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

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.

Figure 1

Map of seaweed samples collection location. 01-Ahangama (N 05⁰ 58.006′ E 080⁰ 22.482′), 02-Thalpe (N 05⁰ 59.792′ E 080⁰ 16.898′), 03-Chilaw (N 07⁰ 36.220′ E 079⁰ 47.120′), 04-Negambo (N 07⁰ 12.170′ E 079⁰ 48.570′), 05-Kankasanthurai (N 09⁰ 48.592′ E 080⁰ 02.546′), 06-Point Pedro (N 09⁰ 49.501′ E 080⁰ 15.119′).

Figure 2

Voucher specimens. SW01-Ulva lactuca, SW02-Caulerpa racemosa, SW03-Gracilaria corticata, SW0-Padina antillarum, SW05-Sargassum illicifolium, SW06-Sargassum polycystem, SW07-Halimeda opuntin, SW08-Caulerpa racemosa, SW09-Turbinaria ornata, SW10-Caulerpa sertularioides, SW11-Stoechospermum polypodioides, SW12-Sargassum illicifolium, SW13-Sargassum illicifolium, SW14-Gracilaria corticata, SW15-Padina antillarum, SW16-Ulva lactuca, SW17-Acanthophora spicifera, SW18-Gelidiopsis variabilis, SW19-Gracilaria corticata, SW20-Chaetomorpha antennina, SW21-Chaetomorpha crassa, SW22-Jania adhaereus, SW23-Sargassum illicifolium.

Figure 3

L929 cell viability and proliferation percentage against aqueous extracts of seaweed samples after 24 h incubation. A; Cell viability is given as a percentage with compared to the control test. B; L929 cell proliferation observed as a percentage of the scratch wound close area compared to the control test. SW01-Ulva lactuca; 19.54 μg/μl, SW02-Caulerpa racemose; 5.48 μg/μl, SW03-Gracilaria corticata; 6.45 μg/μl, SW04-Padina antillarum; 13.60 μg/μl, SW05-Sargassum illicifolium; 16.04 μg/μl, SW06-Sargassum polycystem; 6.40 μg/μl, SW07-Halimeda opuntin; 3.82 μg/μl, SW08-Caulerpa racemose; 8.66 μg/μl, SW09-Turbinaria ornate; 11.68 μg/μl, SW10-Caulerpa sertularioides; 8.50 μg/μl, SW11-Stoechospermum polypodioides; 5.86 μg/μl, SW14-Gracilaria corticata; 20.56 μg/μl, SW15-Padina antillarum; 18.24 μg/μl, SW16-Ulva lactuca; 47.72 μg/μl, SW17-Acanthophora spicifera; 0.10 μg/μl, SW18-Gelidiopsis variabilis; 7.44 μg/μl, SW19-Gracilaria corticata; 11.42 μg/μl, SW20-Chaetomorpha antennina; 17.02 μg/μl, SW21-Chaetomorpha crassa; 9.77 μg/μl, SW22-Jania adhaereus; 42.10 μg/μl, All values are expressed as mean±SEM (n=08). One-way analysis of variance (ANOVA) Tukey’s comparisons test. ∗indicates comparison with the Control, ∗p<0.05.

Figure 4

The migration and proliferation ability on L929 cells against the seaweed extracts. Microscopic inspection (40×magnifications) at 12 h and 24 h of scratch wound healing. SW01-Ulva faciata, SW02-Caulerpa racemosa, SW03-Gracilaria corticata, SW04-Padina antillarum, SW05-Sargassum illicifolium, SW06-Sargassum polycystem, SW07-Halimeda opuntin, SW08-Caulerpa racemosa, SW09-Turbinaria ornata, SW10-Caulerpa sertularioides, SW11-Stoechospermum polypodioides, SW14-Gracilaria corticata, SW15-Padina antillarum, SW16-Ulva lactuca, SW17-Acanthophora spicifera, SW18-Gelidiopsis variabilis, SW19-Gracilaria corticata, SW20-Chaetomorpha antennina, SW21-Chaetomorpha crassa,SW22-Jania adhaereus. Cell proliferation is given as a percentage compared to the control test. All values are expressed as mean ± SEM (n = 08). One-way analysis of variance (ANOVA) Tukey's comparisons test. ∗indicates significantly difference with Control, ∗p < 0.05.

Figure 5

Changes in wound area at each time point to the original wound area of mice in control and treatment group over a period of 12 days. A; Wound healing area (mm²) of test groups, B; Wound healing percentages (%) of test and control groups, C; Digital photographs of mice showing various stages of wound healing. Day 0 picture was taken immediately after injury. Values of diameter of wounds are expressed as mean ± SEM (n = 12). a = when compared with control group, (∗) indicates statistically significant difference from respective group using ANOVA, followed by Tukey's comparisons test (p > 0.05). (†) indicates statistically no significant difference from respective group using ANOVA, followed by Tukey's comparisons test (p > 0.05). Control group received an equal volume of distilled water, orally; untreated wounds and Treatment group received S. illicifolium extracts (400 mg/kg BW/day for 12 days, orally).

Figure 6

Photomicrographs epidermis sections of wound tissues; hematoxylin and eosin stain staining (H & E; 40 and 100). A; Histopathological skin sections determine the changes in wound healing events B; Granulation tissue of the wounded skin. Arrows pointing events during wound healing; s, scab; re, re- epithelialization; IC, inflammatory cells; nv, neovascularization, GT: granulation tissue, mnc: mononuclear; F: fibroblasts; CF: collagen fiber, NE: new epithelium. Control (C): received an equal volume of distilled water, orally; untreated wounds, Treatment (T): received S. illicifolium extracts (400 mg/kg BW/day for 12 days, orally).

Figure 7

RT-PCR analysis for mRNA expression of mice wound tissue samples over a period of 12th days. A; TGF-β expression during the wound healing of treatment and control (Ratios of TGF-β on 3,6,9 and 12 days post injury treatment and control). B; TNF-α expression during the wound healing of treatment and control (Ratios of TNF-α on 3,6,9 and 12 days post injury treatment I and control). C; β-Actin expression during the wound healing of treatment and control (Ratios of β-Actin on 3,6,9 and 12 days post injury treatment I and control). Control: Wound were created and received an equal volume of distilled water, orally, Treatment: Wound were created and received S. illicifolium extracts (400 mg/kg BW/day for 14 days, orally). Values are expressed as mean ± SEM (n = 4 animals); Data is compared against control group. One way analysis of variance (ANOVA) Tukey-Kramer multiple comparisons test ∗p < 0.05. 3T–3 Day Treatment, 3C- 3 Day Control, 6T - 6 Day Treatment, 6C - 6 Day Control, 9T - 9 Day Treatment, 9C - 9 Day Control, 12T - 12 Day Treatment, 12C - 12 Day Control, N – Negative Control, L - Ladder. Full, non-adjusted images of RT-PCR blots include in Supplementary Figure 1.

Figure 8

Biochemical values of mice of each test group over a period of the 26^th day. A; Serum aspartate aminotransferase (AST) levels in mice, B; Alanine aminotransferase (ALT) levels in mice, C; Serum creatinine levels in mice. Data are expressed as mean ± SEM (n = 12 mice); Data is compared against control group. a = when compared with control group, b = when compared with treatment II Group, (∗) indicates statistically significant difference from respective group using ANOVA, followed by Tukey's comparisons test (p > 0.05). (†) indicates statistically no significant difference from respective group using ANOVA, followed by Tukey's comparisons test (p > 0.05). Control group received an equal volume of distilled water, orally; untreated wounds and Treatment group received S. illicifolium extracts (400 mg/kg BW/day for 12 days, orally).

Figure 9

Histopathological sections of heart, liver, lungs, spleen, and kidney in mice treated with seaweed extracts and the control. Vital organs of mice stained with hematoxylin and eosin stain (H & E; 40×). Control (C): received an equal volume of distilled water, orally, Treatment (T): received S. illicifolium extracts (400 mg/kg BW/day for 12 days, orally).