and 86.5% mortality at a volume of 500µl for 24 hrs respectively. On the other hand methanolic extract S.isoetifolium has shown 100% mortality against Sitophilus oryzae and 98% mortality against Sitophilus zeamais at 500µl for 24 hrs. This reveals that C.rotundata and S.isoetifolium have a great potent of insecticidal property [32].
Methanolic extract of S. isoetifolium and C. rotundata were tested against gram-positive bacteria and fungi. The investigation also explored the preliminary phytochemical analysis of the active biogenic compounds. Tannins, terpenoids, saponins, phenols, Alkaloids and glycosides were present in both S.isoetifolium and C.rotundata, while Flavonoids and steroids were only present in C.rotundata. Antimicrobial analysis of C.rotundata have shown maximum activity against the G + Staphylococcus aureus (17mm), G -Escherichia coli (15mm) and fungi, Aspergillus niger (15mm). On the other hand S.isoetifolium showed maximum activity against G + Staphylococcus aureus (15mm), G -Escherichia coli (8mm) and fungi, Aspergillus niger(12mm). Overall, higher antimicrobial activity was seen by C.rotundata to S.isoetifolium. This was suggested due to lack or presence of traces of saponins, tannins, phenolic compounds and flavanoids which are responsible for the antimicrobial action [31].
The ethanol extract of C. rotundata has shown lower antibacterial activity against Shigella (7mm) and P.fluorescence (7mm) when compared to the positive control streptomycin sulphate (9mm). Conversely the butanolic extract showed maximum activity against S. aerous (6mm) followed by P. vulgaris (5mm). Tannins, saponins, resins, proteins, acidic compounds, reducing sugars, terpenoids, cardiac glycosides and alkaloids were present in phytochemical analysis of the ethanolic and methanolic extracts, but phenols, steroids, catachols and flavonoids were absent [33].
Dihydrochaleone diglycoside has been isolated from the EtOAc of the Egyptian seagrass T.ciliatum. The antiinfluenza A virus activity of the new compound and asebotin was evaluated. The obtained result have shown that the inhibition doze concentration of asebotin was more than that of Thalassedendrone with IC50 = 2.00 and 1.96 µg/ml respectively, and the cytotoxic concentration (CC50) of 3.36 and 3.14 µg/ml respectively [35].

Antioxidant Activity of Seagrass
Studies have shown that there is a positive correlation between the increased dietary intake of natural antioxidants and the reduced coronary heart disease, cancer mortality, and longer life expectancy. Halophila ovalis, Syringodium isoetifolium and Halodule pinifolia have antioxidant property due to the presence of phenolic compound [36].
Danaraj Jeyapragash et al. (2016) evaluated the antioxidant activity of methanolic extracts from the rhizome and leave portion of six seagrass species. Among evaluated species higher DPPH radical scavenging activity was found in leaf (78.84 ± 0.87) and rhizome extracts (75.480 ± 0.97) of C. rotundata and the lowest scavenging activity was found in the leaf and rhizome extracts of H. ovate (12.01 ± 0.63 and 5.769 ± 1.14). Generally, maximum phenolic content and antioxidant activity were shown by C. rotundata followed by H. uninervis [37].
Ace Baehaki et al. (2016) investigated the phytochemistry and antioxidant activity of the methanol extracts from Halodule uninervis. It was found that the methanol extract of these seagrass produces interesting phytochemical compounds: flavonoids, alkaloids, steriod and phenols. While the free radical scavenging potential (DPPH method (IC50)) and the highest reducing power of H.uninervis was found to be 1.575 ppm and 1.381 ppm respectively. In addition they have suggested that the free radical scavenging activity of H. uninervis could be due to their high content of flavonoids components which interrupts the propagation of autoxidation by donating hydrogen atom of some hydroxyl (OH) bases that are attached outside the benzene rings, resulting in the formation of stable free radical [7]. Neelima (2015) determined the antioxidant radical scavenging activity of seven seagrass species from Mandapam coast of South India. Ethyl acetate extracts fractions of Thalassia hemprichii have exhibited high DPPH radical scavenging activity (94.34 %), which is comparable to the positive controls highest being 95.6±0.94. This ascertained that, this activity is due to the presence of phenolic compounds which act as free radical scavengers and antioxidants. While the the aqueous fractions showed little or negligible DPPH scavenging activity that suggestes the active DPPH radical scavengers of these seagrass are seem to be found in the polar and semi polar extract in nature [39].
Methanolic extract of C.rotundata was evaluated to have the smallest value in comparison to three solvents (nhexane, ethyl acetate and methanol). Methanol has found to be the best solvent for the extraction of phenols, while n-hexane has the lowest and ethyl acetate moderate extraction capacity. So the methanol extract of T.hempricii has large amount of phenol content, and the lowest n-hexane extract for the C. rotundata. In general methanol is able to extract the component phenol in high quantities, as ethyl acetate solvent produced high quantities in S.isoetifolium. Strongest DPPH radical scavenging activity was observed in ethyl acetate extract of S.isoetifolium with 96.34 ppm. The methanol extract of T.hempricii, C.rotundata and E.acoroides has DPPH radical scavenging activity of 214.68, 123.72 and115.79 ppm respectively. Besides, higher fiber content was registered by the spp E.acoroides (8.93g/100gm) and lowest content by S.isoetifolium (621gm/100gm). To wrap up all the seagrass spp have shown a promising antioxidant property [40].
Eight seagrass spp were tested for the search of safe and cheap antioxidant source. As a result, higher antioxidant activity was observed in H. pinifolia, H. stipulacea, and C. serrulata of 132.382, 75.027, and 32.415 mg ascorbic 312 acid equivalent/g respectively. When tested by thin layer chromatography, H. pinifolia showed strong antioxidant activity. Higher DPPH radical scavenging activities were recorded in H. pinifolia (68.066%) followed by H. stipulacea (67.413%) and C. serrulata (61.853%). The minimum DPPH radical scavenging activity was recorded in H. ovata (16.926%). The scavenging effect of standards on the DPPH radical decreased in the order: ascorbic acid > gallic acid, which was 57.28 and 55.753%, respectively. Highest ability for reducing Fe 3+ was observed in H. stipulacea (46.289 ± 1.002), and H. pinifolia(42.611 ± 0.800) compared to the other seagrass studied. Finally, from this particular study it has been concluded that seagrass have strong antioxidant properties due to the total phenolic content [41].

Conclusion
The Eritrean Red Sea contains diversified seagrass species which possess a plethora of bioactive compounds that can be candidates for drug development and other purposes. Hence, extensive research is required in these areas to create a safe and better future.