In Vitro Anti-Oxidant, In Vivo Anti-Hyperglycemic, and Untargeted Metabolomics-Aided-In Silico Screening of Macroalgae Lipophilic Extracts for Anti-Diabetes Mellitus and Anti-COVID-19 Potential Metabolites.

COVID-19 patients with comorbid DM face more severe outcomes, indicating that hyperglycemic conditions exacerbate SARS-CoV-2 infection. Negative side effects from existing hyperglycemia treatments have urged the need for safer compounds. Therefore, sourcing potential compounds from marine resources becomes a new potential approach. Algal lipids are known to possess beneficial activities for human health. However, due to limitations in analyzing large amounts of potential anti-hyperglycemic and anti-COVID-19-related marine metabolites, there is an increasing need for new approaches to reduce risks and costs. Therefore, the main aim of this study was to identify potential compounds in macroalgae Sargassum cristaefolium, Tricleocarpa cylindrica, and Ulva lactuca lipophilic extracts for treating DM and COVID-19 by an integrated approach utilizing in vitro anti-oxidant, in vivo anti-hyperglycemic, and metabolomic-integrated in silico approaches. Among them, S. cristaefolium and T. cylindrica showed potential anti-hyperglycemic activity, with S. cristaefolium showing the highest anti-oxidant activity. A GC-MS-based untargeted metabolomic analysis was used to profile the lipophilic compounds in the extracts followed by an in silico molecular docking analysis to examine the binding affinity of the compounds to anti-DM and anti-COVID-19 targets, e.g., α-amylase, α-glucosidase, ACE2, and TMPRSS2. Notably, this study reveals for the first time that steroid-derived compounds in the macroalgae T. cylindrica had higher binding activity than known ligands for all the targets mentioned. Studies on drug likeliness indicate that these compounds possess favorable drug properties. These findings suggest the potential for these compounds to be further developed to treat COVID-19 patients with comorbid DM. The information in this study would be a basis for further in vitro and in vivo analysis. It would also be useful for the development of these candidate compounds into drug formulations.

Brown Macroalgae Sargassum cristaefolium Extract Inhibits Melanin Production and Cellular Oxygen Stress in B16F10 Melanoma Cells.

The brown macroalgae Sargassum has been reported for its anti-UV and photoprotective potential for industrial applications. This study evaluated the melanin inhibition activity of Sargassum cristaefolium (SCE) ethanol extract. Melanogenesis inhibition by SCE was assessed in vitro with B16-F10 melanoma cell models and in silico against melanin regulatory proteins Tyrosinase (TYR) and Melanocortin 1 Receptor (MC1R). The regulatory properties evaluated were the melanin content, intracellular tyrosinase activity and cellular antioxidant activities. In addition, the bioactive compounds detected in SCE were subjected to molecular docking against TYR and MC1R. Based on the results, 150 µg/mL SCE effectively inhibited the production of melanin content and intracellular tyrosinase activity. Cellular tyrosinase activity was reduced by SCE-treated cells in a concentration-dependent manner. The results were comparable to the standard tyrosinase inhibitor kojic acid. In addition, SCE effectively decreased the intracellular reactive oxygen species (ROS) levels in B16-F10 cells. The antioxidant properties may also contribute to the inhibition of melanogenesis. In addition, LCMS UHPLC-HR-ESI-MS profiling detected 33 major compounds. The results based on in silico study revealed that the bioactive compound putative kaurenoic acid showed a strong binding affinity against TYR (-6.5 kcal/mol) and MC1R (-8.6 kcal/mol). However, further molecular analyses are needed to confirm the mechanism of SCE on melanin inhibition. Nevertheless, SCE is proposed as an anti-melanogenic and antioxidant agent, which could be further developed into cosmetic skin care products.

Multivariate Analysis Revealed Ultrasonic-Assisted Extraction Improves Anti-Melanoma Activity of Non-Flavonoid Compounds in Indonesian Brown Algae Ethanol Extract.

Indonesia has high biodiversity of algae that are under-utilised due to limitations in the processing techniques. Here, we observed the effect of two different extraction methods (cold maceration and ultrasonic-assisted extraction (UAE)) on multiple variables of Indonesian brown algae ethanol extracts (Sargassum polycystum, Sargassum cristaefolium, Sargassum aquifolium and Turbinaria ornata). The variables observed included metabolites screening by untargeted metabolomics liquid chromatography-high-resolution mass spectrometry (LC-HRMS), observation of total phenolic content (TPC), total flavonoid content (TFC), anti-oxidant and B16-F10 melanoma cells cytotoxicity. UAE extracts had higher extraction yield and TPC, but no TFC difference was observed. UAE extract had more lipophilic compounds, such as fatty acids (Palmitic acid, Oleamide, Palmitoleic acid, Eicosapentaenoic acid, α-Linolenic acid, Arachidonic acid), lipid-derived mediators (11,12-Epoxyeicosatrienoic acid ((±)11(12)-EET)), steroid derivatives (Ergosterol peroxide), lipophilic metabolite (Fucoxanthin), and lipid-soluble vitamins (all-trans-retinol). Principle component analysis (PCA) revealed that TPC, not TFC, in the UAE extracts was correlated with the anti-oxidant activities and cytotoxicity of the extracts towards B16-F10 melanoma cells. This means other non-flavonoid phenolic and lipophilic compounds may have contributed to its bioactivity. These results suggest that out of the two methods investigated, UAE could be a chosen method to extract natural anti-melanogenic agents from brown algae.

The Antiproliferative and Apoptosis-Inducing Effects of the Red Macroalgae Gelidium latifolium Extract against Melanoma Cells.

The red macroalga Gelidium latifolium is widely distributed in the coastal areas of Indonesia. However, current knowledge on its potential biological activities is still limited. In this study, we investigated the potential bioactive compounds in Gelidium latifolium ethanol extract (GLE), and its cytotoxic effects against the murine B16-F10 melanoma cell line. GLE shows high total phenolic content (107.06 ± 17.42 mg GAE/g) and total flavonoid content (151.77 ± 3.45 mg QE/g), which potentially contribute to its potential antioxidant activity (DPPH = 650.42 ± 2.01 µg/mL; ABTS = 557.01 ± 1.94 µg/mL). ESI-HR-TOF-MS analysis revealed large absorption in the [M-H]- of 327.2339 m/z, corresponding to the monoisotopic molecular mass of brassicolene. The presence of this compound potentially contributes to GLE's cytotoxic activity (IC50 = 84.29 ± 1.93 µg/mL). Furthermore, GLE significantly increased the number of apoptotic cells (66.83 ± 3.06%) compared to controls (18.83 ± 3.76%). Apoptosis was also confirmed by changes in the expression levels of apoptosis-related genes (i.e., p53, Bax, Bak, and Bcl2). Downregulated expression of Bcl2 indicates an intrinsic apoptotic pathway. Current results suggest that components of Gelidium latifolium should be further investigated as possible sources of novel antitumor drugs.

Macroalgae Sargassum cristaefolium Extract Inhibits Proinflammatory Cytokine Expression in BALB/C Mice.

Ultraviolet radiation (UVR) which could induce skin damage and skin disease is a growing concern due to the increase in global warming. Brown macroalgae Sargassum cristaefolium has been recognized to exhibit UV protective activities. However, the mechanism of its photoprotective activity remains unclear. The purpose of this study is to investigate the potential mechanism of S. cristaefolium's photoprotective activity against UV radiation. Phytochemical analyses revealed valuable bioactive compounds in SCE, such as fucoxanthin which is widely known as an anti-inflammatory carotenoid. Treatment with SCE before UV-A radiation show reduced levels of wrinkles and desquamation. Interestingly, SCE treatment induces the skin healing process after UV radiation. SCE effectively inhibited proinflammatory TNF-α and IL-6 expression while increasing IL-10 production in the BALB/c mice skin. Current results suggest that SCE potentially protects the skin by attenuation of inflammatory cytokines. In addition, SCE demonstrates promising antibacterial activity (MIC = 1.302 µg/mL) against Staphylococcus aureus. Overall, SCE could be a source of an effective anti-inflammatory agent protecting against UV irradiation-induced skin damages.

Hypoglycemic Effect of Combination of Azadirachta indica A. Juss. and Gynura procumbens (Lour.) Merr. Ethanolic Extracts Standardized by Rutin and Quercetin in Alloxan-induced Hyperglycemic Rats.

PURPOSE: Exploration of plant combinations could be an alternative approach for diabetes treatment. The aim of this study is to evaluate the hypoglycemic effect of combination of A. indica and G. procumbens ethanolic extracts in alloxan-induced diabetic rats. METHODS: Powder of A. indica and G. procumbens leaves were macerated with ethanol 70%. Determination of rutin in A. indica and quercetin in G. procumbens were performed by TLC-densitometry. Hyperglycemia in rats was induced by an intraperitoneal injection of alloxan monohydrate at a single dose of 150 mg/kgBW. The rats were treated with 3 dosage variation of combinations for 15 days. Hypoglycemic effect was evaluated by estimating the blood glucose levels and the rats pancreas histological study. RESULTS: A. indica contained 2.90±0.15% of rutin and G. procumbens contained 18.86±0.86% of quercetin. Combination at the ratio of 50mg/kgBW A. indica:112.5mg/kgBW G. procumbens showed the highest hypoglycemic effect: 68.74±4.83% (preprandial) and 73.91±3.18% (postprandial). Histological studies indicated that this combination improved the morphology of the islets of Langerhans and ß cells. It also increased insulin expression and decreased the elevated-glucose concentrations. CONCLUSION: This study showed that combination of both extracts has better hypoglycemic effect than the single treatment of A. indica or G. procumbens. Combination of both extracts was potential to develop as a blood glucose-lowering agent for diabetic patients.