Preliminary screening of the aqueous extracts of twenty-three different seaweed species in Sri Lanka with in-vitro and in-vivo assays

Amal D Premarathna; T H Ranahewa; S K Wijesekera; D L Harishchandra; K J K Karunathilake; Roshitha N Waduge; R R M K K Wijesundara; Anura P Jayasooriya; Viskam Wijewardana; R P V J Rajapakse

doi:10.1016/j.heliyon.2020.e03918

Preliminary screening of the aqueous extracts of twenty-three different seaweed species in Sri Lanka with in-vitro and in-vivo assays

Heliyon. 2020 Jun 5;6(6):e03918. doi: 10.1016/j.heliyon.2020.e03918. eCollection 2020 Jun.

Affiliations

¹ Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka.
² Department of Zoology, Faculty of Natural Sciences, Open University, Kandy Regional Center, Polgolla, Sri Lanka.
³ Department of Pathobiology, Faculty of Medicine, University of Peradeniya, Sri Lanka.
⁴ Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka.
⁵ Department of Basic Veterinary Sciences, Faculty of Medical Sciences, University of West Indies, Trinidad and Tobago, West Indies.
⁶ Department Atomic Energy Agency (IAEA) Vienna, Austria.

Abstract

Background: Seaweeds are an important source of bioactive compounds which are applied in various aspects of medicinal investigations. The present study was conducted to investigate cytoxicity (in-vitro and in-vivo) and wound healing activity of different seaweed species in Sri Lanka.

Methods: Twenty-three seaweed samples, belonging to Phaeophyta (Brown), Chlorophyta (Green) and Rhodophyta (Red) were used for the experiments. Samples were collected from the inter-tidal and the sub-tidal habitats around Sri Lankan coast (Southern, Northern and North-western). Aqueous seaweed extracts were tested for cytotoxic and wound healing activity; in-vitro and in-vivo. To determine toxicity of aqueous seaweed extracts, brine shrimp lethality assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on mouse fibroblasts (L929) cell line were performed. Cell migration induction of seaweed extracts was assessed by scratch wound healing assay using L929 cell line. Based on the our previous experiments S.ilicifolium (SW23) was selected for the in vivo study to confirm our hypothesis. Albino mice (BALB/c) were divided into three groups (12 in each) and a circular area (44.07 ± 02.51 mm²) of full skin was excised to create a wound in mice group II and III. Group III received aqueous extract of Sargasum illicifolium (400 mg/kg BW/day for 12 days, orally), Group II received distilled water for 12 days whereas Group I was used as the control group and it was tested without forming wounds and without providing any treatment. Further, the expression level of Tumor Necrosis Factor (TNF-α) and Transforming Growth Factor-β (TGF-β) via RT-PCR were measured every three days until the end of the experiment.

Results: Phytochemical tests showed positive results to flavonoids in all the selected green seaweeds and alkaloids were observed in red seaweeds. In the toxicity assay, red seaweed, Acanthophora spicifera (SW17) was found to be highly effective on nauplii of brine shrimp (LC₅₀ = 0.072 μg/μl). LC₅₀ value of green seaweed species, Caulerpa racemosa (SW02 and SW08) and Caulerpa sertularioides (SW10) was not found within the tested concentration series. The highest cytotoxic effect on L929 cell line was exhibited by aqueous extracts of red seaweed; Jania adhaereus with 50.70 ± 7.304% cell viability compared with control group. The highest cell migration activity was observed in L929 cell line group treated with extracts of green seaweed namely; Halimeda opuntin (SW07) and extracts of brown seaweed namely; Stoechospermum polypodioides (SW11). Extracts of S. illicifolium (SW23) exhibited a significantly enhanced wound healing activity in mice group III within three days (P < 0.05) with an open wound area of 17.35 ± 1.94 mm² compared with control group (26.29 ± 2.42 mm²). TGF-β gene expression peaked on 6^th day of post-wound and subsequently decreased on 9^th day of post-wound in mice group III. TNF-α expression was suppressed in mice group III whereas it was elevated in group II. TGF-β expression is enhanced in the treatment group compared to the control group.

Conclusions: Aqueous extracts of selected seaweeds are a significant source of potential compounds with wound healing properties, which might be helpful in the healing of various wounds. This also infers that many species of brown and red seaweeds have the potential of wound healing, specifically, Sargasum illicifolium and Jania adhaereus could be a potential candidate for in-vivo studies related to wound healing and cancer therapy in the near future.

Keywords: Albino mice; Alternative medicine; Animal behavior; Biochemistry; Brine shrimp; Cell viability; Immunology; L929; Laboratory medicine; Molecular biology; Pharmacology; Phytochemical; Seaweeds; Surgery; TGF-β; TNF-α; Toxicity; Toxicology; Wound healing; m-RNA expression.